人CD59基因活性位点突变对肿瘤逃逸相关分子补体及caspase-3的作用研究
发布时间：2018-12-25 12:03
【摘要】： 目的构建两种人CD59基因突变的重组体pALTER-MAX-CD59,转染入HeLa细胞,筛选高表达人突变CD59的阳性细胞克隆,检测HeLa细胞中突变CD59对肿瘤逃逸相关分子(caspase-3)的抑制效应及突变人CD59的补体抑制活性,研究CD59基因突变与肿瘤逃逸的相关性,进一步证实人CD59与肿瘤逃逸相关的活性位点,为肿瘤逃逸机制的进一步阐明提供新的理论依据。 方法用重组聚合酶链反应定点诱变技术构建第40位色氨酸缺失的人的CD59突变体(突变1)和第39-41位氨基酸突变为色氨酸的人的CD59突变体(突变2);与pALTER-MAX质粒连接后,用阳离子脂质体法转染入HeLa细胞,以新霉素类似物G418筛选阳性细胞克隆,以免疫荧光、免疫酶标、ELISA、Western-blot、流式细胞术进一步筛选高表达突变人CD59的细胞株,用免疫组化法检测转染人突变CD59的细胞中肿瘤逃逸相关分子caspase-3的表达变化,用乳酸脱氢酶(LDH)测试法检测突变人CD59的抗补体活性。 结果酶切鉴定及序列测定均证实成功构建了两种人CD59基因突变的重组质粒;免疫荧光、免疫酶标、ELISA、Western-blot、流式细胞术筛选出高表达突变人CD59的细胞株;免疫组化检测发现转染突变1后的HeLa细胞中caspase-3的含量较转染野生型人CD59的HeLa细胞中的caspase-3的含量明显增加,转染突变2后的HeLa细胞中caspase-3的含量较转染野生型人CD59的HeLa细胞中的caspase-3的含量明显减少。LDH法检测转染突变CD59后HeLa细胞具有抗补体活性,但其活性减弱。 结论成功构建了两种CD59突变的重组质粒,并获得高表达两种突变CD59的HeLa细胞株,初步研究了CD59基因突变后抗补体活性改变及caspase-3的变化,证实了CD59引起肿瘤逃逸的途径除了通过调节其抗补体活性来影响MAC的形成外还能通过影响caspase-3的表达来实现。
[Abstract]:Objective to construct two recombinant human CD59 gene mutants pALTER-MAX-CD59, and transfect them into HeLa cells to screen the positive cell clones expressing human mutant CD59. The inhibitory effect of mutated CD59 on tumor escape associated molecule (caspase-3) and complement inhibitory activity of mutant human CD59 in HeLa cells were detected, and the correlation between CD59 gene mutation and tumor escape was studied. The active sites associated with tumor escape were further confirmed by human CD59, which provided a new theoretical basis for further elucidation of the mechanism of tumor escape. Methods Human CD59 mutants (mutant 1) and CD59 mutants (mutagen2) were constructed by site-directed mutagenesis of recombinant polymerase chain reaction (RPCR) for the 40 th position tryptophan deficient human and the 39-41 amino acid mutagenesis for tryptophan. After ligation with pALTER-MAX plasmid, HeLa cells were transfected into HeLa cells by cationic liposome method. Positive cell clones were screened by neomycin analogue G418. Immunofluorescence, immunoenzyme labeling and ELISA,Western-blot, were used to screen the positive cells. Flow cytometry was used to screen the cell lines with high expression of human CD59, and the expression of tumor escape related molecule caspase-3 was detected by immunohistochemical method in the cells transfected with human mutant CD59. The anti complement activity of mutant human CD59 was detected by lactate dehydrogenase (LDH) assay. Results two recombinant plasmids of human CD59 gene mutation were successfully constructed by restriction endonuclease digestion and sequence analysis, immunofluorescence, immunoenzyme labeling and ELISA,Western-blot, flow cytometry were used to screen the cells with high expression of human CD59. Immunohistochemical analysis showed that the content of caspase-3 in HeLa cells transfected with mutant 1 was significantly higher than that in HeLa cells transfected with wild type CD59. The content of caspase-3 in HeLa cells after transfection of mutant 2 was significantly lower than that in HeLa cells transfected with wild type CD59. LDH assay showed that HeLa cells had anti-complement activity after transfection of mutant CD59, but its activity decreased. Conclusion two recombinant plasmids with CD59 mutation were successfully constructed and HeLa cell lines with high expression of CD59 were obtained. The changes of complement resistance and caspase-3 after CD59 gene mutation were preliminarily studied. It is confirmed that the pathway of escape induced by CD59 can not only affect the formation of MAC by regulating its anticomplement activity, but also affect the expression of caspase-3.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.12;R730.3
