Rac1信号通路在滋养细胞侵袭行为中的作用及机制研究

发布时间：2018-05-22 08:31

本文选题：Racl + β-catenin　；参考：《山东大学》2016年博士论文

【摘要】：第一部分Rac 1信号通路在正常妊娠及子痫前期中表达的研究研究目的检测Rac1信号通路在正常妊娠及子痫前期产妇胎盘的表达变化,探讨其与滋养细胞侵袭的关系。研究方法从2010年6月至2014年12月,我们在山东大学第二医院收集的就诊孕妇,其中,正常妊娠组共50例,分为早孕组及足月妊娠组,早孕组为25例6-9周妊娠者,行人工流产术,年龄(28.5±3.8)岁；足月妊娠组为25例37-40周妊娠者,行择期剖宫产术,年龄(27.9±3.3)岁；子痫前期组共计67例行择期剖宫产的37-40周子痫前期患者,其中子痫前期轻度组37例,年龄(28.9±3.25)岁；子痫前期重度组30例,年龄(26.9±2.8)岁。各组孕妇的孕周、年龄、孕次以及基础血压等无明显差异性(P0.05)。分别采用Real-time PCR法、免疫组织化学法、GTPase pull down、Western blot法检测50例正常妊娠组(包括早孕25例、足月妊娠25例)以及67例子痫前期组(包括子痫前期轻度37例、子痫前期重度30例)胎盘组织GTP-Rac1、Rac1与其下游的靶基因β-catenin、 Snail的蛋白表达及MMP9的蛋白定位、表达和mRNA的变化。结果我们应用GTPase pull down以及Western blot法对正常妊娠组以及子痫前期组胎盘组织GTP-Rac1的表达水平进行检测,结果显示早孕组胎盘绒毛组织中GTP-Rac1的表达水平显著高于足月妊娠组(P0.05)；子痫前期组GTP-Rac1的表达水平明显低于足月妊娠组(P0.05),其中,子痫前期重度组GTP-Rac1表达水平低于子痫前期轻度组,差异有统计学意义(P0.05)；而各组间Rac1蛋白表达水平未见明显的差异(P0.05)。Western blot检测发现,早孕组胎盘组织核内β-catenin及Snail表达水平显著高于足月妊娠组(P0.05)；子痫前期组胎盘组织核内β-catenin及Snail表达水平低于足月妊娠组,差异有统计学意义(P0.05),其中子痫前期重度组的β-catenin及Snail表达水平显著低于子痫前期轻度组(P0.05)。免疫组化结果显示,MMP9主要在细胞滋养细胞、合体滋养细胞中表达,可见其主要分布于胞浆,呈棕黄色或深棕黄色,足月妊娠组较早孕组MMP9阳性细胞数量明显减少；子痫前期组MMP9阳性细胞数量明显低于足月妊娠组,且染色明显减弱,子痫前期重度组与子痫前期轻度组比较,MMP9阳性细胞数量明显降低,染色明显减弱。Western blot及Real-time PCR结果示,早孕组MMP9蛋白及mRNA的表达水平明显高于足月妊娠组(P0.05)；子痫前期组胎盘组织的MMP9蛋白及mRNA的表达水平显著低于足月妊娠组(P0.05),且子痫前期重度组与子痫前期轻度组比较,胎盘组织中MMP9蛋白及mRNA的表达水平显著降低(P0.05)。结论(1) GTP-Rac1在正常妊娠胎盘滋养细胞中的表达随孕周增加而降低,且Rac1下游重要的靶基因β-catenin Snail在胎盘组织核内的表达及与滋养细胞侵袭力密切相关的MMP9的表达与Rac1的活性变化一致。Rac1信号通路的变化与正常早期妊娠滋养细胞侵袭能力最强,而至晚期妊娠几乎无侵袭能力的变化相同,这提示了Rac1信号通路可能通过对MMP9表达的调控,参与了滋养细胞侵袭的调控。(2)子痫前期组与足月妊娠组比较,胎盘组织中GTP-Rac1及MMP9的表达与核内β-catenin以及Snail的表达明显降低,且在子痫前期重度组中,其表达水平最低,这提示Rac1信号通路可能通过调控MMP9的表达在子痫前期胎盘滋养细胞浅侵袭的病理过程中发挥关键作用。第二部分Racl信号通路对绒毛外滋养细胞侵袭行为的影响研究目的探讨Rac1信号通路对体外培养人绒毛外滋养细胞系HTR-8/SVneo由MMP9的活化以及细胞侵袭力的影响。研究方法体外培养人绒毛外滋养细胞系,使用脂质体将重组质粒Rac1 shRNA转染HTR-8/SVneo细胞,检测]Rac1 shRNA对HTR-8/SVneo细胞中Rac1表达的抑制作用。将HTR-8/SVneo细胞分为：正常对照组(normal control,只加脂质体)、Cont-shRNA组f转染重组质粒Cont-shRNA), Rac1 shRNA组(转染重组质粒Rac1 shRNA)及β-catenin阻断组(给予β-catenin阻断剂IWP-2)。根据Invitrogen提供的试剂盒说明书选择优化转染条件,稳定转染HTR-8/SVneo细胞。应用Western blot法检测Rac1的活化及表达水平变化；Western blot法测定HTR-8/SVneo细胞β-catenin及Snail的蛋白表达变化；细胞免疫荧光法检测MMP9的表达；Transwell invasion实验检测HTR-8/SVneo细胞侵袭力变化；Real-time PCR法检测Rac1mRNA和MMP9 mRNA的表达。结果Western blot法检测显示,shRNA-Rac1重组质粒能够成功在HTR-8/SVneo细胞表达,并能抑制Rac1基因的表达。Western blot检测发现,正常对照组及Cont-shRNA组HTR-8/SVneo细胞核内表达β-catenin、Snail,而Rac1 shRNA组与β-catenin阻断组的细胞核内表达的β-catenin、Snail水平明显比正常对照组低(P0.05)。细胞免疫荧光结果示,MMP9在正常对照组及Cont-shRNA组的HTR-8/SVneo细胞内染色明显,而在Rac1 shRNA组及β-catenin阻断组,染色明显减弱。Western blot及Real-time PCR结果进一步证实,shRNA-Rac1能显著抑制HTR-8/SVneo细胞MMP9蛋白及mRNA的表达(P0.05)；Transwell invasion实验结果显示：正常对照组与Cont-shRNA组的穿透细胞数目无明显差异(P0.05)；Rac1 shRNA组以及β-catenin阻断组的穿透细胞数目显著低于正常对照组(P0.05)。结论在HTR-8/SVneo细胞中,Rac1可能通过诱导β-catenin核内表达而使Snail活化,并明显增加HTR-8/SVneo细胞中MMP9表达,从而调控绒毛外滋养细胞的侵袭行为。
[Abstract]:The first part Rac 1 signal pathway was used to detect the expression of Rac1 signaling pathway in normal pregnancy and pre - eclampsia . The relationship between the Rac1 signaling pathway and the invasion of trophoblasts was investigated . From June 2010 to December 2014 , we collected 50 cases of pregnant women collected from the Second Hospital of Shandong University . Among them , 50 of the normal pregnancy groups were divided into early pregnancy group and term pregnancy group . The early pregnancy group was 25 patients with 6 - 9 weeks of pregnancy , and the age was 28.5 卤 3.8 years .
