本文選題：TDRG1基因　切入點(diǎn)：NTERA-2細(xì)胞　出處：《中南大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：TDRG1基因是本課題組電子克隆的人睪丸特異表達(dá)基因。前期的研究提示該基因可能與睪丸腫瘤的發(fā)生發(fā)展相關(guān),并利用RNA干擾技術(shù)(RNAi)成功在人睪丸生殖細(xì)胞腫瘤NTERA-2細(xì)胞中構(gòu)建了下調(diào)TDRG1基因mRNA表達(dá)的穩(wěn)定系統(tǒng)。本研究擬在前期工作基礎(chǔ)上,進(jìn)一步驗(yàn)證靶向TDRG1基因mRNA的shRNA重組質(zhì)粒對(duì)NTERA-2細(xì)胞中TDRG1基因表達(dá)的干擾作用,探討TDRG1基因?qū)TERA-2細(xì)胞生物學(xué)特性的影響。 方法：1.RT-PCR及細(xì)胞免疫熒光(IF)檢測(cè)TDRG1基因mRNA及蛋白在NTERA-2細(xì)胞和小鼠睪丸畸胎瘤F9細(xì)胞中的表達(dá)。2.利用已構(gòu)建的TDRGl-shRNA重組質(zhì)粒在體外轉(zhuǎn)染NTERA-2細(xì)胞,實(shí)時(shí)定量PCR (qRT-PCR)及IF檢測(cè)轉(zhuǎn)染后NTERA-2細(xì)胞TDRG1基因mRNA及蛋白表達(dá)的變化。3.采用MTT實(shí)驗(yàn)、Tranwell實(shí)驗(yàn)及流式細(xì)胞儀檢測(cè)轉(zhuǎn)染后NTERA-2細(xì)胞體外增殖、侵襲能力及凋亡潛能的變化。 結(jié)果：TDRG1-shRNA486及TDRG1-shRNA/control重組質(zhì)粒體外成功轉(zhuǎn)染NTERA-2細(xì)胞。相對(duì)于空白對(duì)照組及陰性轉(zhuǎn)染組,TDRG1-shRNA486重組質(zhì)粒體外顯著降低NTERA-2細(xì)胞TDRG1基因mRNA及蛋白的表達(dá)(P0.05)。TDRG1基因mRNA及蛋白表達(dá)顯著降低后,實(shí)驗(yàn)組NTERA-2細(xì)胞體外增殖、侵襲能力下降,而凋亡潛能增加(P0.05)。 結(jié)論：1.前期構(gòu)建的TDRGl-shRNA486重組質(zhì)粒載體可穩(wěn)定、高效干擾NTERA-2細(xì)胞TDRG1基因,降低TDRG1基因mRNA及蛋白的表達(dá),成功構(gòu)建了體外TDRG1基因低表達(dá)的睪丸生殖細(xì)胞腫瘤細(xì)胞模型。2.TDRG1基因正向調(diào)控NTERA-2細(xì)胞的增殖、侵襲能力,負(fù)向調(diào)控凋亡潛能,具有癌基因性質(zhì)。
[Abstract]:Objective\\\. RNA interference technique was used to construct a stable system for down-regulating mRNA expression of TDRG1 gene in NTERA-2 cells of human testicular germ cell tumor. To further verify the interference of shRNA recombinant plasmid targeting mRNA of TDRG1 gene on the expression of TDRG1 gene in NTERA-2 cells, and to explore the effect of TDRG1 gene on the biological characteristics of NTERA-2 cells. Methods: 1. The expression of TDRG1 gene mRNA and protein in NTERA-2 cells and mouse testicular teratoma F9 cells was detected by RT-PCR and immunofluorescence assay. The constructed TDRGl-shRNA recombinant plasmid was used to transfect NTERA-2 cells in vitro. Real-time quantitative PCR qRT-PCR and if were used to detect the changes of mRNA and protein expression of TDRG1 gene in NTERA-2 cells after transfection. MTT assay and flow cytometry were used to detect the proliferation, invasiveness and apoptosis potential of NTERA-2 cells after transfection. Results compared with control group and negative transfection group, the expression of mRNA and protein of TDRG1 gene in NTERA-2 cells was significantly decreased. The expression of mRNA and protein of TDRG1-shRNA486 gene was significantly decreased after transfection of TDRG1-shRNA486 and TDRG1-shRNA/control recombinant plasmid into NTERA-2 cells in vitro, and the expression of mRNA and protein of TDRG1-shRNA486 gene was significantly decreased compared with that of control group and negative transfection group. In the experimental group, NTERA-2 cells proliferated in vitro, and the invasiveness decreased, while the apoptotic potential increased (P 0.05). Conclusion the recombinant plasmid vector constructed in the early stage of TDRGl-shRNA486 can be stable and interfere with the TDRG1 gene of NTERA-2 cells, and reduce the expression of mRNA and protein of TDRG1 gene. The model of testicular germ cell tumor cells with low expression of TDRG1 gene in vitro was successfully constructed. 2. TDRG1 gene positively regulated the proliferation, invasion and apoptosis potential of NTERA-2 cells, and had the character of oncogene.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.21

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