CASK过表达慢病毒载体的构建及其对H1299细胞生物学功能特性的影响

发布时间：2018-03-23 21:55

本文选题：CASK　切入点：H1299　出处：《广西医科大学》2017年硕士论文

【摘要】：目的肺癌是我国最常见的恶性肿瘤之一,而非小细胞肺癌(non-small cell lung cancer,NSCLC)约占肺癌总数的80%-85%,是临床最常见的一种类型。由于肺癌在早期缺乏明显的特异性症状,同时易发生血行转移,因此大多数患者在确诊时已进入中晚期阶段,治疗效果及预后均不佳。本课题组在前期通过生物信息学方法筛选出11个基因,其中人钙/钙调蛋白依赖性丝氨酸蛋白激酶(calcium/calmodulin-dependent serine protein kinase,CASK)在报道中与肺癌发病有重要关系。故本研究通过构建能高效表达人CASK基因的慢病毒载体并对H1299细胞系稳定转染后进行鉴定,并通过对人非小细胞肺癌H1299细胞株进行感染后,研究CASK过表达对其增殖能力和迁移能力的影响。方法1.CASK过表达慢病毒载体的构建采用聚合酶链反应(PCR)扩增CASK基因片段,通过重组反应完成目的基因扩增产物和线性化GV358载体的体外环化。对重组产物进行转化后,挑取单克隆进行PCR鉴定,并对阳性克隆进行测序分析。最后将正确克隆菌液扩大培养、抽提以获得高纯度质粒。将重组质粒和包装质粒共转染至293T细胞。Western Blot检测转染后293T细胞蛋白表达水平;用酶联免疫吸附实验(ELISA)法进行滴度检测。2.细胞模型效能检测将成功包装的CASK过表达慢病毒LV-CASK(OE组)和阴性对照病毒CON238(NC组)分别感染人非小细胞肺癌H1299细胞,同时设置空白对照组(MOCK组),用荧光定量PCR(FQ-PCR)和Western Blot法检测CASK表达水平。3.CASK过表达对H1299细胞生物学功能特性的影响实验分组:空白对照组(MOCK组)、CASK过表达组(OE组)和阴性对照组(NC组),NC组和OE组分别用CON238和LV-CASK进行感染,MOCK组不予任何处理,分别使用MTT法和细胞划痕实验观察CASK过表达对H1299细胞增殖能力和迁移能力的影响。结果1.CASK过表达慢病毒载体的构建通过PCR技术成功地扩增CASK基因片段并连接到GV358病毒载体上,PCR及DNA测序鉴定结果证明CASK-GV358质粒构建正确。重组慢病毒转染293T细胞后可观察到绿色荧光及蛋白表达,包装过表达慢病毒并测定其浓缩滴度为2×108 TU/m L。2.细胞模型效能检测FQ-PCR和Western Blot检测结果显示,与NC组相比,OE组CASK m RNA和蛋白的表达均显著上调(P0.05)。3.CASK过表达对H1299细胞生物学功能特性的影响MTT法结果表明,OE组细胞增殖能力与NC组相比有明显差异(P0.05),提示CASK过表达对H1299细胞增殖能力有抑制作用;细胞划痕实验结果表明OE组细胞迁移能力与NC组无明显差异(P0.05),CASK过表达对H1299细胞迁移能力无明显作用。结论成功构建了CASK基因过表达慢病毒载体,可在H1299细胞中稳定表达,CASK过表达可抑制H1299细胞增殖能力,为进一步研究CASK在肺癌中的机制和作用奠定了基础。
[Abstract]:Objective Lung cancer is one of the most common malignant tumors in China, and non-small cell lung cancer (NSCLC) accounts for 80% -85% of the total number of lung cancer. Therefore, most of the patients had entered the stage of middle and late stage of diagnosis, and the therapeutic effect and prognosis were not good. 11 genes were screened out by bioinformatics in the early stage. Calcium / calmodulin-dependent serine protein kinase (CASKK) is associated with the pathogenesis of lung cancer. Therefore, a lentivirus vector expressing human CASK gene was constructed and identified after stable transfection of H1299 cell line. The effect of CASK overexpression on the proliferation and migration of human non-small cell lung cancer cell line H1299 was studied. Methods the construction of 1.CASK overexpression lentivirus vector was used to amplify the CASK gene fragment by polymerase chain reaction (PCR). In vitro cyclization of the target gene amplification product and linearized GV358 vector was completed by recombination reaction. After the recombinant product was transformed, the monoclonal was selected for PCR identification. The positive clones were sequenced. Finally, the recombinant plasmid and packaging plasmid were cotransfected into 293T cells. Western Blot was used to detect the protein expression level of 293T cells after transfection. Elisa assay was used to detect the titer of human non-small cell lung cancer H1299 cells. 2. The efficacy of the cell model was detected. The CASK overexpressed lentivirus LV-CASK(OE group and the negative control virus CON238(NC group were respectively infected with H1299 cells in human non-small cell lung cancer (NSCLC). At the same time, a blank control group was set up to detect the expression level of CASK. 3. The effect of over expression of CASK on the biological function of H1299 cells was detected by fluorescence quantitative PCR FQ-PCRR) and Western Blot. The experimental group was divided into two groups: the blank control group (mock group) and the control group (mock overexpression group) and negative group (OE group). The control group, NC group and OE group were treated with CON238 and LV-CASK respectively. The effects of CASK overexpression on the proliferation and migration of H1299 cells were observed by MTT assay and scratch assay respectively. Results the construction of 1.CASK overexpression lentivirus vector successfully amplified the CASK gene fragment and ligated to GV358 by PCR technique. The construction of CASK-GV358 plasmid was confirmed by PCR and DNA sequencing. Green fluorescence and protein expression were observed after the recombinant lentivirus was transfected into 293T cells. The lentivirus was overexpressed and its concentration titer was determined to be 2 脳 10 ~ 8 TU/m L. 2.The results of FQ-PCR and Western Blot detection showed that, Effect of CASK m RNA and protein expression on the biological function of H1299 cells in OE group compared with NC group the results of MTT assay showed that the proliferation ability of H1299 cells in OE group was significantly different from that in NC group, indicating the overexpression of CASK in H1299 cells. 3. The proliferation of H1299 cells was inhibited. The results of cell scratch test showed that there was no significant difference between OE group and NC group in cell migration ability. There was no significant effect of P0.05CSK overexpression on H1299 cell migration. Conclusion the lentivirus vector of CASK gene overexpression was constructed successfully. Overexpression of Cask in H1299 cells could inhibit the proliferation of H1299 cells, which laid a foundation for further study of the mechanism and role of CASK in lung cancer.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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