Expression and significance of interleukin-18.12 and tumor necrosis factor-α in intrahepatic cholestasis of pregnancy

Objective To investigate the effect of Interleukin(IL)-18,IL-12 and tumor necrosis factor-α(TNF-α)in hepatic injury in intrahepatic cholestasis of pregnancy(ICP).Methods Sixty-two cases of ICP patients(ICP group),30 cases of normal pregnant women(control group)and 30 cases of hepatitis B(HBV) women (hepatitis group) were recruited. Serum IL-18, IL-12 and TNF-α were examined by ELISA. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined by automatic biochemical analysis instrument. Results ( 1 ) In hepatitis group, serum concentrations of IL-18,IL-12 and TNF-α were (256±51 ) ng/L, ( 122±96) ng/L and (207±3) ng/L; serum levels of ALT and AST were(363±174) U/L and (359 ±237) U/L, respectively. In ICP group, serum concentrations of IL18, IL-12 and TNF-α were (72±32) ng/L, (42 ±28) ng/L and (48±14) ng/L; serum levels of ALT and AST were (201 ±128) U/L and ( 132±87) U/L, respectively. While in control group, serum concentrations of IL-18, IL-12 and TNF-α were (43 ± 13) ng/L, ( 10±3) ng/L and (33±9) ng/L; serum levels of ALT and AST were (13 ~ 4) U/L and (15 ± 3) U/L, respectively. Serum IL-18, IL-12, TNF-α, ALT and AST levels in hepatitis group were significantly higher than those in ICP group and control group ( P ＜0. 05 ).Serum IL-18, IL-12, TNF-α, ALT and AST levels in ICP group were significantly higher than those in control group(P ＜ 0. 05 ). (2) In severe ICP subgroup, serum concentrations of IL-18, IL-12 and TNF-α were (81 ±32) ng/L, (50 ±25) ng/L and(50 ± 14) ng/L; serum levels of ALT and AST were (269 ± 111 ) U/L and (181±73) U/L In mild ICP subgroup, serum concentrations of IL-18, IL-12 and TNF-α were (48 ±18 ) ng/L, (17 ± 4 ) ng/L and (40 ± 10 ) ng/L; serum levels of ALT and AST were (87±46) U/L and (50 ±21 ) U/L, respectively. Serum IL-18, IL-12, TNF-α, ALT and AST levels in severe ICP subgroup were significantly higher than those in mild ICP subgroup and control group (P ＜ 0. 05). And serum ALT and AST levels in mild ICP subgroup were significantly higher than those in control group(P ＜0. 05). (3) There were 16 cases with preterm birth (50%, 16/32 ) and 10 cases with meconium-stained amniotic fluid( 31%, 10/32 ) in severe ICP subgroup, significantly higher than those in mild ICP subgroup ( P＜ 0. 05 ), which contained 2 preterm births ( 7%, 2/30) and 1 meconium-stained amniotic fluid (3%, 1/30). While in control group, the numbers were 1(3%, 1/30)and 1(3%, 1/30),respectively. As for the cases of neonates whose 1 minute Apgar score were not more than 7, there were 2 cases, 1 case and 1 case in severe ICP subgroup, mild ICP subgroup and control group, respectively,which showed no significant difference(P＞ 0. 05). Conclusion Serum IL-18, IL-12 and TNF-α may be involved in the process of hepatic injury of ICP.     

Expression and significance of interleukin-18.12 and tumor necrosis factor-α in intrahepatic cholestasis of pregnancy

Objective To investigate the effect of Interleukin(IL)-18,IL-12 and tumor necrosis factor-α(TNF-α)in hepatic injury in intrahepatic cholestasis of pregnancy(ICP).Methods Sixty-two cases of ICP patients(ICP group),30 cases of normal pregnant women(control group)and 30 cases of hepatitis B(HBV) women (hepatitis group) were recruited. Serum IL-18, IL-12 and TNF-α were examined by ELISA. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined by automatic biochemical analysis instrument. Results ( 1 ) In hepatitis group, serum concentrations of IL-18,IL-12 and TNF-α were (256±51 ) ng/L, ( 122±96) ng/L and (207±3) ng/L; serum levels of ALT and AST were(363±174) U/L and (359 ±237) U/L, respectively. In ICP group, serum concentrations of IL18, IL-12 and TNF-α were (72±32) ng/L, (42 ±28) ng/L and (48±14) ng/L; serum levels of ALT and AST were (201 ±128) U/L and ( 132±87) U/L, respectively. While in control group, serum concentrations of IL-18, IL-12 and TNF-α were (43 ± 13) ng/L, ( 10±3) ng/L and (33±9) ng/L; serum levels of ALT and AST were (13 ~ 4) U/L and (15 ± 3) U/L, respectively. Serum IL-18, IL-12, TNF-α, ALT and AST levels in hepatitis group were significantly higher than those in ICP group and control group ( P ＜0. 05 ).Serum IL-18, IL-12, TNF-α, ALT and AST levels in ICP group were significantly higher than those in control group(P ＜ 0. 05 ). (2) In severe ICP subgroup, serum concentrations of IL-18, IL-12 and TNF-α were (81 ±32) ng/L, (50 ±25) ng/L and(50 ± 14) ng/L; serum levels of ALT and AST were (269 ± 111 ) U/L and (181±73) U/L In mild ICP subgroup, serum concentrations of IL-18, IL-12 and TNF-α were (48 ±18 ) ng/L, (17 ± 4 ) ng/L and (40 ± 10 ) ng/L; serum levels of ALT and AST were (87±46) U/L and (50 ±21 ) U/L, respectively. Serum IL-18, IL-12, TNF-α, ALT and AST levels in severe ICP subgroup were significantly higher than those in mild ICP subgroup and control group (P ＜ 0. 05). And serum ALT and AST levels in mild ICP subgroup were significantly higher than those in control group(P ＜0. 05). (3) There were 16 cases with preterm birth (50%, 16/32 ) and 10 cases with meconium-stained amniotic fluid( 31%, 10/32 ) in severe ICP subgroup, significantly higher than those in mild ICP subgroup ( P＜ 0. 05 ), which contained 2 preterm births ( 7%, 2/30) and 1 meconium-stained amniotic fluid (3%, 1/30). While in control group, the numbers were 1(3%, 1/30)and 1(3%, 1/30),respectively. As for the cases of neonates whose 1 minute Apgar score were not more than 7, there were 2 cases, 1 case and 1 case in severe ICP subgroup, mild ICP subgroup and control group, respectively,which showed no significant difference(P＞ 0. 05). Conclusion Serum IL-18, IL-12 and TNF-α may be involved in the process of hepatic injury of ICP.     

细胞因子信号传导负调控因子3在妊娠期肝内胆汁淤积症孕妇胎盘组织中的表达及其意义

Objective To detect the expression and significance of suppressor of cytokine signaling 3(SOCS3) in the placentas of pregnant women with intrahepatic cholestasis of pregnancy (ICP). Methods Totally, 44 ICP gravidas, including 21 severe ICP and 23 mild ICP who delvered through cesarean section at the Second Xiangya Hospital of Central South University from April 2008 to January 2009, were selected as the ICP group, and another 25 healthy pregnant women were chosen as control. Placentas of the above gravidas were collected and the expression and localization of SOCS3 were determined by immunohistochemical peroxidase streptomyces-avidin link (SP) method (indicated by the percentage ofpositive cells and average gray scale) and the levels of interleukin-10 (IL-10) and interferon-γ (IFN-γ)from placenta homogenation were measured by enzyme linked immunoadsorbent assay (ELISA). Results(1) SOCS3 were expressed in placentas of both groups mainly in the intracytoplasma of trophocyte.However, weakly positive, positive, and strongly positive expressions were found in the severe ICP, mild ICP and the control group, respectively. Almost no expression was detected in membrane and nucleus of the trophoblasts. (2) The percentage of SOCS3 positive cells in the severe ICP group was significantly lower than in the control and the mild ICP group, respectively [ (0.15±0.08)% vs (0.69±0.12)% and (0.42±0.09) % , P < 0.01 ]. The average gay SOCS3 in placental tissue in the severe ICP group was significantly higher than that in control and mild ICP group, respectively (204 ±7 vs 81 ±7 and 147 ± 7, P <0.01 ).(3) Significant lower level of IL-10 in placenta homogenation was found in the severe ICP group than in the control and mild ICP group [ ( 1.16 ± 0.68) μg/L, vs ( 1.39 ± 0.08) μg/L and ( 1.22 ± 0.75 ) μg/L,P < 0.01 ]. (4) The opposite results were found in the level of IFN-γin trophoblasts and the ratio of IFN-γ/IL-10 [ severe ICP group: ( 16.8 ± 0.7 ) μg/L and 16.02 ± 2.79; control group: ( 10.5 ± 0.3 ) μg/L and8.56 ±0.14; mild ICP group: ( 13.4 ±0.5) μg/L and 8.56 ±0.14, P <0.01]. (5) Negative correlation was shown between the percentage of SOCS3 positive cells in trophoblasts and the ratio of IFN-γ/IL-10 ( r =-0.685 and -0.702, P < 0.01 ), and the average gay SOCS3 was positively correlated with the ratio of IFN-γ/IL-10 (r=0.621 and 0.891, P<0.01) in both mild and severe ICP group. Conclousions SOCS3 may participate in the pathogenesis of ICP and its expression may affected by the severity of ICP, and SOCS3may also play a role in the immunological regulation in ICP patients.     

