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.     

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.     

细胞外信号调节激酶信号转导通路活化在新生大鼠缺氧缺血性脑损伤中的作用

目的 观察细胞外信号调节激酶(extracellular-signal regulated kinase,ERK)在新生大鼠缺氧缺血性脑损伤中的作用.方法 Wistar大鼠随机分为假手术组、缺氧缺血组和PD98059(ERK抑制剂)组,通过结扎右侧颈总动脉,恢复2 h后,吸入含8%氧气的氧氮混合气体2 h的方法 建立新生大鼠缺氧缺血性脑损伤模型,PD98059组结扎右侧颈总动脉前10 min股静脉注射PD980592 mg/kg.各组新生大鼠于模型制作后6 h剥离结扎侧海马及皮层脑组织,分别测定脑组织丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活性和细胞凋亡水平.Western印迹法检测ERK1和ERK2,Bcl-2和Bax蛋白的表达.组间差异比较采用方差分析及q检验.结果 缺氧缺血组细胞凋亡率明显高于假手术组[(18.80±1.37)%和(3.53±0.34)%](q=6.06,P＜0.01),PD98059组细胞凋亡率[(15.53±0.64)%]低于缺氧缺血组(q=3.87,P＜0.01).缺氧缺血组MDA含量为(342.9±10.8)μmol/L,明显高于假手术组[(181.5±17.0)μmol/L](q=6.35,P＜0.01)和PD98059组[(252.0±17.1)μmol/L](q=5.28,P＜0.01),而SOD活性[(34.8±4.3)U/ml]明显低于假手术组[(63.4±4.3)U/ml](q=4.99,P＜0.01)和PD98059组[(51.5±3.8)U/ml](q=4.17,P＜0.01).3组总ERK1和ERK2蛋白表达差异无统计学意义.缺氧缺血组磷酸化ERK1和磷酸化ERK2蛋白表达明显高于假手术组(q分别=3.82和4.08,P均＜0.01)和PD98059组(q分别=4.79和5.12,P均＜0.01).缺氧缺血后Bcl-2和Bax蛋白表达明显高于假手术组(q分别=3.55和3.42,P均＜0.01);PD98059组较缺氧缺血组抗凋亡蛋白Bcl-2表达增加,促凋亡蛋白Bax表达降低(q分别=3.71和5.86,P均＜0.01).结论 ERK激活通过调节凋亡蛋白表达参与了新生大鼠缺氧缺血性脑损伤的发生.

Abstract：

Objective To investigate the effects of extracellular-signal regulated kinase (ERK) on hypoxic-ischemic brain damage of neonatal rats.Methods The hypoxic-ischemic brain damage model of neonatal Wistar rats was established as following:first the right common carotid artery of the rats was ligated;2 h after operation,the rats began to inhale 8%-oxygen oxygen-nitrogen gas mixture lasting for 2 h.Rats were randomly divided into three groups:sham-group,hypoxic-ischemic group and ERK inhibitor PD98059 group (the rats were injected PD98059 2 mg/kg 10 min before the ligation).Six hours after the models were done,hippocampi and cortex of the ligation side of rats in the three groups were collected,and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured.Apoptosis of neuron was assessed by TUNEL staining.The expression of ERK1,ERK 2,Bcl-2 and Bax were examined by Western blot.The differences among the groups were analyzed with ANOVA and q test.Results Compared with the sham-group,the MDA level [(342.9± 10.8) μmol/L vs (181.5± 17.0) μmol/L,q= 6.35,P＜0.01) and the apoptosis rate of neuron [(18.80±1.37)% vs (3.53±0.34)%,q=6.06,P＜0.01) of hypoxic-ischemic group was higher,and SOD level was lower [(34.8±4.3) U/ml vs (63.4±4.3) U/ml,q=4.99,P＜0.01].While the apoptosis rate of neuron [(15.53±0.64) %] and MDA level [(252.0± 17.1) μmol/L] of PD98059 group were lower than those of hypoxic-ischemic group(q=3.87 and 5.28,P＜0.01respectively),the SOD level [(51.5 ± 3.8) U/ml] was higher than that of hypoxic-ischemic group (q=4.17,P＜0.01).There were no differences of ERK1 and ERK2 expressions among the three groups.The phosphorylated ERK1 and ERK2 levels of hypoxic-ischemic group were higher than those of sham-group (q=3.82 and 4.08,P＜0.01) and PD98059 group (q=4.79 and 5.12,P＜0.01).The expression of Bcl-2 and Bax of hypoxic-ischemic group were higher than those of sham-group (q=3.55 and 3.42,P＜0.01).Compared with hypoxic-ischemic group,Bcl-2 expression (q=3.71,P＜0.01) of PD98059 group was higher,and Bax expression (q=5.86,P ＜ 0.01) was lower.Conclusions ERK is involved in hypoxic-ischemic brain damage of neonatal rats through regulating the expression of apoptosis protein.     

