摘要
第一部分子痫前期患者胎盘及血清中IGF-Ⅱ及MMP-9表达变化的研究
目的通过测定子痫前期(pre-eclampsia，PE)孕妇胎盘组织及血清中胰岛素样生长因子Ⅱ(Insulin like growth factorⅡ，IGF-Ⅱ)及基质金属蛋白酶9(matrix metallop-roteinase 9，MMP-9)的表达，探讨IGF-Ⅱ及MMP-9与子痫前期发病的关系。
方法以30例子痫前期孕妇为子痫前期组(其中轻度子痫前期20例，重度子痫前期10例)，15例同期分娩的正常孕妇为对照组。逆转录-聚合酶链反应(RT-PCR)检测各组胎盘组织中IGF-ⅡmRNA及MMP-9mRNA的表达并进行半定量分析；Westernblot法检测各组胎盘组织中IGF-Ⅱ及MMP-9蛋白质的表达并进行半定量分析；通过免疫组化染色方法检测IGF-Ⅱ及MMP-9在各组胎盘组织中的的表达部位和表达水平，通过高清晰度彩色医学图文分析系统对其进行半定量分析；ELISA法检测各组血清中IGF-Ⅱ及MMP-9的浓度。
结果①RT-PCR结果显示两组胎盘组织中均有IGF-ⅡmRNA及MMP-9mRNA的表达，子痫前期组IGF-ⅡmRNA及MMP-9mRNA水平均明显低于对照组，差异有统计学意义(P＜0．05，P＜0．05)。②Western blot结果显示两组胎盘组织中均有6．7kDa的IGF-Ⅱ蛋白质表达，但子痫前期组表达量明显降低(P＜0．05)，同时也有92kDa的MMP-9蛋白质表达，子痫前期组表达量明显低于对照组(P＜0．05)。③免疫组化染色定量分析发现，IGF-Ⅱ及MMP-9在子痫前期组胎盘组织中的表达明显低于对照组(P＜0．05，P＜0．05)。④ELISA结果显示子痫前期组血清中IGF-Ⅱ及MMP-9水平均明显低于对照组(P＜0．05，P＜0．05)。
结论子痫前期患者胎盘及血清中IGF-Ⅱ及MMP-9表达均明显降低，且随病情加重递减，相应部位IGF-Ⅱ和MMP-9水平呈正相关，提示IGF-Ⅱ与MMP-9与子痫前期发病密切相关。
第二部分IGF-Ⅱ及MMP-9在子痫前期患者胎盘滋养细胞内的表达及其相互关系的研究
目的通过测定正常孕妇及子痫前期孕妇胎盘滋养细胞中IGF-Ⅱ和MMP-9的表达及在IGF-Ⅱ不同作用浓度和作用时间作用下MMP-9水平的变化，从细胞水平探讨IGF-Ⅱ及MMP-9在子痫前期中的表达及IGF-Ⅱ对MMP-9表达的影响。
方法原代培养30例子痫前期孕妇胎盘滋养细胞作为子痫前期组(其中轻度子痫前期20例，重度子痫前期10例)及15例同期分娩的正常孕妇胎盘滋养细胞为对照组，采用RT-PCR法和Western blot法检测各组IGF-Ⅱ及MMP-9的表达水平并检查在IGF-Ⅱ不同的作用时间及不同的作用浓度下各组胎盘滋养细胞MMP-9的表达水平。
结果①子痫前期组患者胎盘滋养细胞中IGF-ⅡmRNA及MMP-9mRNA水平明显低于对照组，二者呈正相关(r=0．467，P＜0．05，P＜0．05)。②子痫前期组患者胎盘滋养细胞中IGF-Ⅱ及MMP-9蛋白水平明显低于对照组，二者呈正相关(r=0．577，P＜0．05，P＜0．05)。③加入IGF-Ⅱ作用胎盘滋养细胞后，各组MMP-9表达均上升，子痫前期组上升更明显，且呈时间和剂量依赖关系。
结论子痫前期患者胎盘滋养细胞中IGF-Ⅱ及MMP-9表达均低于正常孕妇，IGF-Ⅱ可上调MMP-9在滋养细胞中的表达，提示IGF-Ⅱ与MMP-9均参与了子痫前期的病理过程，子痫前期时IGF-Ⅱ含量减少下调MMP-9的表达可能是子痫前期发病的重要因素。
第三部分子痫前期患者胰岛素样生长因子结合蛋白-1的表达及其对IGF-Ⅱ和MMP-9表达的影响
目的检测子痫前期患者胎盘滋养细胞中胰岛素样生长因子结合蛋白-1(IGFBP-1)的表达及其对IGF-Ⅱ及MMP-9表达的影响。
方法原代培养30例子痫前期孕妇胎盘滋养细胞作为子痫前期组(其中轻度子痫前期20例，重度子痫前期10例)及15例同期分娩的正常孕妇胎盘滋养细胞为对照组，采用RT-PCR法检测各组IGFBP-1的表达水平并检测在IGFBP-1不同的作用时间及不同的作用浓度下各组胎盘滋养细胞IGF-Ⅱ及MMP-9的表达水平。
结果①子痫前期组患者胎盘滋养细胞中IGFBP-1 mRNA水平明显高于对照组(P＜0．05)。②加入IGFBP-1作用胎盘滋养细胞后，各组IGF-Ⅱ及MMP-9表达均下降，子痫前期组下降更明显，且呈时间和剂量依赖关系。
结论IGFBP-1可抑制IGF-Ⅱ及MMP-9在人胎盘滋养细胞中的表达，提示IGFBP-1下调IGF-Ⅱ可能参与了子痫前期的病理过程。用IGF-Ⅱ增强MMP-9基因的表达将是预防和治疗子痫前期的一个新思路。
PartⅠThe Research of Changes of Insulin-Like Growth Factor-ⅡandMatrix Metalloproteinase-9 in Maternal Serum and Placenta andPathogenesis of Patients with Pre-eclampsia.
