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.     

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.     

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.     

自噬基因beclin1对子宫颈癌HeLa细胞生长的作用及其机制

目的 研究自噬基因beclin 1对宫颈癌细胞株HeLa细胞生长的抑制作用,并探讨其可能的机制.方法 实验分为5组:(1)pcDNA3.1(+)-beclin 1组:转染重组载体pcDNA3.1(+)-beclin 1质粒(过度表达beclin 1基因)的HeLa细胞;(2)pSUPER-beclin 1组:转染重组载体pSUPER-beclin 1质粒的HeLa细胞(抑制Beclin 1基因表达);(3)pcDNA3.1(+)组:转染空载体pcDNA3.1(+)质粒的HeLa细胞;(4)pSUPER组:转染空载体pSUPER质粒的HeLa细胞;(5)空白对照组:未转染质粒的HeLa细胞.采用逆转录(RT)PCR技术和蛋白印迹法检测各组细胞中beclin 1 mRNA和蛋白的表达以及凋亡因子--半胱氨酸天冬氨酸蛋白酶9(caspase-9)mRNA及蛋白的表达;四甲基偶氮唑蓝(MTT)比色法检测各组细胞的生长情况;流式细胞仪检测各组细胞的凋亡率;电镜观察和流式细胞仪检测各组细胞的自噬情况.将各组细胞分别接种于裸鼠皮下,观察各组裸鼠体内的成瘤性及肿瘤生长情况.结果 (1)各组细胞中beclin 1、caspase-9 mRNA和蛋白的表达:pcDNA3.1(+)-beclin 1组beclin 1和caspase-9 mRNA的表达水平分别为994.72±468.76和12.88±2.71,pSUPER-beclin 1组分别为0.18±0.63和0.11±0.08,pcDNA3.1(+)组分别为0.57±0.12和4.28±3.25,pSUPER组分别为0.67±0.29和2.77±1.27,空白对照组分别为0.74±0.25和3.67±3.78,pcDNA3.1(+)-beclin 1组均明显高于pcDNA3.1(+)组、pSUPER组和空白对照组,pSUPER-beclin 1组均明显低于pcDNA3.1(+)组、pSUPER组和空白对照组,差异均有统计学意义(P＜0.05).各组细胞中beclin 1和caspase-9蛋白的表达情况与mRNA相似.(2)各组细胞的生长情况:pcDNA3.1(+)-beclin 1组细胞的生长明显慢于空白对照组及pcDNA3.1(+)组,而pSUPER-beclin 1组细胞的生长明显快于空白对照组及pSUPER组,差异均有统计学意义(P＜0.05).(3)各组细胞的凋亡率:pcDNA3.1(+)-beclin 1组为(28.2±2.3)%,pcDNA3.1(+)组为(14.6±4.6)%,pSUPER-beclin 1组为(5.7±2.0)%,pSUPER组为(16.2±3.1)%,空白对照组为(11.2±3.0)%,pcDNA3.1(+)-beclin 1组和pSUPER-beclin 1组分别与另外3组比较,差异均有统计学意义(P＜0.05).(4)各组细胞的自噬情况:pcDNA3.1(+)-beclin 1组细胞内可见自噬体的形成,而其余各组自噬体形成不明显.pcDNA3.1(+)-beclin 1组自噬率为(10.3±1.5)%,pcDNA3.1(+)组为(3.6±0.8)%,pSUPER-beclin 1组为(1.2±0.3)%,pSUPER组为(3.2±1.2)%,空白对照组为(2.2±1.1)%,pcDNA3.1(+)-beclin 1组和pSUPER-beclin 1组分别与另外3组比较,差异均有统计学意义(P＜0.05).(5)各组裸鼠的成瘤情况:pcDNA3.1(+)-beclin 1组裸鼠成瘤时间长于空白对照组、pcDNA3.1(+)组和pSUPER组.pSUPER-beclin 1组裸鼠肿瘤体积第7天开始明显大于空白对照组、pcDNA3.1(+)组和pSUPER组(P＜0.05);pcDNA3.1(+)-beclin 1组第21天开始明显小于空白对照组、pcDNA3.1(+)组和pSUPER组(P＜0.05).接种第28天,pSUPER-beclin 1组肿瘤质量为(0.52±0.08)g,明显高于空白对照组的(0.37±0.12)g和pSUPER组的(0.34±0.24)g(P＜0.05);pcDNA3.1(+)-beclin 1组肿瘤质量为(0.18±0.12)g,明显低于空白对照组和pcDNA3.1(+)组的(0.34±0.18)g(P＜0.05).结论 自噬基因beclin 1可以抑制宫颈癌HeLa细胞体内、外的生长,这种作用不仅与自噬调控通路有关,而且可能通过调控caspse-9基因的表达参与内源性细胞凋亡通路的调节,为宫颈癌基因治疗提供了新途径.

