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Effects of Constitutively Overexpressing hTERT on the Growth and Metabolism of Vero Cells Cultured in Vitro

Author: WangHaiTao
Tutor: ChenZhaoLie
School: PLA Military Academy of Medical Sciences
Course: Cell Biology
Keywords: Vero cells hTERT Overexpression Cell culture Cell growth
CLC: Q813.1
Type: Master's thesis
Year: 2010
Downloads: 26
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Abstract


Vero cells are acknowledged by WHO and China biological products procedures used in human vaccine production. With the outstanding advantages of broad virus infection spectrum, high efficiency of virus colonization, non-tumorigenic and handy operation of the rapid amplification of cultivation, Vero cells are treated as one of the most widely used cell line in the world in human vaccine production. However, the high attachment to the culture appliance and dependence on serum and the massive apoptosis in the large bioreactor seriously restricts the use of Vero cells in current vaccine production processes.hTERT (human telomerase reverse transcriptase), which is considered as an important component and limiting factor of telomerase. Recently, more and more evidences have proved that hTERT, beyond the ability of constructing the telomerase complex, extending the length of telomere and guarantee stability of genomes in the process of cell division. It can also effectively reduce cell apoptosis caused by bad environment and improve cell survival ability. These findings suggest that the method of constitutive overexpression of hTERT in cell line to improve its biological properties is of great significance.The goal of this research is to establish a stable and efficient hTERT expressing Vero cell line, and study the influences of hTERT overexpression with different levels on the biological characteristics of Vero cells cultured in vitro.With EGFP as a reporter gene, we confirmed the high efficiency of eukaryotic expression vector phEF-ChMAR-hyg in Vero cell line before its being used in h-TERT expression. Both RT-PCR and Real-Time PCR were carried to amount hTERT mRNA. We got hTERT ectopic expression cell lines whose relative expression level were as high as 35.26 times and 6.14 times to wildtype, named T1 and T3. The consistency of hTERT in mRNA, protein, and activity was investigated by Western blot and TRAP-PCR (telomeric repeat amplification protocol-PCR), and the investigation in stability of hTERT expression was followed.With the cell density and viability as the major indicator as well as cell morphology and dynamic stretching accompany, we inspect the hTERT overexpression cell (T1 and T3) in different conditions,such as still attachment culture, microimmobilized culture and suspension culture. T1 and T3, especially the T1 cell line, got the ability of nonadhesion dependent growth, which is followed with the poorer capability of adhesion and extension. Meanwhile, constitutively hTERT overexpression in Vero cells reduced serum dependence in vitro, and increased cell viability and viable cell density in late stage of batch culture.Using the glucose consumption rate (qGlu), lactate production rate (qLac), glutamine consumption rate (qGln) and conversion rate of glucose to lactate (YLac/Glu) as the indexes, the metabolism of T1, T3 and wildtype Vero cellswere compared in both attachment and microcarrier cultures. T1, T3 and wildtype Vero cells showed the same metabolic characteristics in glucose consumption, lactate production and amino acids consumption. It was shown that the constitutive overexpression of hTERT may not dramatically affect the metabolism in Vero cells.The results suggest that constitutive overexpression of hTERT reduces the dependency on serum and adhesion, increases the viable cell density and cell viability of Vero cells in late stage of the batch culture. It is a potential technical approach to optimize the characteristics of animal cells cultured in vitro.

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CLC: > Biological Sciences > Bioengineering ( Biotechnology ) > Cell Engineering > Cells, tissue culture techniques
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