Objects The purpose of the present study was to investigate the expression ofinsulin-like growth factorⅡ(IGF-Ⅱ)and matrix metalloproteinase 9(MMP-9)in theplacenta and the serum of women with pre-eclampsia(PE)and discuss their role in thepathogenesis of pre-eclampsia.
Methods Select 30 examples of pre-eclampsia in pregnant women as the case group(20 examples as mild pre-eclampsia group and 10 examples as severe pre-eclampsia group),15 examples of normal pregnant women giving birth in the same period as the controlgroup.Reverse transcript polymerase chain reaction(RT-PCR)was used to detect theexpression of IGF-ⅡmRNA and MMP-9mRNA in placental tissue on each group;Westernblot analysis was applied to detect the expression of IGF-Ⅱand MMP-9;Immunohis-tochemistry was used to detect the location and expression of IGF-Ⅱand MMP- 9 inplacental tissue and HMIAS-2000 was used to determine the quantity of them;Enzyme linked immunosorbent assay was used to detect the concentration of IGF-Ⅱand MMP-9 inserum of each group.
Results①The expressive quantity of IGF-ⅡmRNA and MMP-9mRNA were bothsignificantly lower in women with pre-eclampsia than in the normal group(P＜0.05,P＜0.05).②Western blot analysis showed that both groups express 6.7kDa IGF-Ⅱand92kDa MMP-9 but the levels were significantly lower in women with pre-eclampsia than inthe normal group(P＜0.05,P＜0.05).③Immunohistochemistry analysis tested that the levelsof IGF-Ⅱand MMP-9 were both significantly lower in women with pre-eclampsia than inthe normal group(P＜0.05,P＜0.05).④Enzyme linked immunosorbent assay showed thatthe levels of IGF-Ⅱand MMP-9 were both lower in women with pre-eclampsia than in thenormal group(P＜0.05,P＜0.05).
Conclusion The levels of IGF-Ⅱand MMP-9 expression in the placenta tissues andserum of women with pre-eclampsia were significantly decreased and there was a positivecorrelation between them,which indicating that IGF-Ⅱand MMP-9 may play importantrole in the pathogenesis of pre-eclampsia.
PartⅡThe Study on Expressions of Insulin-Like Growth Factor-ⅡandMatrix Metalloproteinase-9 in Placental Trophoblast Cells and TheirMutual Relationship in Patients with Pre-eclampsia.
