本文選題：鼻咽癌　切入點(diǎn)：CNE2細(xì)胞系　出處：《南華大學(xué)》2011年碩士論文

【摘要】：目的:采用基因定點(diǎn)突變技術(shù),將鼻咽癌候選抑瘤基因STGC3中層粘連蛋白G結(jié)構(gòu)域(LG domain)缺失掉,通過觀察LG缺失的STGC3基因?qū)θ吮茄拾┘?xì)胞系CNE2生長增殖的影響,探討STGC3蛋白質(zhì)在細(xì)胞生長增殖中發(fā)揮負(fù)性調(diào)控作用的結(jié)構(gòu)域。 方法:我們前期研究結(jié)果提示,LG domain位于STGC3基因所編碼蛋白的1～42位氨基酸,應(yīng)用PCR定點(diǎn)突變方法,缺失掉STGC3基因的LG domain,成功構(gòu)建pcDNA3.1(+)-STGC3 ~(△1~42AA)真核表達(dá)載體,將重組pcDNA3.1(+)-STGC3 ~(△1~42AA)轉(zhuǎn)染入CNE2細(xì)胞系,G418篩選,陽性細(xì)胞克隆的RT-PCR鑒定,獲得穩(wěn)定轉(zhuǎn)染的CNE2-pcDNA3.1(+)-STGC3 ~(△1~42AA)細(xì)胞系。通過CNE2-pcDNA3.1(+)-STGC3△1~42AA細(xì)胞系生長曲線的繪制、細(xì)胞克隆形成率檢測、細(xì)胞周期分析,觀察STGC3基因LG domain缺失突變對CNE2細(xì)胞生長增殖的影響。 結(jié)果:重組質(zhì)粒DNA經(jīng)酶切和測序鑒定,結(jié)果均證實(shí)pcDNA3.1(+)-STGC3 ~(△1~42AA)真核表達(dá)載體構(gòu)建成功。經(jīng)RT-PCR鑒定,成功建立穩(wěn)定表達(dá)STGC3△1~42AA的CNE2細(xì)胞系。細(xì)胞生長曲線結(jié)果表明:從第3天開始轉(zhuǎn)STGC3 ~(△1~42AA)組和轉(zhuǎn)野生型STGC3基因組細(xì)胞生長速度較未轉(zhuǎn)染組及轉(zhuǎn)空載體組減慢(p0.05),轉(zhuǎn)STGC3 ~(△1~42AA)組細(xì)胞生長速度較轉(zhuǎn)STGC3基因組快(p0.01)。平皿克隆形成實(shí)驗(yàn)結(jié)果顯示:與兩對照組相比,轉(zhuǎn)基因組集落生長速度慢、數(shù)目少、體積小,克隆形成能力明顯降低,轉(zhuǎn)STGC3 ~(△1~42AA)組與轉(zhuǎn)野生型基因組相比,其形成的細(xì)胞集落大,數(shù)量更多,表明轉(zhuǎn)STGC3 ~(△1~42AA)組細(xì)胞生長增殖速度快于轉(zhuǎn)野生型基因組,兩組間的差異均有顯著性( p0.01)。經(jīng)流式細(xì)胞儀檢測,結(jié)果顯示:轉(zhuǎn)野生型STGC3基因組和轉(zhuǎn)STGC3 ~(△1~42AA)基因組G_0/G_1期細(xì)胞比例顯著高于未轉(zhuǎn)染組和轉(zhuǎn)空載體組(p0.01),但轉(zhuǎn)STGC3 ~(△1~42AA)組G1期阻滯作用較轉(zhuǎn)野生型基因組下降(p0.01)。 結(jié)論: 1.成功構(gòu)建了pcDNA3.1(+)-STGC3 ~(△1~42AA)真核表達(dá)載體。 2. STGC3基因LG domain的缺失突變,導(dǎo)致該基因的細(xì)胞生長增殖負(fù)調(diào)控作用降低。 3. LG domain是STGC3蛋白質(zhì)的重要功能結(jié)構(gòu)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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 鄧敏;賀修勝;羅橋;趙帥;曾超;李艷蘭;;Tet調(diào)控STGC3基因表達(dá)CNE2細(xì)胞系的建立及其功能初步研究[J];生物化學(xué)與生物物理進(jìn)展;2006年01期

2 李桂源;劉華英;周鳴;周后德;李小玲;;鼻咽癌癌變的分子機(jī)理[J];生物化學(xué)與生物物理進(jìn)展;2006年10期

3 邱青朝;胡波;賀修勝;羅橋;龍治峰;唐國華;廖銀花;;Tet調(diào)控STGC3基因表達(dá)的CNE2細(xì)胞系裸鼠成瘤實(shí)驗(yàn)性研究[J];生物化學(xué)與生物物理進(jìn)展;2007年04期

4 胡波;邱青朝;賀修勝;羅橋;唐國華;龍治峰;廖銀花;;雌激素對轉(zhuǎn)STGC3基因CNE2細(xì)胞系生長增殖的影響[J];生物化學(xué)與生物物理進(jìn)展;2007年05期

5 谷化平;尚培中;徐志勇;;P63和PTEN蛋白在鼻咽癌中的表達(dá)及臨床意義[J];山西醫(yī)藥雜志;2007年01期

，

本文編號：1653400