本文编号：2391084
[Abstract]:Objective to construct two recombinant human CD59 gene mutants pALTER-MAX-CD59, and transfect them into HeLa cells to screen the positive cell clones expressing human mutant CD59. The inhibitory effect of mutated CD59 on tumor escape associated molecule (caspase-3) and complement inhibitory activity of mutant human CD59 in HeLa cells were detected, and the correlation between CD59 gene mutation and tumor escape was studied. The active sites associated with tumor escape were further confirmed by human CD59, which provided a new theoretical basis for further elucidation of the mechanism of tumor escape. Methods Human CD59 mutants (mutant 1) and CD59 mutants (mutagen2) were constructed by site-directed mutagenesis of recombinant polymerase chain reaction (RPCR) for the 40 th position tryptophan deficient human and the 39-41 amino acid mutagenesis for tryptophan. After ligation with pALTER-MAX plasmid, HeLa cells were transfected into HeLa cells by cationic liposome method. Positive cell clones were screened by neomycin analogue G418. Immunofluorescence, immunoenzyme labeling and ELISA,Western-blot, were used to screen the positive cells. Flow cytometry was used to screen the cell lines with high expression of human CD59, and the expression of tumor escape related molecule caspase-3 was detected by immunohistochemical method in the cells transfected with human mutant CD59. The anti complement activity of mutant human CD59 was detected by lactate dehydrogenase (LDH) assay. Results two recombinant plasmids of human CD59 gene mutation were successfully constructed by restriction endonuclease digestion and sequence analysis, immunofluorescence, immunoenzyme labeling and ELISA,Western-blot, flow cytometry were used to screen the cells with high expression of human CD59. Immunohistochemical analysis showed that the content of caspase-3 in HeLa cells transfected with mutant 1 was significantly higher than that in HeLa cells transfected with wild type CD59. The content of caspase-3 in HeLa cells after transfection of mutant 2 was significantly lower than that in HeLa cells transfected with wild type CD59. LDH assay showed that HeLa cells had anti-complement activity after transfection of mutant CD59, but its activity decreased. Conclusion two recombinant plasmids with CD59 mutation were successfully constructed and HeLa cell lines with high expression of CD59 were obtained. The changes of complement resistance and caspase-3 after CD59 gene mutation were preliminarily studied. It is confirmed that the pathway of escape induced by CD59 can not only affect the formation of MAC by regulating its anticomplement activity, but also affect the expression of caspase-3.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.12;R730.3
【参考文献】
相关期刊论文 前10条
1 杨崇礼,卢学春,刘慧英;再生障碍性贫血、骨髓增生异常综合征、阵发性睡眠性血红蛋白尿症演变的问题[J];白血病;2000年06期
2 杜红俊,惠延年,王雨生,韩泉洪,马吉献;Caspase-3在柔红霉素诱导的人视网膜色素上皮细胞凋亡中作用[J];第四军医大学学报;2001年07期
3 高珏华;CD59和阵发性睡眠性血红蛋白尿[J];国外医学.输血及血液学分册;1998年06期
4 王海滨,郭峰;红细胞参与细胞因子调控的研究进展[J];国外医学(免疫学分册);1999年05期
5 张剑波,董巍,江南;贫血患者外周血CD55~-和CD59~-细胞检测结果及其意义[J];临床检验杂志;2003年S1期
6 韩永胜,翟志敏,丁邦胜,吴竞生,何晓东;CD59检测在阵发性睡眠性血红蛋白尿症诊断中的意义[J];临床输血与检验;2003年03期
7 周珞平,翟志敏,韩永胜,潘理明,朱薇波,蔡晓燕,吴竞生,孙自敏;CD59抗原在血液系统恶性肿瘤患者中的表达及意义[J];临床输血与检验;2004年01期
8 周镜然,白云,姜曼,黎万玲;CD59融合蛋白表达载体构建及其在大肠杆菌中的表达[J];免疫学杂志;2000年02期
9 陈政良,朱锡华;天然免疫系统的“分子模式识别作用”及其免疫生物学意义[J];免疫学杂志;2000年03期
10 张丽,高美华;突变人CD59基因在真核细胞中的表达活性[J];免疫学杂志;2005年04期
，本文编号：2391084
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2391084.html
最近更新
教材专著