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.     

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人脐静脉内皮细胞氧化应激反应的影响

目的 探讨脂氧素对脂多糖诱导的脐静脉内皮细胞氧化应激反应的影响.方法 切取正常妊娠剖宫产手术4 h内的新生儿脐带,从中分离出脐静脉内皮细胞并进行传代培养,将培养的脐静脉内皮细胞分成4组:空白对照组,脂多糖处理组(10 μg/ml脂多糖),脂多糖+脂氧素处理组(10μg/ml脂多糖+100 nmol/L脂氧素),脂氧素处理组(100 nmol/L脂氧素).分别作用12 h和24 h后进行检测.采用免疫细胞荧光法检测核因子E2相关因子2(Nrf2)的表达及核转位情况、内皮细胞特异性Ⅷ因子的表达;采用逆转录(RT)PCR检测Nrf2、血红素加氧酶(HO)1和还原型辅酶Ⅰ醌类氧化还原酶(NQO1)mRNA的表达.结果 (1)荧光显微镜下观察到脐静脉内皮细胞胞质内有黄绿色荧光反应,证实有内皮细胞特异性Ⅷ因子抗原存在.(2)免疫细胞荧光染色显示,空白对照组内皮细胞中Nrf2大部分表达在胞质,胞核内基本不表达.12 h后,脂多糖处理组Nrf2胞核内的表达增加,出现了由胞质向胞核的转位;但24 h后,Nrf2在胞质和胞核内的表达都明显下降.脂多糖+脂氧素处理组在12 h和24 h,Nrf2在胞质和胞核中的表达均明显高于脂多糖处理组;脂氧素处理组Nrf2大部分表达在胞质中.(3)脂多糖处理组和脂多糖+脂氧素处理组在12 h后,Nrf2和HO-1 mRNA表达水平分别为0.581±0.019、0.081±0.009及0.692±0.048、0.136±0.018,均明显高于空白对照组,分别比较,差异均有统计学意义(P＜0.05);脂多糖处理组在12 h后,NQO1 mRNA表达为0.381±0.009,虽高于空白对照组的0.355±0.044,但差异无统计学意义(P＞0.05),而脂多糖+脂氧素处理组NQO1 mRNA表达为0.574±0.034,与空白对照组比较,差异有统计学意义(P＜0.05);脂多糖处理组24 h后,Nrf2和NQO1 mRNA的表达水平分别为0.180±0.017、0.472±0.064,与空白对照组比较,差异均有统计学意义(P＜0.05);脂多糖+脂氧素处理组Nrf2、HO-1、NQO1 mRNA的表达均较脂多糖处理组明显增加,差异均有统计学意义(P＜0.05);脂氧素处理组Nrf2、HO-1、NQO1 mRNA表达水平无论12 h还是24 h,与空白对照组比较,差异均无统计学意义(P＞0.05).结论 脂氧素通过激活脐静脉内皮细胞胞质内的Nrf2,使其向胞核内转位,进而上调Nrf2下游对细胞有保护作用的抗氧化酶,如HO-1、NQO1,抑制脂多糖诱导的氧化应激,从而保护脐静脉内皮细胞免受损伤.     