Abstract：

Objective To investigate the inhibitory effects and the mechanism of autophagy gene beclin 1 on cervical cancer HeLa cells. Methods The eukaryotic expression vector and short hairpin RNA (shRNA) expression vector of beclin 1 were transfected via lipofectamine into HeLa cells. Experimental cells were classified into 5 groups: pcDNA3. 1 ( + )-beclin 1 group, pSUPER-beclin 1 group, pcDNA3.1 ( + )group, pSUPER group and HeLa group. Real time-PCR and western blot were used for detecting expression of mRNA and protein of beclin 1 and caspase-9 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis, and autophagy of HeLa, while proliferation was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The ultrastructural analysis of autophagic vacuoles was under the electron microscope. Five groups cells were seeded subcutaneously on nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed, and immunohistochemistry was used to detect the protein expression of beclin 1 in tumor tissue. Results ( 1 ) The mRNA expression of beclin 1 and caspase-9: pcDNA3. 1 ( + )-beclin 1 group were 994.72 ±468.76 and 12. 88 ±2. 71, pSUPER-beclin 1 group were 0. 18 ± 0. 63 and 0. 11 ± 0. 08, pcDNA3. 1 ( + ) group were 0. 57 ± 0. 12 and 4. 28 ± 3. 25,pSUPER group were 0. 67 ± 0. 29 and 2. 77 ± 1.27, and HeLa group were 0. 74 ± 0. 25 and 3.67 ± 3.78,respectively. The eukaryotic expression vector pcDNA3. 1 ( + ) -beclin 1 significantly improved the expression of mRNA of beclin 1 and caspase-9 in HeLa cells( P ＜0. 05 ), and the shRNA expression vector inhibited the expression of mRNA of beclin 1 and caspase-9 ( P ＜ 0.05 ). (2) The cell proliferations: pcDNA3.1 ( + ) -beclin 1 vector significantly inhibited the growth of HeLa cells, while pSUPER-beclin 1 vector significantly improved the growth of HeLa cells(P ＜0. 05). (3) The rate of apoptosis: pcDNA3.1 ( + )-beclin 1 group was (28.2 ±2.3)%, pcDNA3. 1( + ) group was(14.6 ±4.6)%,pSUPER-beclin 1 group was(5.7 ±2. 0) %, pSUPER group was( 16. 2 ± 3.1 ) %, and HeLa group was( 11.2 ± 3. 0) %. The pcDNA3. 1 ( + ) -beclin 1 vector significantly increased the apoptosis rate, while the pSUPER-beclin 1 vector significantly decreased the apoptosis rate(P ＜0. 05 ). (4)The activity of autophagy: more autophagy cells were identified in pcDNA3.1( + )-beclin 1 group; the rate of autophagy of five group were( 10. 3 ± 1.5)% in pcDNA3. 1 ( + ) -beclin 1 group, ( 3.6 ± 0. 8 ) % in pcDNA3. 1 ( + ) group, ( 1.2 ± 0. 3 ) % in pSUPER-beclin 1 group, (3.2 ± 1.2)% in pSUPER group and (2.2 ± 1. 1)% in HeLa group, there was statistical significances between test groups and control groups( P ＜ . 05 ). (5)Carcinogenic activity of HeLa cells in nude mice: the duration of tumorigenesis was the longest in pcDNA3.1 ( + )-beclin 1 group and the shortest in pSUPER-beclin 1 group among all groups. The tumor size began to grow larger from 7th day after injection in pSUPER-beclin 1 group than in control groups( P ＜ 0. 05 ). The tumor size was smaller from 21st day after injection in pcDNA3.1( + )-beclin 1 group than in control groups(P ＜0. 05). From 28th day after injection,the tumor weigh was (0. 52 ± 0. 08 )g in pSUPER-beclin 1 group, apparently more than HeLa group (0. 37 ±0. 12) g and pSUPER group (0. 34 ± 0. 24 ) g ( P ＜ 0. 05 ). While in pcDNA3. 1 ( + )-beclin 1 group the tumor weighed (0. 18 ±0. 12) g, which was lower than HeLa group and pcDNA3. 1 ( + ) group (0. 34 ± 0. 18 ) g ( P ＜ 0. 05 ) . Conclusions Autophagy gene beclin 1 overexpression can inhibit proliferation and growth of HeLa cells in vitro and in vivo. Beclin 1 not noly participate in the regulation of autophagy signaling, but also play an important role in the regulation of endogenous apoptosis signaling through caspase-9. So it might be one of the new strategies for gene therapy of cervical carcinoma.     

