STGC3基因缺失突变对CNE2细胞生长增殖的影响

发布时间：2018-03-23 12:11

本文选题：鼻咽癌　切入点：CNE2细胞系　出处：《南华大学》2011年硕士论文

【摘要】：目的:采用基因定点突变技术,将鼻咽癌候选抑瘤基因STGC3中层粘连蛋白G结构域(LG domain)缺失掉,通过观察LG缺失的STGC3基因对人鼻咽癌细胞系CNE2生长增殖的影响,探讨STGC3蛋白质在细胞生长增殖中发挥负性调控作用的结构域。方法:我们前期研究结果提示,LG domain位于STGC3基因所编码蛋白的1～42位氨基酸,应用PCR定点突变方法,缺失掉STGC3基因的LG domain,成功构建pcDNA3.1(+)-STGC3 ~(△1~42AA)真核表达载体,将重组pcDNA3.1(+)-STGC3 ~(△1~42AA)转染入CNE2细胞系,G418筛选,阳性细胞克隆的RT-PCR鉴定,获得稳定转染的CNE2-pcDNA3.1(+)-STGC3 ~(△1~42AA)细胞系。通过CNE2-pcDNA3.1(+)-STGC3△1~42AA细胞系生长曲线的绘制、细胞克隆形成率检测、细胞周期分析,观察STGC3基因LG domain缺失突变对CNE2细胞生长增殖的影响。结果:重组质粒DNA经酶切和测序鉴定,结果均证实pcDNA3.1(+)-STGC3 ~(△1~42AA)真核表达载体构建成功。经RT-PCR鉴定,成功建立稳定表达STGC3△1~42AA的CNE2细胞系。细胞生长曲线结果表明:从第3天开始转STGC3 ~(△1~42AA)组和转野生型STGC3基因组细胞生长速度较未转染组及转空载体组减慢(p0.05),转STGC3 ~(△1~42AA)组细胞生长速度较转STGC3基因组快(p0.01)。平皿克隆形成实验结果显示:与两对照组相比,转基因组集落生长速度慢、数目少、体积小,克隆形成能力明显降低,转STGC3 ~(△1~42AA)组与转野生型基因组相比,其形成的细胞集落大,数量更多,表明转STGC3 ~(△1~42AA)组细胞生长增殖速度快于转野生型基因组,两组间的差异均有显著性( p0.01)。经流式细胞仪检测,结果显示:转野生型STGC3基因组和转STGC3 ~(△1~42AA)基因组G_0/G_1期细胞比例显著高于未转染组和转空载体组(p0.01),但转STGC3 ~(△1~42AA)组G1期阻滞作用较转野生型基因组下降(p0.01)。结论: 1.成功构建了pcDNA3.1(+)-STGC3 ~(△1~42AA)真核表达载体。 2. STGC3基因LG domain的缺失突变,导致该基因的细胞生长增殖负调控作用降低。 3. LG domain是STGC3蛋白质的重要功能结构域。
[Abstract]:Objective: to study the effect of LG deletion on the growth and proliferation of nasopharyngeal carcinoma (NPC) cell line CNE2 by using locus mutagenesis technique, which is a candidate tumor suppressor gene for nasopharyngeal carcinoma (NPC). To investigate the domain of STGC3 protein which plays a negative role in cell growth and proliferation. Methods: our previous study suggested that LG domain was located at the 1G 42 amino acid of the protein encoded by the STGC3 gene. Using the PCR site-directed mutation method, the LG domain of the STGC3 gene was deleted, and the eukaryotic expression vector pcDNA3.1 (1 + STGC3 (1: 42AA)) was successfully constructed. The recombinant pcDNA3.1was transfected into CNE2 cell line G418 for screening. The stable transfected CNE2-pcDNA3.1cell line was obtained by RT-PCR identification. The cell clone formation rate was detected by drawing the growth curve of CNE2-pcDNA3.1cell line (1-STGC3 1~42AA cell line). Cell cycle analysis was performed to observe the effect of LG domain deletion mutation of STGC3 gene on the growth and proliferation of CNE2 cells. Results: the recombinant plasmid DNA was identified by restriction endonuclease digestion and sequencing. The results confirmed that the eukaryotic expression vector of pcDNA3.1 (1-STGC3 (1h42AA)) was successfully constructed and identified by RT-PCR. CNE2 cell lines expressing STGC3 1~42AA stably were successfully established. The results of cell growth curve showed that the growth rate of STGC3 (1h42AA) group and wild type STGC3 genome cell line was slower than that of untransfected group and empty vector group, and the cell growth rate was lower than that of untransfected group and empty vector group. The cell growth rate of STGC3 group was faster than that of STGC3 genome. The results of plate clone formation test showed that compared with two control groups, the growth rate of the cells in the STGC3 group was higher than that in the control group. The colony growth rate of transgenic group is slow, the number is small, the volume is small, and the clone forming ability is obviously reduced. Compared with the transgenic wild type genome, the transgenic group has a larger colony and more cells. The results showed that the growth and proliferation rate of the transfected STGC3 42AA group was faster than that of the transgenic wild-type genome, and the difference between the two groups was significant (p0.01). The results showed that the percentage of cells in the G_0/G_1 phase of the transgenic STGC3 genome and the transgenic STGC3 42AA group was significantly higher than that of the untransfected group and the empty vector group, but the G1 phase arrest effect of the transgenic STGC3 group was lower than that of the wild type group. Conclusion:. 1. The eukaryotic expression vector of pcDNA3.1- STGC3 (1: 42AA) was successfully constructed. 2. The deletion mutation of LG domain in STGC3 gene resulted in the decrease of negative regulation of cell growth and proliferation of the gene. 3. LG domain is an important functional domain of STGC3 protein.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

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