本文關(guān)鍵詞： KiSS-1 kisspeptin GPR54 復(fù)發(fā)性流產(chǎn)　出處：《蘇州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：探討KiSS-1基因?qū)θ焉镒甜B(yǎng)細(xì)胞生物學(xué)行為的影響及其在復(fù)發(fā)性流產(chǎn)中的作用機(jī)制。 方法：①免疫組織化學(xué)方法檢測(cè)kisspeptin、GPR54在復(fù)發(fā)性流產(chǎn)患者絨毛及蛻膜組織中表達(dá)水平；②構(gòu)建重組質(zhì)粒pcDNA3.1（+）-KiSS-1；③將重組質(zhì)粒pcDNA3.1（+）-KiSS-1轉(zhuǎn)染至人滋養(yǎng)層腫瘤JAR細(xì)胞株，建立單克隆細(xì)胞系；④應(yīng)用RT-PCR及Western Blot方法分別檢測(cè)轉(zhuǎn)染細(xì)胞KiSS-1的mRNA及編碼多肽kisspeptin表達(dá)；⑤檢測(cè)KiSS-1基因及外源性kisspeptin對(duì)JAR細(xì)胞生長(zhǎng)增殖、軟瓊脂克隆形成能力及遷移侵襲能力的影響。 結(jié)果：①?gòu)?fù)發(fā)性流產(chǎn)患者絨毛kisspeptin、GPR54的表達(dá)均明顯低于正常妊娠者（P0.05），蛻膜組織kisspeptin的表達(dá)明顯低于正常妊娠者（P0.05），蛻膜組織中GPR54的表達(dá)在兩組間無(wú)明顯差異；②成功構(gòu)建重組質(zhì)粒pcDNA3.1（+）-KiSS-1，并經(jīng)PCR和測(cè)序鑒定無(wú)誤；③將重組質(zhì)粒pcDNA3.1（+）-KiSS-1成功轉(zhuǎn)染至人滋養(yǎng)細(xì)胞腫瘤株JAR細(xì)胞；④篩選出穩(wěn)定轉(zhuǎn)染的細(xì)胞，并通過(guò)RT-PCR和Western Blot鑒定目的基因KiSS-1mRNA和編碼多肽kisspeptin均成陽(yáng)性表達(dá)；⑤KiSS-1基因和kisspeptin對(duì)JAR細(xì)胞的生長(zhǎng)增殖及克隆形成能力無(wú)影響，但明顯抑制其遷移和侵襲能力。 結(jié)論： Kisspeptin在復(fù)發(fā)性流產(chǎn)患者母胎界面絨毛和蛻膜組織的表達(dá)均低下，GPR54在復(fù)發(fā)性流產(chǎn)患者母胎界面絨毛組織表達(dá)低下。KiSS-1基因及其產(chǎn)物kisspeptin能顯著抑制滋養(yǎng)細(xì)胞遷移及侵襲能力，，推測(cè)KiSS-1基因在妊娠期母胎界面的表達(dá)調(diào)控對(duì)妊娠的維持起重要作用，其表達(dá)異常與復(fù)發(fā)性流產(chǎn)有關(guān)。
[Abstract]:Objective: to investigate the effect of KiSS-1 gene on the biological behavior of gestational trophoblastic cells and its mechanism in recurrent abortion. Methods the expression level of kisspeptinine GPR54 in villi and decidua of recurrent abortion patients was detected by immunohistochemical method. The recombinant plasmid pcDNA3.1 (pDNA3.1- KiSS-1) was constructed and transfected into human trophoblastic tumor JAR cell line. RT-PCR and Western Blot were used to detect the expression of mRNA and kisspeptin, respectively. The effects of KiSS-1 gene and exogenous kisspeptin on the growth and proliferation of JAR cells, the ability of soft Agar clone formation and migration and invasion were detected. Results the expression of GPR54 in chorionic villi of patients with recurrent abortion was significantly lower than that in normal pregnant women (P 0.05), and the expression of kisspeptin in decidua was significantly lower than that in normal pregnancy (P 0.05). There was no significant difference between the two groups in the expression of GPR54 in decidual tissues. The recombinant plasmid pcDNA3.1was successfully transfected into human trophoblastic tumor cell line JAR cell line, and stable transfected cells were screened by PCR and sequencing. By RT-PCR and Western Blot, both the target gene KiSS-1mRNA and the encoding polypeptide kisspeptin were identified as positive expression of p5KiSS-1 gene and kisspeptin, which had no effect on the growth, proliferation and clone formation of JAR cells, but significantly inhibited the migration and invasion of JAR cells. Conclusion: the expression of Kisspeptin in the villi and decidua of the maternal and fetal interface of recurrent abortion patients is low. The expression of GPR54 gene and its product kisspeptin can significantly inhibit the migration and invasion of trophoblast cells in the villus tissues of recurrent abortion patients. It is speculated that the regulation of KiSS-1 gene expression at maternal and fetal interface plays an important role in the maintenance of pregnancy. The abnormal expression of KiSS-1 gene is related to recurrent abortion.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.21

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本文編號(hào)：1495875