摘要
第一部分子痫前期患者胎盘组织中Cyr61及CTGF表达研究
目的
检测在正常孕妇和子痫前期患者胎盘组织中Cyr61和CTGF的表达情况,探讨它们在子痫前期发病机制中的作用及两者间的关联。
方法
采用免疫组化SP法和实时荧光定量PCR法检测34例重度子痫前期孕妇和16例正常晚期妊娠孕妇胎盘组织中Cyr61和CTGF蛋白定位表达、mRNA水平表达。
结果
1.Cyr61在胎盘绒毛合体滋养细胞、细胞滋养细胞和内皮细胞中均有表达,主要表达在合体滋养细胞的胞浆内；CTGF在胎盘绒毛合体滋养细胞、细胞滋养细胞和内皮细胞中均有表达,主要表达在细胞滋养细胞的胞浆内。重度子痫前期患者胎盘中Cyr61蛋白表达水平明显低于正常组(P<0.01)。CTGF在重度子痫前期组胎盘中蛋白表达水平明显高于正常组(P<0.05)。2. Cyr61 mRNA在重度子痫前期组的表达水平较正常组明显下降,差别有统计学意义(P<0.05);CTGF mRNA在重度子痫前期组的表达水平较正常妊娠晚期组明显增加,差别有统计学意义(P<0.01)。3.在重度子痫前期患者胎盘组织中Cyr61与CTGF表达水平呈负相关(r=-0.34,P<0.01)。
结论1.子痫前期胎盘滋养细胞中Cyr61与CTGF的表达异常,导致VEGF的生成减少和ECM的重塑障碍,可能与子痫前期的发病有关。
2.子痫前期胎盘滋养细胞中的Cyr61与CTGF的表达水平呈负相关,外源性加入Cyr61或VEGF可能拮抗CTGF在子痫前期发病中的作用。
第二部分缺氧条件下绒毛外滋养细胞增殖及凋亡情况
目的
通过二氯化钴化学物质来诱导绒毛外滋养细胞缺氧过程的出现,探讨缺氧条件下滋养细胞的增殖能力及细胞凋亡情况。
方法
1.MTT法检测滋养细胞增殖能力：滋养细胞在二氯化钴终浓度为150μmol/L、300μmol/L的培养基中分别培养：6h、12h、24h、48h和72h。常氧组细胞在不含COCl2的培养基中培养。
2.流式细胞仪检测滋养细胞的凋亡情况：滋养细胞在二氯化钴终浓度为：300μmol/L的培养基中培养6h、12h、24h、48h和72h。
结果
1.与正常组相比,滋养细胞在经过CoCl2处理后细胞增殖活力受到抑制；且当COCl2的浓度为300μmol/L时,对滋养细胞增殖的抑制效应较150μmol/更为明显(P<0.05)。当滋养细胞在300μmol/L COCl2作用24h时、细胞增殖能力受到显著抑制(P<0.01)。2.与正常对照组相比,滋养细胞在终浓度为300μmol/L CoCl2预下,其细胞凋亡水平随着时间的延长增加,在缺氧12h凋亡增加明显(10.7+/-0.8-fold；P<0.01)且在缺氧24h细胞凋亡水平达峰值(24.0+/-3.4-fold:P<0.05)。
结论
采用CoCl2模拟的化学缺氧能抑制滋养细胞的增殖并促进其细胞凋亡,随着缺氧时间的延长细胞凋亡增加且在缺氧24h细胞凋亡最显著。
第三部分缺氧作用下绒毛外滋养细胞中Cyr61表达在子痫前期发病中的作用研究
目的
研究滋养细胞在CoCl2模拟的化学缺氧条件下,细胞基质蛋白Cyr61在滋养细胞中的表达情况；探讨Cyr61的表达与子痫前期发病机制的相关研究。
方法
1.采用细胞免疫荧光法检测Cyr61蛋白在滋养细胞中的定位表达。
2.实时荧光定量PCR检测缺氧条件下滋养细胞中Cyr61转录水平的表达情
况。3.蛋白印迹法检测缺氧条件下滋养细胞中Cyr61的蛋白水平的表达。
结果
1.细胞基质蛋白Cyr61主要表达在滋养细胞的胞浆中,部分表达在细胞核中。
2.在缺氧6h和12h时,滋养细胞中的Cyr61表达增加；随着缺氧时间延长达24h和48h后,该蛋白表达下调(P<0.05)。
结论
低氧环境可以诱导滋养细胞中Cyr61的产生；但是随着缺氧时间的延长,滋养细胞中的Cyr61表达下调。
第四部分子痫前期患者外周血清干预下绒毛外滋养细胞中Cyr61表达下调在子痫前期发病中的相关研究
目的
研究绒毛外滋养细胞在正常孕妇和子痫前期患者外周血清干预下,该细胞中Cyr61的表达情况。
方法
1.采用细胞免疫荧光法检测滋养细胞中Cyr61的表达定位。
2.实时荧光定量PCR检测子痫前期患者外周血清处理24h后,滋养细胞中Cyr61转录水平的表达情况。
3.蛋白印迹法检测子痫前期患者外周血清处理24h后,滋养细胞中Cyr61的蛋白水平的表达。
结果
1.细胞基质蛋白Cyr61主要表达在滋养细细胞的胞浆中,部分表达子在细胞核中。2.与正常孕妇外周血清干预相比,滋养细胞经子痫前期外周血清处理后,该细胞中Cyr61转录和蛋白水平均下降(P<0.05)。
结论
子痫前期孕妇外周血清可下调滋养细胞中Cyr61的表达,从而说明该蛋白的下调可能与子痫前期发病有关。
Part one
Study expression of Cyr61 and CTGF in placentas from preeclamptic women
Objective:
To detect the expression of Cyr61 and CTGF in placentas from normal term pregnancy and preeclampsia women, and explore their roles in the pathogenesis of preeclampsia.