In the term pregnancy group , 25 cases ( 37 - 40 weeks ) were pregnant and elective cesarean section was performed , and the age was ( 27 . 9 卤 3.3 ) years .
In the pre - eclampsia group , there were 67 cases of pre - eclampsia in 37 - 40 weeks of elective cesarean section , in which 37 cases were mild in the early stage of eclampsia , and the age was 28 . 9 卤 3.25 years .
The expression levels of GTP - Rac1 , Rac1 and the target gene 尾 - catenin in the placenta were measured by Real - time PCR , immunohistochemical method and Western blot . The results showed that the level of GTP - Rac1 was significantly higher in the placental villi of the early pregnancy group than in the term pregnancy group ( P0.05 ) .
The expression level of GTP - Rac1 was significantly lower in the pre - eclampsia group than in the term pregnancy group ( P0.05 ) .
The expression level of Rac1 protein in early pregnancy group was significantly higher than that in term pregnancy group ( P0.05 ) .
The expression levels of 尾 - catenin in the placenta were significantly lower than those in the pre - eclampsia group ( P0.05 ) . The results showed that MMP - 9 was mainly expressed in the trophoblasts of the cells and the trophoblasts , which was mainly distributed in the cytoplasm , brown - yellow or deep - brown - yellow , and the number of MMP - 9 - positive cells in the early pregnancy group was significantly decreased .
The number of MMP - 9 - positive cells in early pregnancy group was significantly lower than that in term pregnancy group , and the number of MMP - 9 - positive cells was significantly decreased . Western blot and Real - time PCR showed that the expression level of MMP - 9 protein and mRNA in early pregnancy group was significantly higher than that in term pregnancy group ( P0.05 ) .
The expression of MMP - 9 protein and mRNA in placental tissue was significantly lower than that of normal control group ( P0.05 ) . Conclusion ( 1 ) The expression of GTP - Rac1 and MMP - 9 in placental tissue was significantly lower than that of normal control group ( P0.05 ) .
Western blot was used to determine the changes of 尾 - catenin and protein expression in HSVneo - 8 / SVneo cells .
The expression of MMP 9 was detected by immunofluorescence assay .
Transwell invasion assay was used to detect the changes in invasion ability of HSVneo cells .
The expression of Rac1 mRNA and MMP 9 mRNA was detected by Real - time PCR . Results Western blot assay showed that shRNA - Rac1 recombinant plasmid was able to successfully express the expression of 尾 - catenin in the nucleus of TR8 / SVneo . The results of Western blot and Real - time PCR showed that shRNA - Rac1 significantly inhibited the expression of MMP - 9 and mRNA in the cells of the control group and Cont - shRNA group ( P0.05 ) .
The results of Transwell invasion showed that there was no significant difference in the number of penetrating cells between the control group and the Cont - shRNA group ( P0.05 ) .
Conclusion : Rac1 may be activated by inducing the expression of 尾 - catenin and increase the expression of MMP - 9 and regulate the invasion behavior of extracellular trophoblasts .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.244

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