过表达人TIGAR的CHO细胞株构建及其抗凋亡机制研究

发布时间：2018-05-08 20:18

本文选题：中国仓鼠卵巢细胞 + TIGAR　；参考：《苏州大学》2013年硕士论文

【摘要】：【目的】细胞凋亡（apoptosis）是哺乳动物细胞生产抗体药物过程中限制活细胞密度提高的关键因素之一，，进而制约了抗体药物的产量。本课题采用基因工程手段在无血清悬浮培养的中国仓鼠卵巢细胞（Chinese hamster ovary, CHO）中过表达一种新型凋亡调控基因TIGAR（TP53-induced glycolysis and apoptosis regulator），分析该CHO-TIGAR细胞株在批次培养中的生长代谢、抑制凋亡等现象，探究TIGAR基因在CHO细胞中抑制凋亡的主要机制，进而阐述该研究相关应用价值及意义。【方法】第一部分构建过表达人TIGAR的CHO细胞株的主要方法有：①设计特异性引物通过PCR扩增得到hTIGAR基因，构建pcDNA3.0-TIGAR真核表达载体后转染CHO-SP细胞并挑选阳性克隆，同时以转染pcDNA3.0空载体的细胞作为阴性对照。利用Real-time PCR检测TIGAR基因mRNA水平表达。②在500mL摇瓶中进行批次培养，获取CHO-TIGAR细胞株和对照细胞CHO-pcDNA3.0的生长曲线、活率、葡萄糖消耗量等参数。本课题的第二部分着重探讨TIGAR基因在CHO细胞中抑制凋亡的主要机制，其主要方法有：①批次培养中通过Annexin V/PI法检测CHO-TIGAR细胞和对照组在第4-7天的凋亡率；利用DCFH-DA染料检测实验组与对照组在批次培养中每天的活性氧族（reactive oxygen species, ROS）水平。②Western blot法检测CHO-TIGAR细胞和CHO-pcDNA3.0细胞在批次培养第5-8天中Caspase-9、Caspase-3、Caspase-7等的表达情况，比较两株细胞的凋亡信号通路中关键分子的差异。【结果】在批次培养中，CHO-TIGAR细胞株的最高细胞密度出现在第四天，达到7.2×106cells/mL，对照组的最高密度出现在同一时间，只有6.5×106cells/mL；细胞活率、培养天数方面，CHO-TIGAR细胞均优于对照细胞株；通过流式细胞术检测细胞凋亡率，实验组细胞在批次培养的第6、7、8天凋亡率分别为5.5%、6.08%和21.92%，在对照组中相对应的为10.4%、20.28%、50.3%；检测批次培养1-5天细胞内ROS水平，CHO-TIGAR细胞均低于对照组；Western blot实验证实对照组中与凋亡发生相关的蛋白质Caspase-9、Caspase-3/7表达水平均高于CHO-TIGAR细胞株。【结论】在CHO-SP细胞中过表达hTIGAR基因可以显著提高其在批次培养及无血清条件培养下的活细胞密度，抑制细胞凋亡，从而在批次培养中延缓细胞生长衰退期，提高了积分活细胞密度（IVCC）和细胞抗凋亡能力。这是由于hTIGAR基因的编码蛋白可以有效减少细胞内ROS水平，维持细胞氧化-还原稳态并抑制了由ROS引发的细胞凋亡。因此利用hTIGAR基因对CHO细胞内ROS水平的调控有望为提高抗体药物产量提供新的途径。
[Abstract]:[objective] apoptosis is one of the key factors limiting the increase of living cell density in mammalian cell production of antibody drugs, which restricts the production of antibody drugs. In this study, a novel apoptosis-regulating gene TIGAR(TP53-induced glycolysis and apoptosis regulator was overexpressed in Chinese hamster ovary, CHO) of Chinese hamster ovarian cells in serum-free suspension culture by genetic engineering. The growth and metabolism of the CHO-TIGAR cell line in batch culture were analyzed. In order to explore the main mechanism of TIGAR gene inhibiting apoptosis in CHO cells, the application value and significance of this study were discussed. [methods] in the first part, the main methods of constructing CHO cell lines expressing human TIGAR were: 1: 1 designed specific primers to amplify hTIGAR gene by PCR, then constructed pcDNA3.0-TIGAR eukaryotic expression vector, then transfected CHO-SP cells and selected positive clones. At the same time, the cells transfected with empty pcDNA3.0 vector were used as negative control. The mRNA expression of TIGAR gene was detected by Real-time PCR in 500mL flask. The growth curve, viability and glucose consumption of CHO-TIGAR cell line and control cell CHO-pcDNA3.0 were obtained. In the second part of this thesis, the main mechanism of TIGAR gene inhibiting apoptosis in CHO cells was discussed. The main methods were to detect the apoptosis rate of CHO-TIGAR cells and control group at 4-7 days by Annexin V/PI assay. The expression of Caspase-9, Caspase-3, Caspase-3 and Caspase-7 in CHO-TIGAR cells and CHO-pcDNA3.0 cells during 5-8 days of batch culture were detected by DCFH-DA dye assay, and the levels of reactive oxygen species, ROS) in Ros group and control group were detected by the method of Western blot. To compare the difference of key molecules in apoptosis signaling pathway between two cell lines. [results] the highest cell density of CHO-TIGAR cell line appeared on the fourth day, reaching 7.2 脳 106 cells / mL. in the batch culture, the highest density of the control group appeared at the same time, only 6.5 脳 106 cells / mL. the cell viability and culture days of CHO-TIGAR cell line were better than that of the control cell line. Apoptosis rate was detected by flow cytometry. The apoptotic rate of the cells in the experimental group was 5.50.08% and 21.922% respectively on the 6th day and 7th day in the batch culture, and the corresponding rate in the control group was 10.4 + 20.28% and 50.3.The level of ROS in the cells cultured in the batch for 1-5 days was lower than that in the control group by Western blot assay. The expression level of Caspase-9, Caspase-3 / 7 was higher than that of CHO-TIGAR cell line. [conclusion] overexpression of hTIGAR gene in CHO-SP cells can significantly increase the density of living cells in batch culture and in serum-free culture, inhibit cell apoptosis, and thus delay the growth and decline of cells in batch culture. The integrated living cell density (IVCC) and the ability of cell anti-apoptosis were improved. This is because the protein encoded by hTIGAR gene can effectively reduce the intracellular ROS level, maintain the redox homeostasis and inhibit the apoptosis induced by ROS. Therefore, the regulation of ROS level in CHO cells by hTIGAR gene is expected to provide a new way to increase the production of antibody drugs.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

【共引文献】