脂氧素A4对重度子(癎)前期患者外周血单核细胞体外分泌白细胞介素-1β的影响及其与胞浆内游离钙浓度的关系

目的 探讨脂氧素A4(lipoxin A4,LXA4)对重度子(癎)前期(severe preeclampsia,SPE)患者外周血单核细胞体外分泌白细胞介素-1β(interleukin,IL-1β)的影响及与胞浆游离钙浓度([Ca2+]i)的关系.方法 收集温州医学院附属第一医院产科2008年10月至2009年5月收治的15例SPE患者(SPE组)和20例正常孕妇(正常妊娠组)的外周静脉血,分离外周血单核细胞进行体外培养.将不同浓度LXA4(0、10、100 nmol/L)加入SPE组单核细胞体外培养后,采用酶联免疫吸附实验测定培养上清液中IL-1β的浓度.钙离子荧光探针Fluo-3/AM负载单核细胞后,用激光共聚焦扫描显微镜动态检测单核细胞内[Ca2+]i的变化.结果 (1)体外在0、10、100 nmol/L浓度LXA4的作用下,SPE组外周血单核细胞培养上清液中IL-1β水平分别为(63.16±8.20)pg/L、(53.71±8.08)pg/L、(3.16±6.89)pg/L.LXA4呈剂量依赖性抑制了SPE单核细胞分泌IL-1β(P<0.05),而其对正常妊娠组IL-1β的分泌无影响[分别为(19.22±7.43)pg/L、(16.99±6.32)pg/L、(15.18±5.24)pg/L](P>0.05).(2)SPE组外周血单核细胞[Ca2+]i为1028.09±160.52,较正常妊娠组(323.61±87.86)明显升高(P<0.05).而在100 nmol/L LXA4作用下SPE组单核细胞[Ca2+]i为409.67±116.73,较0 nmol/L LXA4作用下SPE组单核细胞[Ca2+]i明显下降(P<0.05).结论 LXA4体外能明显抑制SPE患者单核细胞分泌IL-1β,其机制可能与降低细胞内[Ca2+]i有关.     

脂氧素A4对缺氧损伤的人脐静脉内皮细胞的保护作用

目的 探讨脂氧素A4(LXA4)对缺氧损伤的人脐静脉内皮细胞(HUVEC)的保护作用及其机制.方法 体外培养HUVEC,培养后的细胞分别以单纯M199培养基培养(对照组)、氯化钴(CoCl2)诱导细胞缺氧(缺氧组)、不同浓度LXA4(1、10、100 nmol/L)加入缺氧组细胞培养液中(药物干预组);倒置相差显微镜下观察各组HUVEC的形态学变化;四甲基偶氮唑蓝(MTT)比色法检测不同浓度LXA4培养24 h和100 nmol/L的LXA4作用不同时间(4、8、12、24 h)后缺氧HUVEC存活率;免疫细胞化学法检测细胞胞质内第Ⅷ因子相关抗原抗体(vWF)水平变化(以灰度值表示),细胞质内出现棕黄色颗粒为阳性;激光共聚焦显微镜观察细胞质内游离Ca2+荧光强度变化.结果 (1)细胞形态:缺氧组HUVEC明显失去原有正常细胞形态,细胞变圆,核固缩;药物干预组正常细胞数量增多,加入100 nmol/L浓度的LXA4后,大部分细胞形态与正常细胞形态基本相似.(2)细胞存活率:缺氧组细胞存活率为(40.1±3.9)%,药物干预组细胞培养液中加入1、10、100 nmol/L浓度的LXA4后,细胞存活率分别为(52.9±1.4)%、(64.1±3.3)%、(76.6±1.6)%,分别与缺氧组比较,差异均有统计学意义(P<0.05).药物干预组细胞培养液中LXA4浓度为100 nmol/L时,作用4、8、12、24 h,细胞存活率分别为(68.2±2.3)%、(82.5±1.4)%、(82.9±1.4)%和(72.2±8.5)%,且在12 h时达峰值;药物干预组各时间点细胞存活率分别与缺氧组比较,差异均有统计学意义(P<0.05).(3)vWF水平:随着药物干预组细胞培养液中LXA4浓度的增加,其胞质内vWF水平逐步降低,LXA4浓度为1、10、100 nmol/L时.vWF水平分别为203.9±0.7、204.6±0.9、191.8±0.5,分别与缺氧组比较,差异均有统计学意义(P<0.05).(4)Ca2+荧光强度:激光共聚焦显微镜观察结果显示,LXA4可提高缺氧HUVEC胞质内Ca2+荧光强度.结论 LXA4对缺氧HUVEC维持正常的细胞形态起重要作用,并可提高其细胞存活率及降低细胞质内vWF水平,其保护机制可能是通过降低细胞内钙超载和转移细胞核内Ca2+,使细胞质内Ca2+增加.     

