本文關鍵詞： 復發(fā)性自然流產(chǎn) 絨毛組織 miR-144 STAT1　出處：《中南大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的：1.探討miR-144在復發(fā)性自然流產(chǎn)與正常早期妊娠絨毛組織中的表達差異。 2.在人滋養(yǎng)細胞HTR-8中探討miR-144對其可能的靶基因STAT1的調(diào)控作用。 3.了解miR-144對人滋養(yǎng)細胞HTR-8凋亡的影響,以探討miR-144在復發(fā)性自然流產(chǎn)發(fā)病過程中的作用。 方法：1.采用實時熒光定量PCR(real-time PCR)技術(shù)檢測miR-144在15例正常早期妊娠和15例復發(fā)性自然流產(chǎn)患者絨毛組織中的表達。 2.采用瞬時轉(zhuǎn)染技術(shù)在人正常滋養(yǎng)細胞系HTR-8細胞中分別轉(zhuǎn)染miR-144mimics與miR-144inhibitor,即過表達與抑制miR-144的表達后,RT-PCR技術(shù)檢測HTR-8細胞中miR-144及STAT1mRNA的表達水平,并采用Western-blot技術(shù)檢測Statl蛋白的表達水平。 3.在HTR-8細胞中過表達與抑制miR-144的表達后,采用流式細胞術(shù)檢測細胞凋亡情況。 結(jié)果：1.實時熒光定量PCR結(jié)果顯示,復發(fā)性自然流產(chǎn)患者絨毛組織中miR-144的表達低于對照組(P0.001)。 2.在人滋養(yǎng)細胞系HTR-8細胞中過表達與抑制miR-144的表達后,RT-PCR技術(shù)檢測miR-144及STAT1mRNA的表達量,結(jié)果顯示與對照組相比,過表達組miR-144的表達量升高,而STAT1mRNA的表達量降低,差異均有統(tǒng)計學意義(P0.001)；抑制組miR-144的表達量降低,而STAT1mRNA的表達量升高,差異均有統(tǒng)計學意義(P0.001); Western-blot技術(shù)檢測STAT1蛋白的表達水平,結(jié)果顯示與對照組相比,過表達組Statl蛋白的表達量降低,抑制組STAT1蛋白的表達量升高,差異均有統(tǒng)計學意義(P0.05)。提示STAT1在mRNA和蛋白水平的表達受到miR-144的負調(diào)控。 3.在HTR-8細胞中過表達與抑制miR-144的表達后,流式細胞術(shù)檢測細胞凋亡指數(shù),結(jié)果顯示,上調(diào)miR-144的表達后細胞凋亡率較對照組降低,差異有統(tǒng)計學意義(P0.05),而下調(diào)miR-144的表達后細胞凋亡率較對照組增加,差異有統(tǒng)計學意義(P0.001)。 結(jié)論：1.復發(fā)性自然流產(chǎn)患者絨毛組織中miR-144的表達降低,提示miR-144可能參與了復發(fā)性自然流產(chǎn)的發(fā)生。 2.STAT1是miR-144的一個作用靶點,且在mRNA和蛋白水平,miR-144均對STAT1有著明顯的負性調(diào)控作用。 3.miR-144可以抑制滋養(yǎng)細胞的凋亡,提示miR-144的低表達可能通過增加滋養(yǎng)細胞的凋亡而參與了復發(fā)性自然流產(chǎn)的發(fā)病過程。
[Abstract]:Objective 1. To investigate the expression of miR-144 in villi of recurrent spontaneous abortion and normal early pregnancy. 2. To investigate the regulation of miR-144 on its possible target gene STAT1 in human trophoblastic HTR-8. 3. To investigate the effect of miR-144 on the apoptosis of human trophoblastic HTR-8 and to explore the role of miR-144 in the pathogenesis of recurrent spontaneous abortion. Method 1. Real-time fluorescence quantitative PCR(real-time PCR was used. The expression of miR-144 in chorionic villi of 15 cases of normal early pregnancy and 15 cases of recurrent spontaneous abortion was detected. 2. Transient transfection technique was used to transfect miR-144mimics and miR-144inhibitor into human normal trophoblast cell line HTR-8 cells. The expression of miR-144 and STAT1mRNA in HTR-8 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) after over-expression and inhibition of miR-144 expression. Western-blot technique was used to detect the expression of Statl protein. 3. After overexpression and inhibition of miR-144 expression in HTR-8 cells, apoptosis was detected by flow cytometry. Results the results of real-time fluorescence quantitative PCR showed that the expression of miR-144 in villi of recurrent spontaneous abortion patients was lower than that of control group (P 0.001). 2. After overexpression and inhibition of miR-144 expression in human trophoblast cell line HTR-8, RT-PCR was used to detect the expression of miR-144 and STAT1mRNA. The results showed that the expression of miR-144 in overexpression group was higher than that in control group, but the expression of STAT1mRNA was decreased, the difference was statistically significant (P 0.001). In the inhibitory group, the expression of miR-144 decreased, but the expression of STAT1mRNA increased, and the difference was statistically significant (P 0.001). The expression level of STAT1 protein was detected by Western-blot. The results showed that the expression of Statl protein in overexpression group was lower than that in control group. The expression of STAT1 protein in the inhibitory group was significantly higher than that in the control group (P 0.05), suggesting that the expression of STAT1 in mRNA and protein level was negatively regulated by miR-144. 3. After overexpression and inhibition of miR-144 expression in HTR-8 cells, apoptosis index was detected by flow cytometry. After upregulating the expression of miR-144, the rate of apoptosis was lower than that of the control group, the difference was statistically significant (P 0.05), while the rate of apoptosis after down-regulating the expression of miR-144 was higher than that of the control group. The difference was statistically significant (P 0.001). Conclusion 1. The expression of miR-144 in villi of recurrent spontaneous abortion patients was decreased, suggesting that miR-144 may be involved in the occurrence of recurrent spontaneous abortion. 2. STAT1 is a target of miR-144 and has a negative regulatory effect on STAT1 at both mRNA and protein levels. 3. MiR-144 can inhibit the apoptosis of trophoblast, suggesting that the low expression of miR-144 may be involved in the pathogenesis of recurrent spontaneous abortion by increasing the apoptosis of trophoblast.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.21

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