本文選題：Racl + β-catenin��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：第一部分Rac 1信號(hào)通路在正常妊娠及子癇前期中表達(dá)的研究研究目的檢測(cè)Rac1信號(hào)通路在正常妊娠及子癇前期產(chǎn)婦胎盤(pán)的表達(dá)變化,探討其與滋養(yǎng)細(xì)胞侵襲的關(guān)系。研究方法從2010年6月至2014年12月,我們?cè)谏綎|大學(xué)第二醫(yī)院收集的就診孕婦,其中,正常妊娠組共50例,分為早孕組及足月妊娠組,早孕組為25例6-9周妊娠者,行人工流產(chǎn)術(shù),年齡(28.5±3.8)歲；足月妊娠組為25例37-40周妊娠者,行擇期剖宮產(chǎn)術(shù),年齡(27.9±3.3)歲；子癇前期組共計(jì)67例行擇期剖宮產(chǎn)的37-40周子癇前期患者,其中子癇前期輕度組37例,年齡(28.9±3.25)歲；子癇前期重度組30例,年齡(26.9±2.8)歲。各組孕婦的孕周、年齡、孕次以及基礎(chǔ)血壓等無(wú)明顯差異性(P0.05)。分別采用Real-time PCR法、免疫組織化學(xué)法、GTPase pull down、Western blot法檢測(cè)50例正常妊娠組(包括早孕25例、足月妊娠25例)以及67例子癇前期組(包括子癇前期輕度37例、子癇前期重度30例)胎盤(pán)組織GTP-Rac1、Rac1與其下游的靶基因β-catenin、 Snail的蛋白表達(dá)及MMP9的蛋白定位、表達(dá)和mRNA的變化。結(jié)果我們應(yīng)用GTPase pull down以及Western blot法對(duì)正常妊娠組以及子癇前期組胎盤(pán)組織GTP-Rac1的表達(dá)水平進(jìn)行檢測(cè),結(jié)果顯示早孕組胎盤(pán)絨毛組織中GTP-Rac1的表達(dá)水平顯著高于足月妊娠組(P0.05)；子癇前期組GTP-Rac1的表達(dá)水平明顯低于足月妊娠組(P0.05),其中,子癇前期重度組GTP-Rac1表達(dá)水平低于子癇前期輕度組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；而各組間Rac1蛋白表達(dá)水平未見(jiàn)明顯的差異(P0.05)。Western blot檢測(cè)發(fā)現(xiàn),早孕組胎盤(pán)組織核內(nèi)β-catenin及Snail表達(dá)水平顯著高于足月妊娠組(P0.05)；子癇前期組胎盤(pán)組織核內(nèi)β-catenin及Snail表達(dá)水平低于足月妊娠組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),其中子癇前期重度組的β-catenin及Snail表達(dá)水平顯著低于子癇前期輕度組(P0.05)。免疫組化結(jié)果顯示,MMP9主要在細(xì)胞滋養(yǎng)細(xì)胞、合體滋養(yǎng)細(xì)胞中表達(dá),可見(jiàn)其主要分布于胞漿,呈棕黃色或深棕黃色,足月妊娠組較早孕組MMP9陽(yáng)性細(xì)胞數(shù)量明顯減少；子癇前期組MMP9陽(yáng)性細(xì)胞數(shù)量明顯低于足月妊娠組,且染色明顯減弱,子癇前期重度組與子癇前期輕度組比較,MMP9陽(yáng)性細(xì)胞數(shù)量明顯降低,染色明顯減弱。Western blot及Real-time PCR結(jié)果示,早孕組MMP9蛋白及mRNA的表達(dá)水平明顯高于足月妊娠組(P0.05)；子癇前期組胎盤(pán)組織的MMP9蛋白及mRNA的表達(dá)水平顯著低于足月妊娠組(P0.05),且子癇前期重度組與子癇前期輕度組比較,胎盤(pán)組織中MMP9蛋白及mRNA的表達(dá)水平顯著降低(P0.05)。結(jié)論(1) GTP-Rac1在正常妊娠胎盤(pán)滋養(yǎng)細(xì)胞中的表達(dá)隨孕周增加而降低,且Rac1下游重要的靶基因β-catenin Snail在胎盤(pán)組織核內(nèi)的表達(dá)及與滋養(yǎng)細(xì)胞侵襲力密切相關(guān)的MMP9的表達(dá)與Rac1的活性變化一致。Rac1信號(hào)通路的變化與正常早期妊娠滋養(yǎng)細(xì)胞侵襲能力最強(qiáng),而至晚期妊娠幾乎無(wú)侵襲能力的變化相同,這提示了Rac1信號(hào)通路可能通過(guò)對(duì)MMP9表達(dá)的調(diào)控,參與了滋養(yǎng)細(xì)胞侵襲的調(diào)控。