二十二碳六烯酸对高氧复苏新生大鼠氧化水平的影响

目的 探讨母鼠孕期和哺乳期补充二十二碳六烯酸(DHA)对高氧或空气复苏的窒息新生大鼠体内氧化和抗氧化水平的影响.方法 孕鼠从妊娠第1天起随机给予不同饮食,从而分为普食组和DHA组(除普通饮食外每日给予100 mg DHA).新生鼠出生24 h内进行缺氧实验2 h,然后随机进入高氧复苏组和空气复苏组.实验共分4组:普食+高氧复苏组、普食+空气复苏组、DHA+高氧复苏组和DHA+空气复苏组.用分光光度法分别检测窒息后即刻、高氧1 d、3 d和7 d时新生鼠血浆中超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)水平.结果 DHA+高氧复苏组新生鼠在高氧1 d、3 d和7 d时的s0D水平分别为(93.7±16.8)U/ml、(128.2±11.5)U/ml和(170.9±9.0)U/ml,显著高于DHA+空气复苏组[(73.7±21.5)U/ml、(112.2±19.9)U/ml和(124.2±24.6)U/ml],差异均有统计学意义(P均<0.05).DHA+高氧复苏组新生鼠的MDA含量在高氧3 d和7 d时分别为(6.0±0.5)nmol/ml和(3.5±0.9)nmol/ml,均显著低于普食+高氧复苏组[分别为(7.5±1.1)nmol/ml和(4.0±0.5)nmol/ml],差异均有统计学意义(P均<0.05).窒息后即刻、高氧1、3和7 d时各组间GSH-Px水平差异均无统计学意义(P>0.05).结论 DHA可能通过提高体内抗氧化酶水平和减少氧化代谢产物,从而对新生鼠窒息后复苏时的高氧性损伤产生一定的保护作用.     

益母草注射液预防剖宫产产后出血的多中心临床研究

目的 探讨剖宫产术中应用益母草注射液预防产后出血的有效性和安全性.方法 采用多中心、随机、单盲、阳性药物对照的前瞻性研究方法,于2007年4至8月,选择全国15所三级医院440例因产科医学指征行剖宫产分娩的产妇为研究对象.其中益母草组147例,于胎儿娩出后子宫肌壁注射益母草注射液40 mg,术后2 h开始,益母草注射液臀部肌内注射,20 mg/12 h,共3次;益母草+缩官素组144例,于胎儿娩出后子宫肌壁注射益母草注射液40 mg及缩宫素10 U,术后2 h开始,益母草注射液臀部肌内注射,20 mg/12 h,共3次;缩宫素组149例,胎儿娩出后子宫肌壁注射缩宫素10 U,同时静脉滴注缩宫素10 U(加入5%葡萄糖液500 ml中),术后2 h开始,缩宫素臀部肌内注射,10 U/12 h,共3次.观察:(1)产时出血量、产后2、6、12、24、48 h的阴道出血量;(2)产后24 h总出血量和产后出血发生率;(3)产前及产后血红蛋白、红细胞计数的差值;(4)不良反应.结果(1)出血量:平均产时出血量益母草组为(368±258)ml,缩宫素组为(269±14t)ml,益母草+缩宫素组为(255±114)ml,3组的产时出血量分别比较,差异均有统计学意义(P<0.01);产后2、6、12、24、48 h各组阴道出血量比较,差异均无统计学意义(P>0.05).(2)产后24 h总出血量:产后24 h总出血量益母草组平均为(480±276)ml、益母草+缩宫素组为(361±179)ml、缩宫素组为(381±179)ml,3组术后24 h总出血量分别比较,差异也有统计学意义(P<0.01).(3)产后出血发生率:产后出血发生率益母草组为32.0%(47/147)、益母草+缩宫素组为11.1%(16/144)、缩宫素组为18.8%(28/149),益母草+缩宫素组产后出血发生率最低,而益母草组产后出血发生率最高,两组比较,差异有统计学意义(P<0.01).(4)产前及产后血红蛋白、红细胞计数:各组产后与产前比较,红细胞计数和血红蛋白水平均有不同程度的下降,其中益母草组红细胞计数差值为(0.3±0.5)×1012/L,血红蛋白差值为(9±13)g/L;益母草+缩宫素组红细胞计数差值为(0.2±0.4)×1012/L,血红蛋白差值为(6±10)g/L;缩宫素组红细胞计数差值为(0.2±0.4)×10124/L,血红蛋白差值为(7±30)g/L,缩宫素组、益母草+缩宫素组分别与益母草组比较,差异均有统计学意义(P<0.05);益母草+缩宫素组血红蛋白下降程度最低.(5)不良反应:3组中共有2例发生轻度过敏反应.结论剖宫产术中应用益母草注射液联合缩宫素,可明显预防产后出血的发生,且药物安全性好.     

