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

【摘要】：【目的】細胞凋亡（apoptosis）是哺乳動物細胞生產抗體藥物過程中限制活細胞密度提高的關鍵因素之一，，進而制約了抗體藥物的產量。本課題采用基因工程手段在無血清懸浮培養(yǎng)的中國倉鼠卵巢細胞（Chinese hamster ovary, CHO）中過表達一種新型凋亡調控基因TIGAR（TP53-induced glycolysis and apoptosis regulator），分析該CHO-TIGAR細胞株在批次培養(yǎng)中的生長代謝、抑制凋亡等現象，探究TIGAR基因在CHO細胞中抑制凋亡的主要機制，進而闡述該研究相關應用價值及意義。 【方法】第一部分構建過表達人TIGAR的CHO細胞株的主要方法有：①設計特異性引物通過PCR擴增得到hTIGAR基因，構建pcDNA3.0-TIGAR真核表達載體后轉染CHO-SP細胞并挑選陽性克隆，同時以轉染pcDNA3.0空載體的細胞作為陰性對照。利用Real-time PCR檢測TIGAR基因mRNA水平表達。②在500mL搖瓶中進行批次培養(yǎng)，獲取CHO-TIGAR細胞株和對照細胞CHO-pcDNA3.0的生長曲線、活率、葡萄糖消耗量等參數。 本課題的第二部分著重探討TIGAR基因在CHO細胞中抑制凋亡的主要機制，其主要方法有：①批次培養(yǎng)中通過Annexin V/PI法檢測CHO-TIGAR細胞和對照組在第4-7天的凋亡率；利用DCFH-DA染料檢測實驗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細胞和CHO-pcDNA3.0細胞在批次培養(yǎng)第5-8天中Caspase-9、Caspase-3、Caspase-7等的表達情況，比較兩株細胞的凋亡信號通路中關鍵分子的差異。 【結果】在批次培養(yǎng)中，CHO-TIGAR細胞株的最高細胞密度出現在第四天，達到7.2×106cells/mL，對照組的最高密度出現在同一時間，只有6.5×106cells/mL；細胞活率、培養(yǎng)天數方面，CHO-TIGAR細胞均優(yōu)于對照細胞株；通過流式細胞術檢測細胞凋亡率，實驗組細胞在批次培養(yǎng)的第6、7、8天凋亡率分別為5.5%、6.08%和21.92%，在對照組中相對應的為10.4%、20.28%、50.3%；檢測批次培養(yǎng)1-5天細胞內ROS水平，CHO-TIGAR細胞均低于對照組；Western blot實驗證實對照組中與凋亡發(fā)生相關的蛋白質Caspase-9、Caspase-3/7表達水平均高于CHO-TIGAR細胞株。 【結論】在CHO-SP細胞中過表達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)下的活細胞密度，抑制細胞凋亡，從而在批次培養(yǎng)中延緩細胞生長衰退期，提高了積分活細胞密度（IVCC）和細胞抗凋亡能力。這是由于hTIGAR基因的編碼蛋白可以有效減少細胞內ROS水平，維持細胞氧化-還原穩(wěn)態(tài)并抑制了由ROS引發(fā)的細胞凋亡。因此利用hTIGAR基因對CHO細胞內ROS水平的調控有望為提高抗體藥物產量提供新的途徑。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R392

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