细胞因子信号传导负调控因子3在妊娠期肝内胆汁淤积症孕妇胎盘组织中的表达及其意义

Objective To detect the expression and significance of suppressor of cytokine signaling 3(SOCS3) in the placentas of pregnant women with intrahepatic cholestasis of pregnancy (ICP). Methods Totally, 44 ICP gravidas, including 21 severe ICP and 23 mild ICP who delvered through cesarean section at the Second Xiangya Hospital of Central South University from April 2008 to January 2009, were selected as the ICP group, and another 25 healthy pregnant women were chosen as control. Placentas of the above gravidas were collected and the expression and localization of SOCS3 were determined by immunohistochemical peroxidase streptomyces-avidin link (SP) method (indicated by the percentage ofpositive cells and average gray scale) and the levels of interleukin-10 (IL-10) and interferon-γ (IFN-γ)from placenta homogenation were measured by enzyme linked immunoadsorbent assay (ELISA). Results(1) SOCS3 were expressed in placentas of both groups mainly in the intracytoplasma of trophocyte.However, weakly positive, positive, and strongly positive expressions were found in the severe ICP, mild ICP and the control group, respectively. Almost no expression was detected in membrane and nucleus of the trophoblasts. (2) The percentage of SOCS3 positive cells in the severe ICP group was significantly lower than in the control and the mild ICP group, respectively [ (0.15±0.08)% vs (0.69±0.12)% and (0.42±0.09) % , P < 0.01 ]. The average gay SOCS3 in placental tissue in the severe ICP group was significantly higher than that in control and mild ICP group, respectively (204 ±7 vs 81 ±7 and 147 ± 7, P <0.01 ).(3) Significant lower level of IL-10 in placenta homogenation was found in the severe ICP group than in the control and mild ICP group [ ( 1.16 ± 0.68) μg/L, vs ( 1.39 ± 0.08) μg/L and ( 1.22 ± 0.75 ) μg/L,P < 0.01 ]. (4) The opposite results were found in the level of IFN-γin trophoblasts and the ratio of IFN-γ/IL-10 [ severe ICP group: ( 16.8 ± 0.7 ) μg/L and 16.02 ± 2.79; control group: ( 10.5 ± 0.3 ) μg/L and8.56 ±0.14; mild ICP group: ( 13.4 ±0.5) μg/L and 8.56 ±0.14, P <0.01]. (5) Negative correlation was shown between the percentage of SOCS3 positive cells in trophoblasts and the ratio of IFN-γ/IL-10 ( r =-0.685 and -0.702, P < 0.01 ), and the average gay SOCS3 was positively correlated with the ratio of IFN-γ/IL-10 (r=0.621 and 0.891, P<0.01) in both mild and severe ICP group. Conclousions SOCS3 may participate in the pathogenesis of ICP and its expression may affected by the severity of ICP, and SOCS3may also play a role in the immunological regulation in ICP patients.     

Interaction among peroxisome proliferators-activated receptor alpha, cytochrome P450 oxysterol 7α-hydroxylase and estrogen receptor and its association with intrahepatic cholestasis in pregnant rats

Objective To investigate the relationship between interaction of peroxisome proliferators-activated receptor alpha (PPARα), cytochrome P450 oxysterol 7α-hydroxylase (CYP7B1) and estrogen receptor (ER) and intrahepatic cholestasis in pregnant rats. Methods Eighty clean SD pregnant rats were selected and divided into four groups randomly with 20 in each. Since the 13th day of pregnancy,rats in the control group was injected subcutaneously with refined vegetable oil 2.0 ml · kg-1 · d -1 , those in the low-dose, moderate-dose and high-dose groups received 17-α-ethynylestradiol (EE) 1.0 mg · kg-1 · d-1,1.25 mg · kg-1 · d-1 and 1.5 mg · kg-1 · d-1, respectively. All rats were sacrificed at the 21at day of pregnancy and maternal hepatic tissues were collected. The serum levels of alanine aminotransferase(ALT), aspartate transaminase (AST), total bile acid (TBA) and bilirubin (BIL) were determined by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of PPARα, CYP7B1, Erα and Erβ in maternal rat livers were examined by real-time PCR. Results (1) Biochemical indicators: the serum levels of ALT,AST, TBA and BIL were significantly lower in the control group than in the rest 3 groups,respectively [ control group: (41.1 ± 2.8 ) U/L, (44.4 ± 3.6) U/L, (26.4 ± 5.6 ) μmol/L and( 2.8 ± 0.2)U/L;low-dose group: (48.2 ±3.4) U/L,(47.9 ±3.7) U/L,(36.4 ±4.2) μmol/L and (4.2 ±0.2) U/L;moderate-dose group: (70.4 ± 5.3 ) U/L, (68.4 ± 5.6) U/L, (64.3 ± 3.8 ) μmol/L and ( 6.2 ± 1.2)U/L; high-dose group: (72.4 ±7.6) U/L, (70.2 ±3.8) U/L, (72.4 ±7.8) μmol/L and (8.2 ±2.2)U/L, P<0.05], and those in the moderate or high-dose groups were higher than in the low-dose group (P<0.05). (2) mRNA expression of Erα and Erβ: the mRNA expression of Erα in pregnant rat livers increased in a dose-dependent manner, which were all significantly higher than that in the control group,respectively ( low-dose group: 0.76 ± 0.02 ); moderate -dose group: ( 0.99 ± 0.04; high-dose group:1.21 ±0.01 ;control group:0.65 ±0.01, P <0.05), but no difference was found among the 4 groups in the mRNA expression of Erβ ( P > 0.05 ). (3) mRNA expression of CYP7B1 and PPARα: the mRNA expression of CYP7B1 in pregnant rat livers increased from the low-dose group to the high-dose group, and were all higher than that of the control group ( low-dose group: 0.93 ± 0.01; moderate-dose group: 0.99 ±0.06; high-dose group: 1.22 ± 0.04; control group: 0.75 ± 0.02, P < 0.05 ). However, the mRNA expression of PPARα decreased from the low-dose group to the high-dose group, and were all lower than that of the control group (low-dose group: 0.83 ± 0.05; moderate-dose group: 0.71 ± 0.02; high-dose group:0.64 ± 0.03; control group: 1.35 ± 0. 05; P < 0.05 ) . Conclusions The down regulated mRNA expression of PPARα, caused by higher dose of estrogen, may increase the expression of CYP7B1 due to the ineffectiveness of the inhibition of PPARα on CYP7B1, which may further stimulate the Erα activity and then induce intrahepatic cholestasis. Abnormal expression of PPARα, CYP7B1 and ER may play a role in the pathogenesis of estrogen-induced intrahepatic cholestasis.     