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.     

妊娠期肝内胆汁淤积症患者血清中白细胞介素18、12及肿瘤坏死因子α的水平变化及其临床意义

目的 探讨白细胞介素(IL)18、IL-12及肿瘤坏死因子α(TNF-α)在妊娠期肝内胆汁淤积症(ICP)患者肝功能异常中的作用.方法 选择2010年4-9月在重庆医科大学附属第一医院就诊的62例ICP患者为ICP组,其中重度患者32例,轻度患者30例;同期就诊的30例健康孕妇为对照组,另选同期在重庆医科大学附属第一医院感染科住院的30例乙型肝炎妇女为肝炎组.采用ELISA 法测定IL-18、IL-12及TNF-α水平.检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平.同时观察ICP组及对照组的围产儿结局.结果 (1)肝炎组血清中IL-18、IL-12、TNF-α水平分别为(256±51)、(122±96)、(207±3)ng/L,ALT、AST水平分别为(363±174)、(359±237)U/L;ICP组IL-18、IL-12、TNF-α水平分别为(72±32)、(42±28)、(48±14)ng/L,ALT、AST水平分别为(201±128)、(132±87)U/L;对照组IL-18、IL-12、TNF-α水平分别为(43±13)、(10±3)、(33±9)ng/L,ALT、AST水平分别为(13±4)、(15±3)U/L.肝炎组血清中IL-18、IL-12、TNF-α及ALT、AST 水平显著高于ICP组和对照组,差异均有统计学意义(P＜0.05);ICP组也显著高于对照组,差异均有统计学意义(P＜0.05).(2)ICP组重度患者血清中IL-18、IL-12、TNF-α水平分别为(81±32)、(50±25)、(50±14)ng/L,ALT、AST水平分别为(269±111)、(181±73)U/L;轻度患者IL-18、IL-12、TNF-α水平分别为(48±18)、(17±4)、(40±10)ng/L,ALT、AST水平分别为(87±46)、(50±21)U/L,ICP组重度患者血清中IL-18、IL-12、TNF-α及AST、ALT水平显著高于轻度患者和对照组,差异均有统计学意义(P＜0.05);轻度患者血清中AST和ALT水平显著高于对照组,差异有统计学意义(P＜0.05).(3)ICP组重度患者的早产儿发生率(50%,16/32)及羊水胎粪污染率(31%,10/32)显著高于轻度患者[分别为7%(2/30)及3%(1/30)]和对照组[分别为3%(1/30)及3%(1/30)],差异均有统计学意义(P＜0.05);重度患者新生儿1分钟Apgar评分≤7分的例数(2例)与轻度患者(1例)和对照组(1例)比较,差异无统计学意义(P＞0.05).结论 IL-18、IL-12和TNF-α可能参与ICP患者肝细胞损害的过程,其水平升高有助于临床诊断ICP患者的肝细胞损害.

Abstract：

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.     

核转录因子-κB与妊娠期糖尿病发病机制的研究

目的:通过调控核转录因子-κB(NF-κB)在细胞信号转导过程中的状态,研究NF-κB与妊娠期糖尿病(GDM)炎症反应发生发展以及终末炎症因子之间的关系,探讨NF-κB在GDM的炎症反应发生中的作用。方法:选择正常孕妇(W组)和GDM孕妇(G组)各30例,检测血清中脂多糖(LPS)的量。将每个样本各分为4个处理组,分别进行未处理、加入LPS(1μg/ml)刺激、加入NF-κB抑制剂毗咯烷二硫代氨基甲酸(PDTC)(50 mmol/L)干预、加入PDTC预处理1小时后再给予LPS刺激,并分别标记为W(G)、W(G)+L、W(G)+P、W(G)+LP组。采用Western blot法检测各组NF-κB蛋白的活化量,采用ELISA法检测各组肿瘤坏死因子(TNF-α)、白介素-1(IL-1)、白介素-10(IL-10)的水平,并行Pearson相关分析。结果:1GDM孕妇组与正常孕妇组比较,血清中LPS的量明显增加(P0.05)。2两组组内的比较:加入LPS刺激后,W(G)+L组、W(G)+LP组中细胞内NF-κB蛋白活化量和炎症因子TNF-α、IL-1、IL-10水平均明显高于W(G)组(P0.05);使用PDTC后,W(G)+LP组中细胞内NF-κB蛋白活化量和炎症因子TNF-α、IL-1、IL-10水平均明显低于W(G)+L组(P0.05)。3GDM组各处理组中NF-κB蛋白活化量和炎症因子TNF-α、IL-1、IL-10的水平均较正常孕妇组中相同处理组明显升高(P0.05)。4Pearson相关分析示:两组细胞中NF-κB的活化量与终末炎症因子TNF-α、IL-1、IL-10水平呈正相关关系(P0.05)。结论:NF-κB作为细胞内多条传导通路的汇合点,介导信号向胞核内转导,通过调控终末炎症因子水平,参与了GDM炎症反应的发生,并可能在其中起着类似"开关点"的作用。     

