shRNA干扰TDRG1表达影响人睾丸生殖细胞肿瘤NTERA-2细胞生物学特性

发布时间：2018-03-22 02:04

本文选题：TDRG1基因　切入点：NTERA-2细胞　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：TDRG1基因是本课题组电子克隆的人睾丸特异表达基因。前期的研究提示该基因可能与睾丸肿瘤的发生发展相关,并利用RNA干扰技术(RNAi)成功在人睾丸生殖细胞肿瘤NTERA-2细胞中构建了下调TDRG1基因mRNA表达的稳定系统。本研究拟在前期工作基础上,进一步验证靶向TDRG1基因mRNA的shRNA重组质粒对NTERA-2细胞中TDRG1基因表达的干扰作用,探讨TDRG1基因对NTERA-2细胞生物学特性的影响。方法：1.RT-PCR及细胞免疫荧光(IF)检测TDRG1基因mRNA及蛋白在NTERA-2细胞和小鼠睾丸畸胎瘤F9细胞中的表达。2.利用已构建的TDRGl-shRNA重组质粒在体外转染NTERA-2细胞,实时定量PCR (qRT-PCR)及IF检测转染后NTERA-2细胞TDRG1基因mRNA及蛋白表达的变化。3.采用MTT实验、Tranwell实验及流式细胞仪检测转染后NTERA-2细胞体外增殖、侵袭能力及凋亡潜能的变化。结果：TDRG1-shRNA486及TDRG1-shRNA/control重组质粒体外成功转染NTERA-2细胞。相对于空白对照组及阴性转染组,TDRG1-shRNA486重组质粒体外显著降低NTERA-2细胞TDRG1基因mRNA及蛋白的表达(P0.05)。TDRG1基因mRNA及蛋白表达显著降低后,实验组NTERA-2细胞体外增殖、侵袭能力下降,而凋亡潜能增加(P0.05)。结论：1.前期构建的TDRGl-shRNA486重组质粒载体可稳定、高效干扰NTERA-2细胞TDRG1基因,降低TDRG1基因mRNA及蛋白的表达,成功构建了体外TDRG1基因低表达的睾丸生殖细胞肿瘤细胞模型。2.TDRG1基因正向调控NTERA-2细胞的增殖、侵袭能力,负向调控凋亡潜能,具有癌基因性质。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.21

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