Interaction among peroxisome proliferators-activated receptor alpha, cytochrome P450 oxysterol 7α-hydroxylase and estrogen receptor and its association with intrahepatic cholestasis in pregnant rats

Objective To investigate the relationship between interaction of peroxisome proliferators-activated receptor alpha (PPARα), cytochrome P450 oxysterol 7α-hydroxylase (CYP7B1) and estrogen receptor (ER) and intrahepatic cholestasis in pregnant rats. Methods Eighty clean SD pregnant rats were selected and divided into four groups randomly with 20 in each. Since the 13th day of pregnancy,rats in the control group was injected subcutaneously with refined vegetable oil 2.0 ml · kg-1 · d -1 , those in the low-dose, moderate-dose and high-dose groups received 17-α-ethynylestradiol (EE) 1.0 mg · kg-1 · d-1,1.25 mg · kg-1 · d-1 and 1.5 mg · kg-1 · d-1, respectively. All rats were sacrificed at the 21at day of pregnancy and maternal hepatic tissues were collected. The serum levels of alanine aminotransferase(ALT), aspartate transaminase (AST), total bile acid (TBA) and bilirubin (BIL) were determined by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of PPARα, CYP7B1, Erα and Erβ in maternal rat livers were examined by real-time PCR. Results (1) Biochemical indicators: the serum levels of ALT,AST, TBA and BIL were significantly lower in the control group than in the rest 3 groups,respectively [ control group: (41.1 ± 2.8 ) U/L, (44.4 ± 3.6) U/L, (26.4 ± 5.6 ) μmol/L and( 2.8 ± 0.2)U/L;low-dose group: (48.2 ±3.4) U/L,(47.9 ±3.7) U/L,(36.4 ±4.2) μmol/L and (4.2 ±0.2) U/L;moderate-dose group: (70.4 ± 5.3 ) U/L, (68.4 ± 5.6) U/L, (64.3 ± 3.8 ) μmol/L and ( 6.2 ± 1.2)U/L; high-dose group: (72.4 ±7.6) U/L, (70.2 ±3.8) U/L, (72.4 ±7.8) μmol/L and (8.2 ±2.2)U/L, P<0.05], and those in the moderate or high-dose groups were higher than in the low-dose group (P<0.05). (2) mRNA expression of Erα and Erβ: the mRNA expression of Erα in pregnant rat livers increased in a dose-dependent manner, which were all significantly higher than that in the control group,respectively ( low-dose group: 0.76 ± 0.02 ); moderate -dose group: ( 0.99 ± 0.04; high-dose group:1.21 ±0.01 ;control group:0.65 ±0.01, P <0.05), but no difference was found among the 4 groups in the mRNA expression of Erβ ( P > 0.05 ). (3) mRNA expression of CYP7B1 and PPARα: the mRNA expression of CYP7B1 in pregnant rat livers increased from the low-dose group to the high-dose group, and were all higher than that of the control group ( low-dose group: 0.93 ± 0.01; moderate-dose group: 0.99 ±0.06; high-dose group: 1.22 ± 0.04; control group: 0.75 ± 0.02, P < 0.05 ). However, the mRNA expression of PPARα decreased from the low-dose group to the high-dose group, and were all lower than that of the control group (low-dose group: 0.83 ± 0.05; moderate-dose group: 0.71 ± 0.02; high-dose group:0.64 ± 0.03; control group: 1.35 ± 0. 05; P < 0.05 ) . Conclusions The down regulated mRNA expression of PPARα, caused by higher dose of estrogen, may increase the expression of CYP7B1 due to the ineffectiveness of the inhibition of PPARα on CYP7B1, which may further stimulate the Erα activity and then induce intrahepatic cholestasis. Abnormal expression of PPARα, CYP7B1 and ER may play a role in the pathogenesis of estrogen-induced intrahepatic cholestasis.     

Effect of lipoxin A4 on interleukin-1β production of human monocytes from severe preeclamptic women and its relationship with cytoplasm free calcium concentration-an in vitro study

Objective To investigate the effect of lipoxin A4(LXA4) on interleukin-1β(IL-1β)production of monocytes in maternal peripheral blood from severe preeelampsia women and its relationship with cytosolic free calcium([Ca2+]i)concentration. Methods Peripheral venous blood was drawn from 15 women with severe preeelampsia(SPE group)and 20 normal pregnant women (control group)who were admitted to the First Affiliated Hospital of Wenzhou Medical College from October 2008 to May 2009.Monoeytes were obtained from peripheral blood with Ficolt density gradient centrifugation and then were suspended in RPMI-1640 culture supplemented with LXA4 at the final concentrations of 0,10 and 100 nmol/L,respectively.IL-1β levels in monocytes supernatant were detected by enzyme-linked immunosorbent assay.The cytoplasma[Ca2+]i of cultured monocytes and its variations affected by LXA4 were measured by laser scanning confocal microscope. Results (1) After incubation with different concentrations of LXA4(0,10,100 nmol/L)for 24 h,the levels of IL-1β in SPE group were(63.16±8.20)pg/L,(53.71±8.08)pg/L and(43.16±6.89)pg/L,respectively, indicating a significant inhibition effect on IL-1β level in a dose-dependent manner (P<0. 05). The IL-1β levels in the control group were (19.22±7.43) pg/L, (16.99±6.32) pg/L and (15. 18±5.24) pg/L, correspondingly (P>0.05). (2) Without LXA4, the [Ca2+ ]i concentrations of monocytes in the SPE group were higher than that of the control (1028.09 ± 160. 52 vs 323.61 ±87.86, P<0. 05). After treatment with 100 nmol/L LXA4, the [Ca2+ ]i concentration of monocytes significantly decreased in the SPE group (409.67± 116.73, P<0. 05). Conclusions In vitro LXA4 may inhibit the IL-1β production in monoeytes of SPE patients through decrease of the cytoplama [Ca2+ ]i concentration.     

Effect of lipoxin A4 on interleukin-1β production of human monocytes from severe preeclamptic women and its relationship with cytoplasm free calcium concentration-an in vitro study

Objective To investigate the effect of lipoxin A4(LXA4) on interleukin-1β(IL-1β)production of monocytes in maternal peripheral blood from severe preeelampsia women and its relationship with cytosolic free calcium([Ca2+]i)concentration. Methods Peripheral venous blood was drawn from 15 women with severe preeelampsia(SPE group)and 20 normal pregnant women (control group)who were admitted to the First Affiliated Hospital of Wenzhou Medical College from October 2008 to May 2009.Monoeytes were obtained from peripheral blood with Ficolt density gradient centrifugation and then were suspended in RPMI-1640 culture supplemented with LXA4 at the final concentrations of 0,10 and 100 nmol/L,respectively.IL-1β levels in monocytes supernatant were detected by enzyme-linked immunosorbent assay.The cytoplasma[Ca2+]i of cultured monocytes and its variations affected by LXA4 were measured by laser scanning confocal microscope. Results (1) After incubation with different concentrations of LXA4(0,10,100 nmol/L)for 24 h,the levels of IL-1β in SPE group were(63.16±8.20)pg/L,(53.71±8.08)pg/L and(43.16±6.89)pg/L,respectively, indicating a significant inhibition effect on IL-1β level in a dose-dependent manner (P<0. 05). The IL-1β levels in the control group were (19.22±7.43) pg/L, (16.99±6.32) pg/L and (15. 18±5.24) pg/L, correspondingly (P>0.05). (2) Without LXA4, the [Ca2+ ]i concentrations of monocytes in the SPE group were higher than that of the control (1028.09 ± 160. 52 vs 323.61 ±87.86, P<0. 05). After treatment with 100 nmol/L LXA4, the [Ca2+ ]i concentration of monocytes significantly decreased in the SPE group (409.67± 116.73, P<0. 05). Conclusions In vitro LXA4 may inhibit the IL-1β production in monoeytes of SPE patients through decrease of the cytoplama [Ca2+ ]i concentration.     