Objective By measuring the changes of expression of insulin-like growth factorⅡand matrix metalloproteinase 9 in the placental trophoblast cells of women withpre-eclampsia and normal pregnant women and the changes of levels of matrixmetalloproteinase 9 under the influence of IGF-Ⅱwith different times and differentconcentrations to discuss the role of them in pre-eclampsia and the influence of insulin-like growth factorⅡon the activity of matrix metalloproteinase 9 in the human placentaltrophoblast of pre-eclampsia.
Methods Primary cultured 30 examples of placental trophoblast cells of pregnantwomen with pre-eclampsia as pre-eclampsia group(20 examples as mild pre-eclampsiagroup and 10 examples as severe pre-eclampsia group),15 examples of normal pregnantwomen giving birth in the same period as the control group.RT-PCR was used to detect theexpressions of IGF-ⅡmRNA and MMP-9mRNA in placental trophoblast cells of eachgroup and to investigate the levels of MMP-9 in each group with the affection of IGF-Ⅱatdifferent times and different concentrations.
Results①The expressive quantity of IGF-ⅡmRNA and MMP-9mRNA were bothsignificantly lower in the placental trophoblast cells of women with pre-eclampsia than inthe normal group and there was a positive correlation between them(r=0.467,P＜0.05,P＜0.05).②Western blot analysis showed that both groups express 6.7kDa IGF-Ⅱand92kDa MMP-9 but the levels were significantly lower in the placental trophoblast cells ofwomen with pre-eclampsia than in the normal group and there was a positive correlationbetween them(r=0.577,P＜0.05,P＜0.05).③After the affection of IGF-Ⅱwith differenttimes and different concentrations,the levels of MMP-9 of each group were increased,ofwitch the pre-eclampsia group rose more obviously and it was showed with time-and dosedependent manner.
Conclusion The expression of IGF-Ⅱand MMP-9 in placental trophoblast cells ofwomen with pre-eclampsia were significantly lower than normal pregnant women,suggesting that both of them are involved in the pathological process of pre-eclampsia.IGF-Ⅱcan increase the expression of MMP-9 in trophoblast cells,suggesting that thedecrease of IGF-Ⅱprobably inhibit the expression of MMP-9 in trophoblast cells witchmay be an important factor in the pathogenesis of pre-eclampsia.
PartⅢThe Expression of Insulin-Like Growth Factor Binding Protein1 in Pre-eclampsia and Its Affection on IGF-Ⅱand MMP-9 Levels.
Objective By measuring the levels of insulin-like growth factor binding protein-1 inthe placental trophoblast cells of women with pre-eclampsia and normal pregnant women todiscuss the role of it in pre-eclampsia and the influence of insulin-like binding protein-1 onthe expressions of IGF-Ⅱand MMP-9 in human placental trophoblast cells.
Methods Primary cultured 30 examples of placental trophoblast cells of pregnantwomen with pre-eclampsia as pre-eclampsia group(20 examples as mild pre-eclampsiagroup and 10 examples as severe pre-eclampsia group),15 examples of normal pregnantwomen giving birth in the same period as the control group.Using RT-PCR assay in eachgroup to detect the expressions of IGFBP-1 in placental trophoblast cells and to investigatethe different levels of IGF-Ⅱand MMP-9 with the affection of IGFBP-1 at different timeand different concentrations.
Results(①The expressive quantity of IGFBP-1 mRNA was significantly higher in theplacental trophoblast cells of women with pre-eclampsia than in the normal group(P＜0.05).②After the affection of IGFBP-1 with different times and different concentrations,thelevels of IGF-Ⅱand MMP-9 of each group were all decreased,of witch the pre-eclampsiagroup declined more obviously and it was showed with time and dose-dependent manner.
Conclusion IGFBP-1 inhibited the expression of IGF-Ⅱand MMP-9 in humantrophoblast cells,suggesting that IGFBP-1 reducing IGF-Ⅱmay be involved in thepathological process of pre-eclampsia.With IGF-Ⅱenhanced MMP-9 gene expression willbe a new train of thought witch may be a prevention and treatment of pre-eclampsia.
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