过氧化物酶增殖体激活受体α、氧固醇7α羟化酶及雌激素受体间调控关系及其与孕鼠肝内胆汁淤积发生的相关性

目的 探讨过氧化物酶增殖体激活受体α(PPARα)、氧固醇7α羟化酶(CYP7B1)、雌激素受体(ER)α及ERβ之间的调控关系与孕鼠肝内胆汁淤积发生的相关性.方法 选择清洁级SD孕鼠80只,随机分为4组,每组20只,自孕第13天起:对照组孕鼠皮下注射精制植物油2.0ml·kg-1·d-1;低剂量组孕鼠皮下注射17-α-乙炔雌二醇(1.0 mg·kg-1·d-1);中剂量组孕鼠皮下注射17-α-乙炔雌二醇(1.25 mg·lg-1·d-1);高剂量组孕鼠皮下注射17-α-乙炔雌二醇(1.5 mg·kg-1·d-1).4组孕鼠于妊娠第21天处死后提取肝脏组织.应用酶联免疫吸附试验检测各组孕鼠血清中丙氨酸转氨酶(ALT)、门冬氨酸转氨酶(AST)、总胆酸(TBA)及胆红素(BIL)水平;应用实时定量PCR技术检测各组孕鼠肝脏组织中PPARα mRNA、CYP7B1 mRNA、ERα mRNA及ERβ mRNA的表达水平.结果 (1)生化指标:对照组孕鼠ALT、AST、TBA及BIL水平分别为(41.1±2.8)U/L、(44.4±3.6)U/L、(26.4±5.6)μmol/L、(2.8±0.2)U/L,低剂量组孕鼠分别为(48.2±3.4)U/L、(47.9±3.7)U/L、(36.4±4.2)μmol/L、(4.2±0.2)U/L,中剂量组孕鼠分别为(70.4±5.3)U/L、(68.4±5.6)U/L、(64.3±3.8)μmol/L、(6.2±1.2)U/L,高剂量组孕鼠分别为(72.4±7.6)U/L、(70.2±3.8)U/L、(72.4±7.8)μmol/L、(8.2±2.2)U/L.低剂量组、中剂量组、高剂量组孕鼠ALT、AST、TBA、BIL水平明显高于对照组(P<0.05);中剂量组、高剂量组孕鼠各生化指标水平明显高于低剂量组(P<0.05).(2)ERαmRNA及ERβmRNA表达水平:ERαmRNA的表达水平在低剂量组(0.76±0.02)、中剂量组(0.99±0.04)和高剂量组(1.21±0.01)孕鼠肝脏组织中呈逐渐升高趋势(P<0.05),并明显高于对照组(0.65±0.01),分别与对照组比较,差异均有统计学意义(P<0.05);ERβ表达水平在4组孕鼠间分别比较,差异均无统计学意义(P>0.05).(3)CYP7B1 mRNA及PPARα mRNA表达水平:CYP7B1 mRNA的表达水平在低剂量组(0.93±0.01)、中剂量组(0.99±0.06)和高剂量组(1.22±0.04)孕鼠肝脏组织中呈逐渐升高趋势(P<0.05),并明显高于对照组(0.75±0.02),分别与对照组比较,差异均有统计学意义(P<0.05);PPARα mRNA表达水平在低剂量组(0.83±0.05)、中剂量组(0.71±0.02)和高剂量组(0.64±0.03)孕鼠肝脏组织中呈逐渐降低趋势(P<0.05),并明显低于对照组(1.35±0.05),分别与对照组比较,差异均有统计学意义(P<0.05).结论 随着雌激素剂量的增加,PPARα表达水平降低,对CYP7B1表达的抑制作用解除,而导致CYP7B1表达水平升高;而CYP7B1有促进ERα高表达的作用,最终由ERα介导了雌激素诱导的肝内胆汁淤积的发生.提示PPARα、CYP7B1及ER的异常表达,是调控雌激素诱导孕鼠肝内胆汁淤积的发生机制之一.     