胎粪吸入综合征患儿支气管肺泡灌洗液肿瘤坏死因子-α、白细胞介素-8变化的临床研究

目的 探讨肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)在胎粪吸入综合征中的作用.方法 对47例胎粪吸入综合征(meeonium aspiration syndrome,MAS)患儿行支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)和血清TNF-α、IL-8检测;同时将MAS患儿随机分成固尔苏治疗组和常规治疗组,分别于治疗前、治疗后24 h检测TNF-α、IL-8水平,并进行比较.TNF-α、IL-8采用酶联免疫吸附法(ELISA)检测.结果 MAS患儿血清TNF-α、IL-8水平分别为(25.97±6.72)ng/L和(18.82±4.89)ng/ml,明显高于对照组(11.94±2.83)ng/L和(10.38±2.28)ng/L(P均<0.01);MAS患儿BALF中TNF-α、IL-8水平为(66.46±12.67)ng/L和(31.17±5.67)ng/L,明显高于其血清水平(25.97±6.72)ng/L和(18.82±4.89)ng/L(P均<0.01);固尔苏治疗组24 h后TNF-α、IL-8水平为(36.97±5.88)ng/L和(21.06±3.99)ng/L,比常规治疗组明显降低(44.38±9.10)ng/L和(26.46±5.01)ng/L(P均<0.01).结论 TNF-α、IL-8在MAS发病过程中起重要作用;检测BALF中TNF-α、IL-8水平比血清更加敏感;固尔苏应用能减少MAS患儿TNF-α、IL-8水平,减轻肺损伤.     

重组人黄体生成素在体外受精-胚胎移植促排卵治疗中的应用

Objective To evaluate application of recombinant human luteinizing hormone (r-hLH)used in ovarian stimulation of assisted reproductive technique and impact on outcome of pregnancy. Methods From Apr. To Jul. 2009, 123 patients with low LH level ( < 1 U/L) at day 3 of menstruation and downregulation of pituitary function undergoing in vitro fertilization-embryo transfer (IVF-ET) in Reproductive Medical Center, Provincial Hospital Affiliated to Shandong University were enrolled in this study, whom were classified into 66 cases treated by r-hLH in r-hLH group and 57 cases without r-hLH treatment in non-r-hLH group. In the mean time, 145 patients with normal level of serum LH ( 1-2 U/L) not given by r-hLH treatment and undergoing IVF-ET were matched as control group. Total amount of gonadotropin, estradiol levels and LH levels on the administration of human chorionic gonadotropin ( hCG), number of oocytes retrieved, number of 2PN zygotes, rate of high quality embryos, the rates of implantation and clinical pregnancy were compared among these three groups. Results The level of serum LH on the day of hCG administration were ( 1.59 ± 0.77 ) U/L in r-hLH group, (0.54 ± 0.25 ) U/L in non-r-hLH group and (2.39 ± 1.01 ) U/L in control group, which reached tatistical difference between every two groups (P < 0.05). The rates of high quality embryo were 59.36％ in r-hLH group, 57.79％ in non-r-hLH group,which were significantly lower than 65.94％ in control group, respectively (P < 0. 05 ). The rates of 2PN were 67.62％ in r-hLH group and 68. 32％ in control group, which were significantly higher than 62. 84％ in non-r-hLH group, respectively ( P < 0.05 ). The rates of implantation of 29.77％ in r-hLH group were significantly higher than 18.26％ in non-r-hLH group ( P < 0.05 ). The total amount of gonadotropin,estradiol level on the day of hCG administration, the number of oocytes retrieved, and clinical pregnancy rate were not significantly different among those three groups ( P > 0.05 ). Conclusion The administration of recombinant human uteinizing hormone in patients who are profoundly suppressed after down-regulation with long protocol can get more quality embryos, the higher rates of 2PN and implantation.     

重组人黄体生成素在体外受精-胚胎移植促排卵治疗中的应用

Objective To evaluate application of recombinant human luteinizing hormone (r-hLH)used in ovarian stimulation of assisted reproductive technique and impact on outcome of pregnancy. Methods From Apr. To Jul. 2009, 123 patients with low LH level ( < 1 U/L) at day 3 of menstruation and downregulation of pituitary function undergoing in vitro fertilization-embryo transfer (IVF-ET) in Reproductive Medical Center, Provincial Hospital Affiliated to Shandong University were enrolled in this study, whom were classified into 66 cases treated by r-hLH in r-hLH group and 57 cases without r-hLH treatment in non-r-hLH group. In the mean time, 145 patients with normal level of serum LH ( 1-2 U/L) not given by r-hLH treatment and undergoing IVF-ET were matched as control group. Total amount of gonadotropin, estradiol levels and LH levels on the administration of human chorionic gonadotropin ( hCG), number of oocytes retrieved, number of 2PN zygotes, rate of high quality embryos, the rates of implantation and clinical pregnancy were compared among these three groups. Results The level of serum LH on the day of hCG administration were ( 1.59 ± 0.77 ) U/L in r-hLH group, (0.54 ± 0.25 ) U/L in non-r-hLH group and (2.39 ± 1.01 ) U/L in control group, which reached tatistical difference between every two groups (P < 0.05). The rates of high quality embryo were 59.36％ in r-hLH group, 57.79％ in non-r-hLH group,which were significantly lower than 65.94％ in control group, respectively (P < 0. 05 ). The rates of 2PN were 67.62％ in r-hLH group and 68. 32％ in control group, which were significantly higher than 62. 84％ in non-r-hLH group, respectively ( P < 0.05 ). The rates of implantation of 29.77％ in r-hLH group were significantly higher than 18.26％ in non-r-hLH group ( P < 0.05 ). The total amount of gonadotropin,estradiol level on the day of hCG administration, the number of oocytes retrieved, and clinical pregnancy rate were not significantly different among those three groups ( P > 0.05 ). Conclusion The administration of recombinant human uteinizing hormone in patients who are profoundly suppressed after down-regulation with long protocol can get more quality embryos, the higher rates of 2PN and implantation.     

高胆红素血症对新生儿T淋巴细胞亚群、血清可溶性白细胞介素-2受体变化趋势的影响和意义

目的 探讨新生儿高胆红素血症(简称高胆)时T淋巴细胞亚群和血清可溶性白细胞介素-2受体(soluble interleukin-2 receptor,sIL-2R)水平的变化趋势及其临床意义.方法 选择2006年12月1日至2007年1月31日住院的31例高胆新生儿作为高胆组,再根据黄疸程度分为重度黄疸组和轻度黄疸组;将其中16例随访病例按照病程分为黄疸高峰期与黄疸恢复期.选取同期与高胆组日龄相匹配的32例健康足月新生儿(无黄疸或血清胆红素水平≤204.0 μmol/L)作为与高胆组相对应的对照组(对照组Ⅰ);选取同期与黄疸恢复期病例日龄相匹配的26例健康足月新生儿(日龄＞7 d)作为与随访病例相对应的对照组(对照组Ⅱ).采用方差分析及两两检验比较各组血清胆红素、T淋巴细胞亚群、sIL-2R水平,并分析其间的相关性.结果 高胆组新生儿的CD3、CD4、CD4/CD8比值分别为(54.0±5.1)%、(26.8±5.0)%和0.8±0.1,较对照组Ⅰ[(62.0±4.7)%、(43.0±4.7)%和1.4±0.2]降低(P＜0.01);而黄疸恢复期较黄疸高峰期增高[(62.4±3.3)%和(55.1±4.2)%、(43.6±2.5)%和(26.1±4.4)%、1.4±0.1和0.8±0.1](P＜0.01);黄疸高峰期血清sIL-2R水平[(319.4±185.2)kU/L]高于黄疸恢复期[(129.7±99.3)kU/L]和对照组Ⅱ[(171.9±102.2)kU/L](P＜0.01).总体的血清胆红素水平与CD4/CD8比值呈负相关(r=-0.99,P＜0.01),与sIL-2R水平呈正相关(r=0.95,P＜0.05),sIL-2R水平与CD4/CD8比值呈负相关(r=-0.92,P＜0.05).结论 新生儿高胆时存在细胞免疫功能抑制状态,该抑制状态有随着黄疸消退而逐渐减轻的趋势.