Methods:
The protein and mRNA expression levels of Cyr61 and CTGF of placenta tissue in 16 cases of normal term pregnancy and 34 cases of severe preeclampsia women were determined by immunohistochemistry and real-time fluorescence quantitative PCR, respectively. Results:
1. Cyr61 was expressed in syncytiotrophoblast, cytotrophoblast and endothelial cell and mainly located in the cytoplasm of the placental villous syncytiotrophoblast. CTGF was expressed in cytotrophoblast and endothelial cell and mainly located in the cytoplasm of the placental villous cytotrophoblast. Compared with control group, the expression of Cyr61 in the severe preeclampsia group was significantly lower (P<0.01), while CTGF in the severe preeclampsia group was significantly higher than that of control group (P<0.05).
2. Cyr61 mRNA expression in the severe preeclampsia group was significantly lower than normal levels, the difference was statistically significant (P<0.05); CTGF mRNA expression in severe preeclampsia were significantly increased comparing with normal levels, the difference was statistically significant (P<0.01).
3. There was a negatively correlation between the expression of Cyr61 and CTGF in placentas from severe preeclampsia (r=-0.34, P<0.01). Conclusions:
1. Abnormal expression of Cyr61 and CTGF in trophoblast might lead toVEGF reduce and the failure of ECM formation and remodeling thus resulted in the pathogenesis of preeclampsia.
2. The expression of Cyr61 and CTGF in preeclampsia is negatively. Therefore, the addition of exogenous VEGF or Cyr61 could be antagonized the effect of CTGF in the pathogenesis of preeclampsia.
Part two
The cell proliferation and apoptosis of extravillous trophoblast under hypoxia
Objective:
To investigate the proliferation and apoptosis of extravillous trophoblast under hypoxic conditions and Cobalt chloride (CoCl2) was used to mimic the effects of hypoxia in villous explants.
Methods:
1. Methl thiazolyl tetrazolium (MTT) assay was implied to evaluate the cell proliferation function. Cells were treated with 150 or 300μmol/L of cobalt chloride. TEV-1 cells were treated with cobalt chloride for 6h,12h,24h,48h and 72h. Normoxic cell lines were cultured in the same way without the use of cobalt chloride.
2. Flow cytometry analysis was used to detect trophoblast apoptosis and the cells were treated with 300μmol/L of cobalt chloride for 6h,12h,24h,48h and 72h.
Results:
1. Compared with the control, CoCl2 inhibited growth parameters of TEV-1 cells.The cell proliferation was remarkably inhibited at a concentration of 300μmol/L CoCl2 in contrast to 150μmol/L CoCl2(P<0.05). When TEV-1 cells were treated with 300μmol/L CoCl2 for 24h, the cell reached the lowest point of proliferation(P<0.01).
2. Annexin-V/PI double staining assay showed that, compared to the cells cultured in normal group, exposure of extravillous trophoblasts to low oxygen concentration(300μmol/L CoCl2), enhanced cell apoptosis in a time-dependent manner, with a significant increase in apoptosis levels after 12h (10.7+/-0.8-fold; P<0.01), reaching a maximum of 24.0+/-3.4-fold at 24h(P<0.05).
Conclusions:
Cobalt chloride, which is used to mimic the effects of hypoxia in TEV-1 cells, inhibits the cell proliferation and promotes cell apoptosis. As the hypoxic condition extends, TEV-1 cells apoptosis increases and peaks, exposure to hypoxic conditions for 24h.
Part three
The expression of Cyr61 in extravillous trophoblast under hypoxia and its role in the pathogenesis of preeclampsia
Objective:
To examine the expression of matricellular protein Cyr61 functional changes involved in adaptation to hypoxia of the TEV-1 cells, using cobalt chloride (CoCl2) as hypoxic mimic and explore Cyr61 activity in the pathological mechanism of preeclampsia.
Methods:
1. Immunofluorescence microscopy analysis was used to detect the location and expression of Cyr61 in extravillous trophoblast.
2. Real-time quantitative PCR was implied to analyze hypoxic regulation of Cyr61 mRNA.
3. The protein level of Cyr61 was investigated by Western blot analysis
Results:
1. The matricellular protein Cyr61 was mainly observed in the cytoplasm and partly in nuclear.
2. Expression of Cyr61 in extravillous trophoblast increased upon hypoxic treatment for 6h and 12h. However, as the hypoxic time extended, Cyr61 expression decreased.
Conclusions:
Although hypoxia transiently induced Cy61 production, as the hypoxic condition extended, the expression of Cyr61 was down-regulated.
Part four
Preeclamptic sera induces Cyr61 decrease in extravillous trophoblast and its relation with preeclampsia
Objective:
To investigate the expression of Cyr61 in extravillous trophoblast and the cells was treated with normal pregnant serum and PE serum, espectively.
Methods:
1. The location and expression of Cyr61 in extravillous trophoblast was demonstrated by immunofluorescence microscopy analysis.
2. The protein and transcriptional level of Cyr61 in extravillous trophoblast, which was cultivated under PE serum for 24h, was investigated by using real-time quantitative PCR and Western blot analysis, respectively.
Results:
1. Cyr61 protein was mainly located in the cytoplasm and partly in nuclear.
2. In contrast to normal pregnant serum, the Cyr61 mRNA expression level as well as protein decreased in extravillous trophoblast exposed to PE serum (P<0.05).
Conclusions:
Preeclamptic sera evoked Cyr61 down regulation and Cyr61 deficiency might play roles in the pathogenesis of preeclampsia.
引文
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