(2)子癇前期組與足月妊娠組比較,胎盤(pán)組織中GTP-Rac1及MMP9的表達(dá)與核內(nèi)β-catenin以及Snail的表達(dá)明顯降低,且在子癇前期重度組中,其表達(dá)水平最低,這提示Rac1信號(hào)通路可能通過(guò)調(diào)控MMP9的表達(dá)在子癇前期胎盤(pán)滋養(yǎng)細(xì)胞淺侵襲的病理過(guò)程中發(fā)揮關(guān)鍵作用。第二部分Racl信號(hào)通路對(duì)絨毛外滋養(yǎng)細(xì)胞侵襲行為的影響研究目的探討Rac1信號(hào)通路對(duì)體外培養(yǎng)人絨毛外滋養(yǎng)細(xì)胞系HTR-8/SVneo由MMP9的活化以及細(xì)胞侵襲力的影響。研究方法體外培養(yǎng)人絨毛外滋養(yǎng)細(xì)胞系,使用脂質(zhì)體將重組質(zhì)粒Rac1 shRNA轉(zhuǎn)染HTR-8/SVneo細(xì)胞,檢測(cè)]Rac1 shRNA對(duì)HTR-8/SVneo細(xì)胞中Rac1表達(dá)的抑制作用。將HTR-8/SVneo細(xì)胞分為：正常對(duì)照組(normal control,只加脂質(zhì)體)、Cont-shRNA組f轉(zhuǎn)染重組質(zhì)粒Cont-shRNA), Rac1 shRNA組(轉(zhuǎn)染重組質(zhì)粒Rac1 shRNA)及β-catenin阻斷組(給予β-catenin阻斷劑IWP-2)。根據(jù)Invitrogen提供的試劑盒說(shuō)明書(shū)選擇優(yōu)化轉(zhuǎn)染條件,穩(wěn)定轉(zhuǎn)染HTR-8/SVneo細(xì)胞。應(yīng)用Western blot法檢測(cè)Rac1的活化及表達(dá)水平變化；Western blot法測(cè)定HTR-8/SVneo細(xì)胞β-catenin及Snail的蛋白表達(dá)變化；細(xì)胞免疫熒光法檢測(cè)MMP9的表達(dá)；Transwell invasion實(shí)驗(yàn)檢測(cè)HTR-8/SVneo細(xì)胞侵襲力變化；Real-time PCR法檢測(cè)Rac1mRNA和MMP9 mRNA的表達(dá)。結(jié)果Western blot法檢測(cè)顯示,shRNA-Rac1重組質(zhì)粒能夠成功在HTR-8/SVneo細(xì)胞表達(dá),并能抑制Rac1基因的表達(dá)。Western blot檢測(cè)發(fā)現(xiàn),正常對(duì)照組及Cont-shRNA組HTR-8/SVneo細(xì)胞核內(nèi)表達(dá)β-catenin、Snail,而Rac1 shRNA組與β-catenin阻斷組的細(xì)胞核內(nèi)表達(dá)的β-catenin、Snail水平明顯比正常對(duì)照組低(P0.05)。細(xì)胞免疫熒光結(jié)果示,MMP9在正常對(duì)照組及Cont-shRNA組的HTR-8/SVneo細(xì)胞內(nèi)染色明顯,而在Rac1 shRNA組及β-catenin阻斷組,染色明顯減弱。Western blot及Real-time PCR結(jié)果進(jìn)一步證實(shí),shRNA-Rac1能顯著抑制HTR-8/SVneo細(xì)胞MMP9蛋白及mRNA的表達(dá)(P0.05)；Transwell invasion實(shí)驗(yàn)結(jié)果顯示：正常對(duì)照組與Cont-shRNA組的穿透細(xì)胞數(shù)目無(wú)明顯差異(P0.05)；Rac1 shRNA組以及β-catenin阻斷組的穿透細(xì)胞數(shù)目顯著低于正常對(duì)照組(P0.05)。結(jié)論在HTR-8/SVneo細(xì)胞中,Rac1可能通過(guò)誘導(dǎo)β-catenin核內(nèi)表達(dá)而使Snail活化,并明顯增加HTR-8/SVneo細(xì)胞中MMP9表達(dá),從而調(diào)控絨毛外滋養(yǎng)細(xì)胞的侵襲行為。
[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 鹵 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 鹵 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 鹵 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 .
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R714.244

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