Abstract：

Objective To investigate the dynamic changes and the clinical significance of T-cell subsets and serum soluble interleukin-2 receptor (sIL-2R)in neonates with hyperbilirubinemia.Methods Thirty-one neonates with hyperbilirubinemia, admitted to the hospital from Decembr 1,2006 to January 31, 2007, were enrolled and divided into two subgroups: severe jaundice group and mild jaundice group according to the bilirubin level. Thirty-two age-mached healty newborns were as controls(control group Ⅰ). The T-cell subsets and sIL-2R of peripheral venous blood samples from these neonates were measured and compared. Sixteen of these 31 neonates with hyperbilirubinemiawere followed up and another twenty-six age-mached healty newborns were as controls(control group Ⅱ ). The level of serum bilirubin in convalescence of sixteen hyperbilirubinemia neonates and control group Ⅱ were tested and analyzed also. Results The levels of CD3, CD4, CD4/CD8 in the neonates with hyperbilirubinemia were lower compared with those of control group Ⅰ [(54.0±5.1)% vs (62.0±4.7)%, (26.8±5.0)% vs (43.0±4.7)%, 0.8±0.1 vs 1.4±0.2] (P＜0.01), but was higher in convalescence than in peak phase[ (62.4±3.3)% vs (55.1±4.2)%, (43.6±2.5)% vs (26.1±4.4)%, 1.4 ± 0.1 vs 0.8±0.1] (P＜0.01). The peak level of sIL-2R in the hyperbilirubinemia group was (319.4± 185.2) kU/L, higher than that in the convalescence [(129.7±99.3) kU/L] and in the control group Ⅱ [(171.9±102.2) kU/L] (P＜0.01). The serum bilirubin level showed negative correlation with CD4/CD8 ( r = -0.99, P ＜ 0.01 ) and positive correlation with sIL-2R (r=0.95, P＜0.05). The sIL-2R level was negatively correlated with CD4/CD8 (r=-0.92, P＜0.05). Conclusions Neonates, when suffering from hyperbilirubinemia, are immunosuppressed which may recover with the alleviation of jaundice.     

脂氧素对脂多糖诱导的脐静脉内皮细胞通透性的影响

目的 研究脂氧素对脂多糖诱导的脐静脉内皮细胞通透性的影响,并进一步探讨其机制.方法 取华中科技大学同济医学院附属同济医院产科因社会因素行剖宫产术的健康产妇分娩的新生儿脐带,分离原代脐静脉内皮细胞后进行传代培养并分组:(1)对照组.(2)脂多糖组:加入10 mg/L脂多糖培养24 h.(3)脂多糖+脂氧素A4组:加入100 nmol/L脂氧素A4和10 mg/L脂多糖共同培养24 h.(4)脂多糖+脂氧素A4+BOC-2组:BOC-2为脂氧素受体拮抗剂,共同培养24 h.计算各组内皮细胞通透系数;逆转录(RT)PCR技术检测内皮细胞中肿瘤坏死因子α(TNF-α)mRNA表达水平;观察不同浓度脂多糖(0、0.1、1、10 mg/L)对对照组内皮细胞功能的影响(以TNF-α mRNA水平表示);蛋白印迹法检测内皮细胞中核因子κB蛋白表达水平;ELISA法检测内皮细胞培养液中TNF-α水平.结果 (1)内皮细胞通透系数:脂多糖组内皮细胞通透系数为(183.1±1.7)%,脂多糖+脂氧素A4组为(103.1±2.2)%,脂多糖+脂氧素A4+BOC-2组为(162.2±2.8)%,对照组为100%,脂多糖+脂氧素A4组通透系数较脂多糖组减小了(80.0±2.2)%,两组比较,差异有统计学意义(P<0.05).脂多糖+脂氧素A4+BOC-2组通透系数与脂多糖+脂氧素A4组相比增加了(59.1±2.8)%,两组比较,差异有统计学意义(P<0.01).脂多糖+脂氧素A4+BOC-2组与脂多糖组比较,差异无统计学意义(P>0.05).(2)TNF-α mRNA表达水平:脂多糖浓度为0、0.1、1、10 mg/L时,对照组脐静脉内皮细胞中的TNF-α mRNA表达水平分别为1.11±0.11、1.27±0.03、1.60±0.06、1.82±0.04,其中,脂多糖浓度为1、10 mg/L时,分别与0浓度比较,差异均有统计学意义(P<0.05).(3)核因子κB蛋白及TNF-αmRNA表达水平:核因子κB蛋白及TNF-αmRNA表达水平,对照组分别为0.24±0.06及0.25±0.05,脂多糖组分别为0.53±0.06及0.81±0.09,脂多糖+脂氧素A4组分别为0.19±0.05及0.41±0.07,脂多糖组核因子κB蛋白及TNF-α mRNA表达水平明显高于对照组,差异有统计学意义(P<0.05);脂多糖+脂氧素A4组核因子κB蛋白及TNF-αmRNA表达水平明显低于脂多糖组,差异有统计学意义(P<0.05).(4)细胞培养液中TNF-α水平:脂多糖组TNF-α水平[(31.94±0.01)ng/L]明显高于对照组[(18.17±0.03)ng/L],两组比较,差异有统计学意义(P<0.05);脂多糖+脂氧素A4组[(15.72±0.07)ng/L]明显低于脂多糖组,两组比较,差异有统计学意义(P<0.05).结论 脂氧素A4可以抑制脂多糖诱导的脐静脉内皮细胞高通透性,其作用可能是首先与其受体结合然后通过抑制核因子κB及其下游细胞因子TNF-α的表达而实现的.

Abstract：

Objective To explore whether lipoxin A4 (LXA4)could prevent lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVEC) monolayer hyperpermeability and its possible mechanism. Methods Human umbilical cords were obtained from women with normal pregnancy immediately after delivery from Tongji Hospital Affiliated of Tongji Medical College. Primary HUVEC were isolated from umbilical veins and subcultured, then, HUVEC were divided into four groups:control group;LPS group (10 mg/L of LPS); LPS + LXA4 group(10 mg/L of LPS and 100 nmol/L of LXA4); LPS +LXA4 + BOC-2 group [10 μmol/L of BOC-2, an effective antagonist of formyl peptide receptor like 1 (FPRL-1)]. All expriments were performed after cells were treated for 24 hours. Endothelial permeability was measured by fluorescein isothiocyan-ate labelled bovine serum albumin (FITC-BSA) clearance across the monolayer; tumor necrosis factor α(TNF-o) mRNA and secretion were detected by reverse transcriplase (RT) -PCR and ELISA assay respectively, and nuclear factor κB(NF-κB) protein change was determined by western blot. Results (1) LPS induced a significant increase in the permeability [Pa value of LPS group was (183.1 ±1.7)%], while co-administrating with LXA4 obviously attenuated this LPS-induced hyperpermeability, Pa value of LPS + LXA4 group was (103.1 ±2.2)%, LPS + LXA4 + BOC-2 group was (162.2 ± 2.8)%, control group was 100%, the permeability of HUVEC monolayer was significantly increased by LPS which was (83.1 ± 1.7)% of control (P <0.01), however, it was notably inhibited by LXA4 (P<0.05); the blockade of FPRL-1 could attenuate the effect of LXA4, that is, there was no difference between the LPS + LXA4 + BOC-2 group and the LPS group. (2) After treatment with different concentration of LPS(0,0.1, 1,10 mg/L), the mRNA expressions of TNF-α were increased (1.11 ±0.11,1.27 ± 0.03, 1.60 ± 0.06, 1.82 ± 0. 04, respectively), compared with the control group, at the concentration of 1,10 mg/L LPS, the difference was statistically significant (P<0. 05). (3) The increased levels of NF-κB and inflammatory mediator TNF-α in the LPS group were both inhibited by LXA4. Levels of NF-κB protein and TNF-o mRNA secretion in LPS treated group (0.53 ±0.06 and 0.81 ±0.09 ,respectively)were both inhibited by LXA4 (0.19 ± 0.05 and 0.41 ± 0.07, respectively, and both had significant difference, P<0.05). (4) Levels of TNF-α in HUVEC culture medium of LPS group [(31.94 ±0.01)ng/L] was significantly higher than the control group [(18.17 ± 0.03) ng/L, P<0.05], LPS + LXA4 group [(15.72 ± 0.07) ng/L] was significantly lower than the LPS group (P<0.05). Conclusion Our findings demonstrated that LXA4 could prevent the endothelial cell hyperpermeability induced by LPS in HUVEC under which the possible mechanism was through inhibiting the expression of NF-κB and its related cytokines through receptor-dependent.     