Nrf_2与足月胎膜早破合并绒毛膜羊膜炎的关系

目的:探讨核因子E2相关因子(Nrf_2)表达与足月胎膜早破(PROM)合并绒毛膜羊膜炎(HCA)的关系。方法:选取足月PROM患者60例,根据胎膜组织病理检查结果分成PROM合并HCA组(HCA组,21例)和未合并HCA组(对照组,39例)。采用免疫组化法、蛋白印迹法和实时荧光定量PCR法分别检测Nrf_2在胎膜组织、母血和脐血中的表达。结果:(1)HCA组胎膜组织、母血和脐血中Nrf_2mRNA表达水平分别为0.48±0.09、0.73±0.11和0.51±0.09,Nrf_2蛋白表达水平分别为0.101±0.019、0.294±0.051和0.175±0.028;HCA组中Nrf_2mRNA和蛋白表达水平均明显低于对照组,差异有统计学意义(P0.05)。(2)Nrf_2在胎膜组织羊膜层、绒毛膜层和底蜕膜层中的细胞核和细胞质均有表达。Nrf_2定位于细胞核的表达在HCA组的每一层表达均低于对照组(P0.05);两组中,Nrf_2在羊膜层、绒毛膜层和底蜕膜层中的表达水平比较,均存在显著差异,底蜕膜最低,羊膜层最高。结论:Nrf_2表达水平显著下降是PROM合并HCA的重要发病机制。Nrf_2含量变化可成为一种新的生物学标记物用于诊断PROM合并HCA的孕妇和评估新生儿预后。     

孕鼠体内高胆酸水平对胎鼠心肌组织的影响

目的 初步探讨孕鼠高胆酸环境对胎鼠心脏结构的影响.方法 选择清洁级雌性SD大鼠30只,随机分成3组,每组各10只.妊娠第13～20天,A组孕鼠每天腹腔内注射胆酸溶液5.5 mg/(kg·d)(高胆酸组),B组注射胆酸溶液1.4 mg/(kg·d)(低胆酸组),C组注射生理盐水1 ml(对照组).3组孕鼠均于孕第21天处死,测定母胎血中总胆汁酸(total bile acid,TBA)和心肌肌钙蛋白I(cardiac troponin I,cTnI)的浓度.取胎鼠的心脏组织,分别行光镜和电镜观察心肌细胞的改变.结果 (1)三组孕鼠和胎鼠血TBA浓度分别A组(22.32±8.12)8mol/L,(28.84±8.06)μmol/L;B组为(9.77±3.56),μmol/L,(9.34±3.54)/μmol/L;C组为(3.60±1.78)μmol/L,(3.95±1.19)μmol/L.三组间分别进行母鼠和胎鼠的两两比较差异均有统计学意义(P<0.01).(2)胎鼠死胎率在A、B、C三组中分别是30.11%,16.85%,7.05%,两两比较有统计学意义(P<0.05).(3)A组胎鼠的cTnI值为(19.98±7.75)ng/ml;B组(11.41±3.64)ng/ml;C组(4.38±1.19)ng/ml,两两比较有统计学意义(P<0.01).(4)光镜下胎鼠心肌病变积分:A组为1.92±0.43,B组为1.36±0.37,C组为0.44±0.12.两两比较有统计学意义(P<0.01).(5)电镜下胎鼠心肌细胞中,A组线粒体的数密度比C组小(P<0.05),而平均体积比C组大(P<0.05).B组线粒体的平均体积比C组大(P<0.05),但数密度与C组相比无差异.A组胎鼠肌原纤维的数密度和平均体积均比C组小(P<0.05).B组胎鼠肌原纤维的数密度比C组小(P<0.05),平均体积无差异.(6)母血TBA、胎鼠TBA、胎血cTnI值及胎鼠心肌病变积分之间两两比较均呈正相关(P<0.01).结论 孕鼠体内高胆酸水平对胎鼠心肌组织损伤明显,孕鼠体内高胆酸可造成胎鼠心肌组织损伤,胎鼠血清cTnI与TBA呈正相关.