PKC抑制剂对子痫前期血清诱导人脐静脉内皮细胞NF-κB核转位及VCAM-1表达的影响

目的:探讨子痫前期血清诱导人脐静脉内皮细胞(HUVEC)蛋白激酶(PKC)活性改变、核因子-κB(NF-κB)核转位及内皮细胞黏附分子-1(VCAM-1)的表达以及PKC抑制剂对它们的影响。方法:用胰酶消化培养法培养正常妊娠HUVEC,传代后待细胞长满至70%~80%后,加或不加PKC抑制剂多黏菌素B(PMB)作用30m in后分别加入正常妊娠及子痫前期血清,培养2h,W estern B lot测定细胞胞浆PKC、胞膜PKC含量、胞浆核因子κB抑制因子(I-κB)、胞核NF-κBp65含量;培养48h,用MTT测定细胞活力,流式细胞学测定细胞凋亡,酶联免疫法测定HUVEC VCAM-1的表达。结果:子痫前期血清培养的HUVEC胞浆PKC及I-κB含量明显低于对照组(P<0.05),胞膜PKC含量、胞核NF-κBp65含量、VCAM-1表达、细胞凋亡率明显高于对照组(P<0.05),细胞活力明显低于对照组(P<0.05)。加PKC抑制剂预处理后,子痫前期组HUVEC胞浆PKC含量及I-κB含量明显增加(P<0.05);胞膜PKC含量、胞核NF-κBp65含量、VCAM-1表达、细胞凋亡率明显下降(P<0.05),细胞活力明显增加(P<0.05)。结论:子痫前期血清可促进HUVEC NF-κB活性及VCAM-1表达,PKC抑制剂可抑制子痫前期患者血清诱导HUVEC的NF-κB活性及VCAM-1表达,PKC、NF-κB在子痫前期内皮细胞损伤过程中可能起重要的桥梁作用。     

川芎嗪对子痫前期脐血清诱导脐静脉内皮细胞NF-κB活性及VCAM-1表达的影响

目的:探讨川芎嗪对子痫前期患者脐静脉血清诱导脐静脉内皮细胞(HU-VEC)核因子-κB(NF-κB)活性及其靶基因血管细胞黏附分子-1(VCAM-1)表达变化的影响。方法:用胰酶消化法培养正常妊娠HUVEC,传代后待细胞长满至70%~80%,加或不加川芎嗪作用30min后分别加入正常妊娠及子痫前期脐静脉血清,培养48h后,MTT测定细胞活力,流式细胞学测定细胞凋亡率,Western-blot测定HUVEC NF-κB的表达,酶联免疫检测VCAM-1的表达。结果:子痫前期患者脐静脉血清培养HUVEC的增殖活力明显低于正常妊娠组,胞核NF-κB及VCAM-1表达明显高于正常妊娠组(P<0.01),子痫前期患者脐静脉血清培养HUVEC的细胞凋亡率明显高于正常妊娠组(P<0.01)。加川芎嗪预处理后,子痫前期组HUVEC增殖活力明显增加,细胞凋亡率、胞核NF-κB及VCAM-1表达明显下降(P<0.01)。结论:川芎嗪可抑制子痫前期患者脐静脉血清诱导的HU-VEC凋亡,NF-κB的核转位及VCAM-1的表达。     