脂氧素对滋养细胞氧化损伤的保护作用及其机制

目的研究脂氧素(lipoxinA4,LXA4)在孕妇血清中的表达及其对滋养细胞氧化损伤的保护作用和作用机制.方法 细胞实验分为6组,对照组;脂多糖(lipopolysaccharides,LPS)组:10 μg/ml LPS作用24 h;干预组:10 μg/ml LPS+100 nmol/L LXA4作用24 h;LXA4组:100 nmol/L LXA4作用24 h;拮抗组:10 μg/ml LPS+100 nmol/L LXA4+100 μmol/L N-叔丁氧羰基吡咯烷(N-tert-butoxycarbonyl-2-pyrrolidine,BOC-2)作用24 h;BOC-2组:10μg/ml LPS+100 μmol/L BOC-2 作用24 h.采用酶联免疫吸附试验检测正常孕妇与子痫前期患者血清LXA4含量;以2,7-二氢二氯荧光素二乙酸酯为荧光探针检测滋养细胞内活性氧(reactive oxygen species,ROS)水平;逆转录-聚合酶链反应检测超氧化物歧化酶(superoxide dismutase,SOD) mRNA的表达;Western印迹分析SOD、转录因子NF-E2相关因子2(NF-E2-related factor,Nrf2)的蛋白表达水平;黄嘌呤氧化酶法测定滋养细胞中SOD活性.结果 (1)子痫前期患者血清LXA4水平为(165.53±18.89) pg/L,与正常孕妇[(545.67±30.91) pg/L]相比明显下降(t=40.64,P<0.01).(2)LPS组与对照组相比,滋养细胞内二氯荧光素(dichlorofluorescein,DCF)密度值明显升高(P<0.01),DCF密度值从53.00±3.08 上升至77.00±5.83( t=8.14,P<0.01);胞核中Nrf2蛋白、SOD mRNA和蛋白表达均显著下降(t分别为34.11、25.61和17.93,P均<0.01);滋养细胞上清中的SOD含量明显下调(t=12.16,P<0.01).(3)干预组与LPS组相比,滋养细胞内DCF密度值明显下降,DCF密度值从77.00±5.83降至62.00±3.39(t=4.97,P<0.01),胞核中Nrf2蛋白、SOD mRNA表达显著上升(t分别为11.74和13.17,P均<0.01),SOD蛋白表达也有所升高(t=4.67,P<0.05),滋养细胞上清中的SOD含量明显增加,酶活力上升至(8.93±0.53) U/ml(t=14.76,P<0.01).(4)拮抗组与干预组相比,滋养细胞上清中SOD含量下降至(6.23±0.41) U/ml(t=9.03,P<0.01),但仍高于LPS组(t=6.10,P<0.01);BOC-2组SOD含量为(4.90±0.32) U/ml,与LPS组相比差异无统计学意义(t=0.79,P>0.05).结论 LXA4可明显减轻LPS引起的胎盘滋养细胞氧化损伤,且LXA4可通过脂氧素受体(lipoxinA4 receptor,ALX-R)激活Nrf2/抗氧化反应元件(antioxidant response element,ARE)信号通路来发挥抗氧化损伤的作用.

Abstract：

Objective To explore lipoxinA4 (LXA4) expression in maternal serum of pregnant women and the protective effect and mechanism of LXA4 on trophoblastic cells from oxidative injury. Methods Trophoblastic cells were randomized into six groups: Control group; Lipopolysaccharides (LPS) group, cells were stimulated by 10 μg/ml LPS for 24 h; Intervention group, cells stimulated by LPS were treated with 100 nmol/L LXA4 for 24 h; LXA4 group, cells were treated with 100 nmol/L LXA4 for 24 h; Antagonistic group, cells stimulated by LPS were treated with 100 nmol/L LXA4 plus 100 μmol/L N-tert-butoxycarbonyl-2-pyrrolidine (BOC-2) for 24 h; BOC-2 group, trophoblastic cells stimulated by LPS were treated with 100 μmol/L BOC-2 for 24 h. The serum concentration of LXA4 in normal group and preeclampsia group was detected by ELISA. The intracellular formation of reactive oxygen species (ROS) was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. SOD mRNA was analyzed by RT-PCR. SOD and Nrf2 protein expressions were analyzed by Western blot. The levels of SOD in trophoblastic cells were detected by using detection kit. Results (1) The serum concentration of LXA4 was significantly lower in preeclampsia group (165.53±18.89) pg/L than in the control [(545.67±30.91) pg/L, P<0.01]. (2) Compared with control group, the levels of ROS in LPS group were significantly higher, DCF density of trophoblastic cells increased from 53.00±3.08 to 77.00±5.83 (P<0.01). The expression of nuclear Nrf2 protein, SOD mRNA and protein in LPS group were obviously decreased (P<0.01). The levels of SOD in LPS group were also significantly lower (P<0.01). (3) Compared with LPS group, the levels of ROS in intervention group were significantly lower, DCF density of trophoblastic cells decreased from 77.00±5.83 to 62.00±3.39 (P<0.01). The expression of nuclear Nrf2 protein, SOD mRNA in intervention group were obviously increased (P<0.01), so did the SOD protein expression (P<0.05). The levels of SOD were significantly increased from (4.77±0.34) U/ml to (8.93±0.53) U/ml (P<0.01). (4) The levels of SOD in antagonistic group were lower than in intervention group, but still higher than LPS group. [(6.23±0.41) U/ml vs (8.93±0.53) U/ml (P<0.01) or (4.77±0.34) U/ml (P<0.01)]. No significant difference was found in the levels of SOD between BOC-2 and LPS group (P>0.05). Conclusions LXA4 can significantly reduce the oxidative stress of placental trophoblastic cells stimulated by LPS. LXA4 can bind to lipoxin receptors and activate Nrf2-ARE signaling pathway playing a protective effect. So LXA4 in pregnant women can affect the oxidative stress of placenta.     

妊娠期肝内胆汁淤积症胎儿胆汁酸水平与肺表面活性物质的相关性研究

目的 探讨妊娠期肝内胆汁淤积症(ICP)胎儿总胆汁酸水平与胎儿肺表面活性物质(PS)的关系.方法 选择2008年4月至2010年2月在中南大学湘雅二医院住院行剖宫产分娩的ICP孕妇55例(ICP组),记录ICP组孕妇的新生儿出生至产后7 d的一般情况,凡符合胎儿窘迫、新生儿窒息、新生儿呼吸窘迫综合征其中1项者为病理围产儿,无上述情况为正常围产儿.另选同期健康孕妇23例为对照组.采用循环酶法测定两组孕妇血、脐血及羊水中总胆汁酸水平;ELISA法测定两组胎儿脐血肺表面活性蛋白A(SP-A)水平;高效液相色谱法测定两组羊水中磷脂酰胆碱(PC)、磷脂酰肌醇(PI)、溶血卵磷脂(LPC)、神经鞘磷脂(SM)的含量.结果 (1)ICP组孕妇血、脐血及羊水中总胆汁酸水平分别为(30.1±7.9)、(9.3±3.3)及(4.4 ±1.5)mmol/L,明显高于对照组的(4.8±2.2)、(4.9±0.9)及(1.4±1.1)mmol/L,两组分别比较,差异均有统计学意义(P＜0.05).(2)ICP 组胎儿脐血SP-A水平为(29.5±6.4)μg/L,明显高于对照组的(22.6±7.4)μg/L,两组比较,差异均有统计学意义(P＜0.05).(3)ICP组中病理围产儿20例,健康围产儿35例,病理围产儿脐血总胆汁酸及SP-A水平分别为(10.9±2.2)mmol/L及(37.0±5.9)μg/L,健康围产儿分别为(8.0±2.8)mmol/L及(26.7±4.8)μg/L,两者比较,差异有统计学意义(P＜0.05).(4)脐血总胆汁酸水平分别与孕妇血、羊水总胆汁酸水平呈正相关(r1=0.706,r2=0.763,P＜0.05);脐血SP-A水平与脐血总胆汁酸水平呈正相关(r3=0.494,P＜0.05).(5)ICP组羊水中PC及PI的含量分别为(65.4±7.2)及(3.8±0.6)mg/L,均明显低于对照组的(69.7±3.7)及(4.3±0.7)mg/L,两组比较,差异有统计学意义(P＜0.05);ICP组羊水LPC的含量为(4.8±0.9)mg/L,明显高于对照组的(4.2±0.6)mg/L,两组比较,差异有统计学意义(P＜0.05);ICP组羊水SM的含量为(3.5±0.8)mg/L,与对照组的(4.0±0.5)mg/L比较,差异无统计学意义(P＞0.05).(6)ICP组羊水PC/LPC比值(14.2±3.2)明显低于对照组(16.9±2.5),差异有统计学意义(P＜0.05).(7)脐血总胆汁酸水平与羊水PC、PI的含量均呈负相关(r1=-0.561,r2=-0.407,P＜0.05);与LPC含量无相关性(r3=0.260,P＞0.05).结论 ICP孕妇的胎儿脐血及羊水中总胆汁酸水平均明显高于健康孕妇,其胎儿PS的改变与胎儿体内高总胆汁酸水平有关.

