敲低STGC3基因表达对NP69细胞生物学行为的影响

发布时间：2018-01-07 23:16

本文关键词：敲低STGC3基因表达对NP69细胞生物学行为的影响　出处：《南华大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：运用shRNA技术，敲低鼻咽癌候选基因STGC3在永生化人鼻咽上皮细胞系NP69的表达，分析STGC3基因表达降低，对NP69细胞系生长、增殖、侵袭、迁移及裸鼠成瘤能力的影响，为揭示STGC3基因在鼻咽癌发病中的作用提供强有力的实验依据。方法：设计与构建STGC3基因的特异性shRNA真核表达载体pRNAT-U6.1/shRNA/STGC3，转染永生化人鼻咽上皮细胞系NP69， G418筛选，建立pRNAT-U6.1/shRNA/STGC3/NP69细胞系，采用荧光显微镜，通过观察转染细胞GFP表达，评价转染效果；RT-PCR技术，检测转染细胞中STGC3基因mRNA的表达水平；通过细胞生长曲线的绘制、细胞集落形成实验及FCM技术，分析STGC3基因表达降低，对NP69细胞生长、增殖及周期分布的影响；施用Transwell侵袭迁移实验，观察转染细胞侵袭与迁移能力；运用裸鼠成瘤实验，观察pRNAT-U6.1/shRNA/STGC3/NP69细胞的成瘤能力。结果：构建真核表达载体pRNAT-U6.1/shRNA/STGC3，经双酶切、测序鉴定、blast比对及RT-PCR鉴定，确定pRNAT-U6.1/shRNA/STGC3真核表达载体成功构建。将pRNAT-U6.1/scramble和pRNAT-U6.1/shRNA/STGC3重组质粒分别转染NP69细胞系，荧光显微镜观察GFP表达，以可见绿色荧光，确定pRNAT-U6.1/scramble、pRNAT-U6.1/shRNA/STGC3导入NP69细胞；RT-PCR验证转染结果；结果表明pRNAT-U6.1/shRNA/STGC3导入NP69细胞，可显著降低STGC3基因在NP69细胞系中的表达。通过细胞生长曲线的绘制、FCM分析，转pRNAT-U6.1/shRNA/STGC3/NP69组细胞生长增殖速度加快；细胞群体中G2期和S期的细胞数目增加，与对照组比较差异具有显著性意义(p0.05)。细胞集落形成实验结果显示，pRNAT-U6.1/shRNA/STGC3/NP69细胞的克隆形成能力增强，所形成的细胞集落数目多，体积大（p0.01）。Transwell侵袭转移实验结果显示：与对照组细胞相比，pRNAT-U6.1/shRNA/STGC3/NP69细胞的侵袭能力增强（p0.001）。将pRNAT-U6.1/shRNA/STGC3/NP69、pRNAT-U6.1/scramble/NP69及NP69三组细胞，分别接种于裸鼠皮下，经8周饲养，均未见肿瘤的形成。结论 1.真核表达载体pRNAT-U6.1/shRNA/STGC3，能有效降低STGC3基因在NP69细胞中的表达。 2.下调STGC3基因在NP69细胞中的表达，其生长增殖速度加快，，呈现部分恶性转化现象，但不足以在裸鼠体内成瘤。
[Abstract]:Objective: to lower the expression of STGC3 candidate gene in immortalized human nasopharyngeal epithelial cell line NP69 by shRNA technique and analyze the decrease of STGC3 gene expression. The effects on the growth, proliferation, invasion, migration and tumorigenesis of NP69 cell line provide a powerful experimental basis for revealing the role of STGC3 gene in the pathogenesis of nasopharyngeal carcinoma. Methods: the specific shRNA eukaryotic expression vector pRNAT-U6.1/shRNA/STGC3 of STGC3 gene was designed and constructed. PRNAT-U6.1/shRNA/STGC3/NP69 cell line was established by transfection of immortalized human nasopharyngeal epithelial cell line NP69 and G418. The effect of transfection was evaluated by observing the expression of GFP in transfected cells. RT-PCR technique was used to detect the expression of STGC3 gene mRNA in transfected cells. By drawing the cell growth curve, colony formation test and FCM technique, the effect of STGC3 gene expression on the growth, proliferation and cycle distribution of NP69 cells was analyzed. The invasion and migration ability of transfected cells was observed by Transwell invasion and migration assay. The tumorigenesis ability of pRNAT-U6.1/shRNA/STGC3/NP69 cells was observed by nude mice tumorigenesis test. Results: the eukaryotic expression vector pRNAT-U6.1 / shRNA-STGC3 was constructed, which was digested by double enzyme, sequenced and compared with RT-PCR. To determine the successful construction of pRNAT-U6.1/shRNA/STGC3 eukaryotic expression vector. PRNAT-U6.1/scramble and pRNAT-U6.1/shR. NA/STGC3 recombinant plasmid was transfected into NP69 cell line. The expression of GFP was observed by fluorescence microscope, and pRNAT-U6.1/scramble was determined by visible green fluorescence. PRNAT-U6.1/shRNA/STGC3 was introduced into NP69 cells. The transfection results were verified by RT-PCR. The results showed that pRNAT-U6.1/shRNA/STGC3 was introduced into NP69 cells. The expression of STGC3 gene in NP69 cell line was significantly decreased, and the cell growth curve was plotted for FCM analysis. In pRNAT-U6.1/shRNA/STGC3/NP69 group, the cell growth and proliferation rate was accelerated. The number of cells in G2 phase and S phase increased significantly compared with the control group. The results of colony formation test showed that the number of cells in G2 phase and S phase was significantly higher than that in control group (P < 0.05). The clone forming ability of pRNAT-U6.1/shRNA/STGC3/NP69 cells was enhanced and the number of colony formation was more. The results of the invasion and metastasis experiment showed that: compared with the control cells. Enhancement of invasion ability of pRNAT-U6.1/shRNA/STGC3/NP69 cells by p0.001). PRNAT-U6.1/shRNA/STGC3/NP69. The cells of pRNAT-U6.1/scramble/NP69 and NP69 were inoculated subcutaneously in nude mice. After 8 weeks of feeding, no tumor formation was found. Conclusion 1. Eukaryotic expression vector pRNAT-U6.1 / shRNA-STGC3 can effectively reduce the expression of STGC3 gene in NP69 cells. 2. Down-regulating the expression of STGC3 gene in NP69 cells, its growth and proliferation was accelerated, and some malignant transformation was observed, but it was not sufficient for tumorigenesis in nude mice.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.63

【共引文献】