本文選題：中國倉鼠卵巢細(xì)胞 + TIGAR��； 參考：《蘇州大學(xué)》2013年碩士論文

【摘要】：【目的】細(xì)胞凋亡（apoptosis）是哺乳動物細(xì)胞生產(chǎn)抗體藥物過程中限制活細(xì)胞密度提高的關(guān)鍵因素之一，，進(jìn)而制約了抗體藥物的產(chǎn)量。本課題采用基因工程手段在無血清懸浮培養(yǎng)的中國倉鼠卵巢細(xì)胞（Chinese hamster ovary, CHO）中過表達(dá)一種新型凋亡調(diào)控基因TIGAR（TP53-induced glycolysis and apoptosis regulator），分析該CHO-TIGAR細(xì)胞株在批次培養(yǎng)中的生長代謝、抑制凋亡等現(xiàn)象，探究TIGAR基因在CHO細(xì)胞中抑制凋亡的主要機(jī)制，進(jìn)而闡述該研究相關(guān)應(yīng)用價(jià)值及意義。 【方法】第一部分構(gòu)建過表達(dá)人TIGAR的CHO細(xì)胞株的主要方法有：①設(shè)計(jì)特異性引物通過PCR擴(kuò)增得到hTIGAR基因，構(gòu)建pcDNA3.0-TIGAR真核表達(dá)載體后轉(zhuǎn)染CHO-SP細(xì)胞并挑選陽性克隆，同時(shí)以轉(zhuǎn)染pcDNA3.0空載體的細(xì)胞作為陰性對照。利用Real-time PCR檢測TIGAR基因mRNA水平表達(dá)。②在500mL搖瓶中進(jìn)行批次培養(yǎng)，獲取CHO-TIGAR細(xì)胞株和對照細(xì)胞CHO-pcDNA3.0的生長曲線、活率、葡萄糖消耗量等參數(shù)。 本課題的第二部分著重探討TIGAR基因在CHO細(xì)胞中抑制凋亡的主要機(jī)制，其主要方法有：①批次培養(yǎng)中通過Annexin V/PI法檢測CHO-TIGAR細(xì)胞和對照組在第4-7天的凋亡率；利用DCFH-DA染料檢測實(shí)驗(yàn)組與對照組在批次培養(yǎng)中每天的活性氧族（reactive oxygen species, ROS）水平。②Western blot法檢測CHO-TIGAR細(xì)胞和CHO-pcDNA3.0細(xì)胞在批次培養(yǎng)第5-8天中Caspase-9、Caspase-3、Caspase-7等的表達(dá)情況，比較兩株細(xì)胞的凋亡信號通路中關(guān)鍵分子的差異。 【結(jié)果】在批次培養(yǎng)中，CHO-TIGAR細(xì)胞株的最高細(xì)胞密度出現(xiàn)在第四天，達(dá)到7.2×106cells/mL，對照組的最高密度出現(xiàn)在同一時(shí)間，只有6.5×106cells/mL；細(xì)胞活率、培養(yǎng)天數(shù)方面，CHO-TIGAR細(xì)胞均優(yōu)于對照細(xì)胞株；通過流式細(xì)胞術(shù)檢測細(xì)胞凋亡率，實(shí)驗(yàn)組細(xì)胞在批次培養(yǎng)的第6、7、8天凋亡率分別為5.5%、6.08%和21.92%，在對照組中相對應(yīng)的為10.4%、20.28%、50.3%；檢測批次培養(yǎng)1-5天細(xì)胞內(nèi)ROS水平，CHO-TIGAR細(xì)胞均低于對照組；Western blot實(shí)驗(yàn)證實(shí)對照組中與凋亡發(fā)生相關(guān)的蛋白質(zhì)Caspase-9、Caspase-3/7表達(dá)水平均高于CHO-TIGAR細(xì)胞株。 【結(jié)論】在CHO-SP細(xì)胞中過表達(dá)hTIGAR基因可以顯著提高其在批次培養(yǎng)及無血清條件培養(yǎng)下的活細(xì)胞密度，抑制細(xì)胞凋亡，從而在批次培養(yǎng)中延緩細(xì)胞生長衰退期，提高了積分活細(xì)胞密度（IVCC）和細(xì)胞抗凋亡能力。這是由于hTIGAR基因的編碼蛋白可以有效減少細(xì)胞內(nèi)ROS水平，維持細(xì)胞氧化-還原穩(wěn)態(tài)并抑制了由ROS引發(fā)的細(xì)胞凋亡。因此利用hTIGAR基因?qū)HO細(xì)胞內(nèi)ROS水平的調(diào)控有望為提高抗體藥物產(chǎn)量提供新的途徑。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R392

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