Abstract：

Objective To explore the relationship between total bile acid(TBA)concentration and fetal pulmonary surfactant in intrahepatic cholestasis of pregnancy(ICP).Methods Fifry five patients with ICP(ICP group)who received cesarean section from April 2008 to February 2010 in Second Xiangya Hospital,Central South University,were recruited.The general conditions of the neonates within 7 days after birth in ICP group were recorded.Those with fetal distress,neonatal asphyxia,or neonatal respiratory distress syndrome were referred as pathological neonates, others were referred as normal neonates. Over the same period, 23 healthy gravidas were recruited as control group. Enzymatic method was used to detect the TBA concentrations in maternal blood, cord blood and amniotic fluid. ELISA was employed to measure the urfactant protein A (SP-A) concentration in cord blood. High performance liquid chromatography system was used to detect the concentrations of phesphatidylcholine (PC),phosphatidylinositol (PI),lysophosphatidylcholine ( LPC), and sphingomyelin(SM) in amniotic fluid. Results ( 1 ) The concentrations of TBA in maternal blood, cord blood and amniotic fluid were ( 30. 1 ± 7.9 ), (9. 3± 3. 3 ) and (4. 4 ± 1.5 ) mmol/L in ICP group, (4. 8 ± 2. 2), (4. 9 ± 0. 9) and ( 1.4 v 1.1 ) mmol/L in control group, respectively. The differences between the two groups were significant ( P ＜ 0. 05 ). ( 2 ) The SP-A concentration in cord blood in ICP group was ( 29. 5 ± 6. 4 ) μg/L, significantly higher than that in control group, which was ( 22. 6 ± 7. 4 )μg/L ( P＜ 0. 05 ). ( 3 ) There were 20 pathological neonates and 35 normal neonates in ICP group. In pathological neonates, the concentrations of TBA and SP-A in cord blood were (10.9 ± 2.2) mmol/L,(37.0 ± 5.9 ) μg/L, respectively; and were ( 8.0 ± 2. 8 ) mmol/L, ( 26. 7 ± 4. 8 ) μg/L in normal neonates. The differences were significant (P＜ 0. 05 ). (4) There was a positive correlation between TBA concentration in cord blood and in maternal blood ( r1 = 0. 706, P＜0. 05 ). The TBA concentration in cord blood was positively correlated with SP-A concentration as well ( r3 = 0. 494,P ＜ 0. 05 ). (5) The PC and PI concentrations in amniotic fluid were (65.4 ± 7.2) mg/L and ( 3. 8 ± 0. 6 ) mg/L in ICP group, ( 69. 7 ±3.7) mg/L and (4. 3 ± 0. 7 ) mg/L in control group, respectively. The differences were significant (P ＜0. 05 ). The concentration of LPC in amniotic fluid in ICP group was (4. 8 ±0. 9) mg/L, significantly higher than that in control group (P＜0. 05), which was (4. 2 ±0. 6) mg/L. The concentration of SM in amniotic fluid was (3.5±0. 8) mg/L in ICP group, (4. 0 ± 0. 5 ) mg/L in control group, with no significant difference ( P＞0. 05 ). (6) The ratio of PC/LPC in ICP group ( 14. 2± 3. 2 ) was significantly lower than that in control group ( 16. 9 ± 2. 5 ) ( P＜ 0. 05 ). ( 7 ) The TBA concentration in cord blood was negatively correlated with PC and PI concentrations (r1 = -0. 561, r2 = -0. 407, P ＜ 0. 05 ), and had no correlation with LPC concentration (r3 = 0. 260, P＞ 0. 05). Conclusions ( 1 ) The fetal TBA concentrations in both cord blood and amniotic fluid of patients with ICP was higher than those of healthy gravidas, they were also positively correlated with maternal TBA concentration. (2) ICP resulted in the change of fetal pulmonary surfactant and this change was associated with TBA concentrations in both cord blood and amniotic fluid.     

子痫前期胎盘中生长阻滞和DNA损伤45α基因及p38丝裂原活化蛋白激酶的表达

目的 探讨子痫前期胎盘组织中生长阻滞和DNA损伤45α(growth arrest and DNA damage-inducible 45 alpha,Gadd45α)基因和p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)信号分子的表达,及其与血清中可溶性血管内皮生长因子受体-1(soluble vascular endothelial growth factor receptor-1,sFlt-1)和可溶性endoglin(soluble endoglin,sEng)的相关性分析.方法 选择2009年9月至2010年3月在重庆医科大学附属第一医院住院分娩的孕妇54例为研究对象,按病情分为子痫前期轻度组20例、子痫前期重度组16例,以足月择期剖宫产孕妇18例为对照组.采用免疫组织化学SP法检测Gadd45α及磷酸化p38 MAPK(phospho-p38 MAPK,p-p38 MAPK)蛋白在各组胎盘组织中的定位;实时荧光定量PCR检测各组胎盘组织中Gadd45α mRNA水平;Western印迹法检测各组胎盘组织中Gadd45α蛋白的表达水平、p38 MAPK及p-p38 MAPK的蛋白表达差异;双抗体夹心酶联免疫吸附法检测各组孕妇血清样本中sFlt-1及sEng的含量,并进行单因素方差及LSD-t检验分析.结果 (1)免疫组织化学检测Gadd45α与p-p38 MAPK蛋白均定位于胎盘滋养层细胞胞质及胞核、血管内皮细胞胞核及少量间质细胞胞核.(2)子痫前期重度及轻度组Gadd45α mRNA相对表达水平(3.33±0.13和2.10±0.11)较正常对照组(1.01±0.18)明显上调,且子痫前期重度组高于轻度组,两两比较差异均有统计学意义(P＜0.05).(3)Western 印迹法检测正常对照组、子痫前期轻度组及重度组患者胎盘组织中Gadd45α蛋白表达水平分别为0.22±0.11、0.65±0.15、1.34±0.17;p-p38 MAPK蛋白的表达水平分别为0.32±0.08、0.72±0.12、1.45±0.21,两两比较差异均有统计学意义(P＜0.05);p38 MAPK蛋白水平组间比较差异无统计学意义(P＞0.05).(4)子痫前期轻度组、重度组孕妇血清sFlt-1、sEng水平明显高于正常对照组,且子痫前期重度组高于轻度组,两两比较差异均有统计学意义(P＜0.05).(5)各组Gadd45α蛋白水平与孕妇血清中的sFlt-1、sEng含量呈正相关(r分别为0.88和0.87,P均＜0.05).结论 Gadd45α在子痫前期患者胎盘组织中的表达明显升高,其可能通过调控p38 MAPK信号转导通路,诱使循环中的sFlt-1、sEng释放增加,从而加重胎盘血管重铸障碍和抑制滋养细胞浸润,参与子痫前期的发病.