清肺通络敷胸膏对流感病毒肺炎大鼠肺组织ERK/NF-κB p65蛋白表达的影响

目的探讨清肺通络膏对流感病毒诱导的肺炎大鼠肺组织中ERK/NF-κB p65信号通路的调控机制。方法将30只Wister大鼠随机分为正常组、模型组、清肺通络敷胸膏组,每组10只。除正常组外,采用鼻腔接种流感病FM1株诱导Wister幼龄大鼠肺炎,在感染后各治疗组采用清肺通络膏贴敷于大鼠背部肺脏投影区治疗。观察各组大鼠的一般状态(饮食量、进水量、毛发、活动量、精神状态、呼吸频率);光镜下观察肺组织形态学;采用免疫组织化学法检测各组大鼠肺组织磷酸化细胞外调节蛋白激酶(p-ERK),核转录因子(NF-κB p65)的表达水平。结果与正常组相比,模型组大鼠肺组织中p-ERK,NF-κB p65蛋白表达显著增高,差异有统计学意义(P0.05),清肺通络膏组p-ERK、NF-κB p65蛋白的表达较模型组明显降低,差异有统计学意义(P0.05)。结论清肺通络膏通过抑制ERK磷酸化,使NF-κB p65表达降低,从而减轻了肺组织的病理损伤,达到治疗目的。     

雌二醇对同型半胱氨酸诱导人脐静脉内皮细胞组织因子表达的影响

目的:探讨雌二醇(E2)对同型半胱氨酸(Hcy)诱导人脐静脉内皮细胞(HU-VEC)组织因子(TF)表达的影响及机制。方法:通过一期凝固法测总细胞促凝活性(PCA);采用RT-PCR测TF表达;用免疫组化法观察NF-κB的变化。结果:Hcy剂量依赖性促进HUVEC的PCA增加(与对照组相比r=0.969,P<0.01);E2单独作用对HUVEC的PCA没有影响(P>0.05);E2抑制Hcy诱导HUVEC表达TF(r=-0.686,P<0.01),最适浓度为1×10-7mol/L;E2可抑制Hcy引起的NF-κB核易位(P<0.01);ICI182,780对E2抑制Hcy诱导HUVEC的TF表达和核易位没有明显的拮抗作用(P>0.05)。结论:E2抑制Hcy诱导HUVEC表达TF;E2降低HUVEC的NF-κB核易位;E2抑制Hcy诱导HU-VEC表达TF作用未通过经典核受体途径。     

原因不明复发性流产患者调节性T淋巴细胞对树突状细胞的调控作用

目的 探讨原因不明复发性流产(URSA)患者外周血和蜕膜组织中CD+4CD+25调节性T淋巴细胞(Tr细胞)对树突状细胞(DC)的调控作用.方法 采集4例URSA患者(流产组)和4例正常早孕妇女(对照组)的外周血和蜕膜组织,密度梯度离心法、免疫磁珠分离法分选出Tr细胞和DC,培养6 d,培养方法分为DC单独培养和Tr细胞与Dc混合培养.酶联免疫吸附试验(ELISA)检测两种方法培养的细胞培养上清液中辅助性T淋巴细胞(Th)1型细胞因子--γ干扰素(IFN-γ)的Th2型细胞因子--白细胞介素10(IL-10)的蛋白含量.结果 (1)外周血:流产组细胞混合培养和单独培养后,细胞培养上清液中IFN-γ蛋白含量为(22.5±3.0)、(23.2±0.7)ng/L,两者比较,差异无统计学意义(P>0.05);IL-10蛋白含量分别为(35±4)、(37±7)ng/L,两者比较,差异也无统计学意义(P>0.05).对照组细胞混合培养和单独培养后,IFN-γ蛋白含量为(36±1.1)、(30.5±4.0)ns/L,两者比较,差异无统计学意义(P>0.05);IL-10蛋白含量分别为(36±9)、(54±20)ng/L,两者比较,差异有统计学意义(P<0.01).(2)蜕膜组织:流产组细胞混合培养和单独培养后,IFN-γ蛋白含量分别为(24.4±2.5)、(23.4±2.6)ng/L,两者比较,差异无统计学意义(P>0.05);IL-10蛋白含量分别为(25±5)、(28±7)ng/L,两者比较,差异也无统计学意义(P>0.05);对照组细胞混合培养后,IFN-γ蛋白含量为(22.6±3.8)ng/L,明显低于单独培养后的(30.7±4.6)ng/L,差异有统计学意义(P<0.01);对照组细胞混合培养后,IL-10蛋白含量为(31±9)ng/L,明显高于单独培养后的(27±6)ng/L,差异也有统计学意义(P<0.05).结论 URSA患者Tr细胞抑制性免疫功能下降,从而导致对DC调控失常,Th1/Th2平衡失调和母胎免疫耐受异常.