Abstract：

Objective To evaluate the expression of Gadd45α and p38 MAPK in placentas and the correlations of Gadd45α protein and serum soluble vascular endothelial growth factor receptor-1 (sFlt-1) and soluble endoglin (sEng) in preeclampsia(PE). Methods Fifty-four pregnant women who delivered from September 2009 to March 2010 in the First Affiliated Hospital of Chongqing Medical University were chosen as the subjects. They were classified into mild preeclampsia group (n=20),severe preeclampsia group (n=16) and the control group (normal pregnant women underwent elective cesarean sections at term without labor and perinatal complications, n=18). Western blot and immunohistochemistry were employed to determine the expression and localization of Gadd45α and p-p38 MAPK protein respectively. Gadd45α mRNA level was determined by quantitative real-time PCR. The levels of seum sFlt-1 and sEng were measured by enzyme-linked immunosorbent assay (ELISA). One-way ANOVA and LSD-t test were applied for statistical analysis. Results (1)Immunohistochemistry identified that the positive stained cells were mostly located in trophoblast cells in normotensive placentas, whereas in preeclamptic placentas Gadd45α protein and p-p38 MAPK protein were detected in trophoblast and endothelial cells, as well as a few stromal cells at increased levels.(2)The mRNA levels of Gadd45α was significantly elevated in mild and severe preeclampsia groups compared to the control group (2.10±0.11 and 3.33±0.13 vs 1.01±0.18, P＜0.05), and Gadd45α mRNA level in severe group was significantly higher than in mild group (P＜0.05).(3)The data of Western blot revealed that the Gadd45α protein levels in each group were 0.22±0.11, 0.65±0.15 and 1.34±0.17, respectively, with significant differences between each group(P＜0.05). The p-p38 MAPK protein levels in each group were 0.32±0.08, 0.72±0.12 and 1.45±0.21, respectively, with significant differences between each group (P＜0.05). p38 MAPK protein levels in the total groups showed no difference(P＞0.05).(4)Compared with the control group, sFlt-1 and sEng concentrations in maternal circulation were significantly increasing in mild and severe preeclampsia groups, and concentrations in severe group were significantly higher than those in mild group (P＜0.05).(5) There were positive correlations between Gadd45α protein levels and the concentrations of serum sFlt-1 and sEng in each group( r=0.88 and 0.87, respectively all P＜0.05). Conclusions Upregulation of Gadd45α in preeclampsia placentas may play an important role in the pathogenesis of preeclampsia. It may induce the increased maternal serum levels of sFlt-l and sEng by activating p38 MAPK signaling pathway, leading to deficient cytotrophoblastic invasion and abnormal placental vascular reconstruction during pregnancy.     

妊娠期糖尿病母鼠血清晚期糖基化终产物与子鼠心脏发育异常的关系

Objective To explore the effect of advanced glycation end product(AGE) in serum of maternal rats with gestational diabetes mellitus (GDM) on the heart development of their offsprings. Methods Fifty-four SD rats were randomly assigned into control group (n= 24) and GDM group (n=30) which were established by administration of streptozotocin intra-abdominally. On the gestational age of 13, 16, 19 days, all rats underwent hysterectomy to obtain the fetal heart tissues. Serum level of AGE and blood glucose level of maternal rats were tested. The expression of receptor AGE (RAGE) in fetal cardiac tissue were detected by immunohistoehemistry. Results The incidence of fetal heart defect in GDM group was significantly higher than the control group at each time point (P<0.01). Rats in GDM group had higher blood glucose level at each time point (P<0.01). The AGE levels of GDM group on gestational age of 13, 16 and 19 day [(5.72±0.68) U/mgpr, (7.31±0.29) U/mgpr and (7.77±0.39) U/mgpr] were significantly higher than those of the control group [(4.45±0.27) U/mgpr, (4.71±0. 35) U/mgpr and (4. 37±0. 44) U/rngpr] (t=6. 142, 16. 295, 0. 399,P<0. 01). The number of heart malformation in fetal rats (r=0.994,P=0. 000) and blood glucose (r=0. 717,P=0. 000) had the positive relationship with the maternal serum AGE level. The expression of RAGE in fetal heart was positively related with the number of fetal heart malformation (r= 0. 638,P= 0. 004). Conclusions The increased maternal serum AGE level in GDM rats may be an important factor in fetal heart dysplasia.     

妊娠期糖尿病母鼠血清晚期糖基化终产物与子鼠心脏发育异常的关系

Objective To explore the effect of advanced glycation end product(AGE) in serum of maternal rats with gestational diabetes mellitus (GDM) on the heart development of their offsprings. Methods Fifty-four SD rats were randomly assigned into control group (n= 24) and GDM group (n=30) which were established by administration of streptozotocin intra-abdominally. On the gestational age of 13, 16, 19 days, all rats underwent hysterectomy to obtain the fetal heart tissues. Serum level of AGE and blood glucose level of maternal rats were tested. The expression of receptor AGE (RAGE) in fetal cardiac tissue were detected by immunohistoehemistry. Results The incidence of fetal heart defect in GDM group was significantly higher than the control group at each time point (P<0.01). Rats in GDM group had higher blood glucose level at each time point (P<0.01). The AGE levels of GDM group on gestational age of 13, 16 and 19 day [(5.72±0.68) U/mgpr, (7.31±0.29) U/mgpr and (7.77±0.39) U/mgpr] were significantly higher than those of the control group [(4.45±0.27) U/mgpr, (4.71±0. 35) U/mgpr and (4. 37±0. 44) U/rngpr] (t=6. 142, 16. 295, 0. 399,P<0. 01). The number of heart malformation in fetal rats (r=0.994,P=0. 000) and blood glucose (r=0. 717,P=0. 000) had the positive relationship with the maternal serum AGE level. The expression of RAGE in fetal heart was positively related with the number of fetal heart malformation (r= 0. 638,P= 0. 004). Conclusions The increased maternal serum AGE level in GDM rats may be an important factor in fetal heart dysplasia.     

妊娠期糖尿病母鼠血清晚期糖基化终产物与子鼠心脏发育异常的关系

Objective To explore the effect of advanced glycation end product(AGE) in serum of maternal rats with gestational diabetes mellitus (GDM) on the heart development of their offsprings. Methods Fifty-four SD rats were randomly assigned into control group (n= 24) and GDM group (n=30) which were established by administration of streptozotocin intra-abdominally. On the gestational age of 13, 16, 19 days, all rats underwent hysterectomy to obtain the fetal heart tissues. Serum level of AGE and blood glucose level of maternal rats were tested. The expression of receptor AGE (RAGE) in fetal cardiac tissue were detected by immunohistoehemistry. Results The incidence of fetal heart defect in GDM group was significantly higher than the control group at each time point (P<0.01). Rats in GDM group had higher blood glucose level at each time point (P<0.01). The AGE levels of GDM group on gestational age of 13, 16 and 19 day [(5.72±0.68) U/mgpr, (7.31±0.29) U/mgpr and (7.77±0.39) U/mgpr] were significantly higher than those of the control group [(4.45±0.27) U/mgpr, (4.71±0. 35) U/mgpr and (4. 37±0. 44) U/rngpr] (t=6. 142, 16. 295, 0. 399,P<0. 01). The number of heart malformation in fetal rats (r=0.994,P=0. 000) and blood glucose (r=0. 717,P=0. 000) had the positive relationship with the maternal serum AGE level. The expression of RAGE in fetal heart was positively related with the number of fetal heart malformation (r= 0. 638,P= 0. 004). Conclusions The increased maternal serum AGE level in GDM rats may be an important factor in fetal heart dysplasia.     

妊娠期糖尿病母鼠血清晚期糖基化终产物与子鼠心脏发育异常的关系

Objective To explore the effect of advanced glycation end product(AGE) in serum of maternal rats with gestational diabetes mellitus (GDM) on the heart development of their offsprings. Methods Fifty-four SD rats were randomly assigned into control group (n= 24) and GDM group (n=30) which were established by administration of streptozotocin intra-abdominally. On the gestational age of 13, 16, 19 days, all rats underwent hysterectomy to obtain the fetal heart tissues. Serum level of AGE and blood glucose level of maternal rats were tested. The expression of receptor AGE (RAGE) in fetal cardiac tissue were detected by immunohistoehemistry. Results The incidence of fetal heart defect in GDM group was significantly higher than the control group at each time point (P<0.01). Rats in GDM group had higher blood glucose level at each time point (P<0.01). The AGE levels of GDM group on gestational age of 13, 16 and 19 day [(5.72±0.68) U/mgpr, (7.31±0.29) U/mgpr and (7.77±0.39) U/mgpr] were significantly higher than those of the control group [(4.45±0.27) U/mgpr, (4.71±0. 35) U/mgpr and (4. 37±0. 44) U/rngpr] (t=6. 142, 16. 295, 0. 399,P<0. 01). The number of heart malformation in fetal rats (r=0.994,P=0. 000) and blood glucose (r=0. 717,P=0. 000) had the positive relationship with the maternal serum AGE level. The expression of RAGE in fetal heart was positively related with the number of fetal heart malformation (r= 0. 638,P= 0. 004). Conclusions The increased maternal serum AGE level in GDM rats may be an important factor in fetal heart dysplasia.