| Recombinant protein drug, which has the property of highly active, strongspecificity, low toxicity, clear biological function and conducive to clinical application,has become an important part of pharmaceutical products, it has also been widelyapplied in the treatment of various diseases including autoimmune diseases, cancer, etc.But insufficient production capacity has been the bottleneck of in the development ofrecombinant drugs. While the growing market needs could be met by amplifying thevolume of reactor to meet the growing market needs and promote the recombinantprotein production. Alternatively, it is preferable to get a stable cell line with prolongedculture time, high yield, less by-product generation and resistance to apoptosis throughgenetic engineering.In the process of fed-batch culture, lactic acid and other metabolic wastes areaccumulated with the extension of incubation time，which would lead to pH decline inculture medium, however, adjusting pH value with alkaline substance may cause higherosmotic pressure, which would suppress the growth of cells, eventually lead to thelower cell viability and protein yield.LDH-C gene exists in the sperm cells, testicular cells and some cancers. It is foundthat in the sperm cells, different from lactic dehydrogenase A and B, LDH-C prefers tousing lactic acid as the substrate and catalyzing lactic acid towards pyruvic acid. It isalso reported that LDH-C can help sperm to survive in the environment of high lacticacid, at the same time it allows lactate to be the source of energy. Although the role ofLDH-C in CHO cells is unclear, the above studies have shown that LDH-C can be usedas an interesting targets for cell engineering, it may make the CHO cell metabolismswitch toward lactic acid aerobic metabolism, improving growth performance. This research was aim to establish a stable gene transfection cell line throughLDH-C gene engineering, and improve the high lactic acid environment of cell culturein the late phase, thus inhibiting apoptosis. The significance of this study is that it canlay the foundation for improving the production of antibody or protein drugs.Objective: In order to construct a stable cell line which has the characteristics oflong culture time, low metabolic by-products and resistance to apoptosis throughgenetic engineering, human LDH-C gene was overexpressed in CHO cell, and the cellline would lay a foundation for the increase of antibody production.Methods: LDH-C gene was amplified from human SKBR-3cells by reversetranscription polymerase chain reaction (RT-PCR), after cut by XhoⅠand HindⅢ, itwas inserted to eukaryotic expression vector pcDNA3.0and then transfected to CHOcells through Lipofectamine2000. A stable monoclonal cell line with LDH-Coverexpression was obtained from G418resistant cell pool. LDH-C gene expressionwas detected by Real-time PCR at mRNA level, then three cell lines were selected tocultivate in fed-batch mode, meanwhile making a record of cells growth, viability andIVCC everyday, and eventually obtained a cell line with the highest IVCC value.Making a record of cell density and counting cell viability with trypan blueexclusion method everyday, meanwhile the concentration of the lactic acid in cellculture supernatant was detected to investigate the effect of LDH-C on CHOmetabolism.To investigate the anti-apoptosis of CHO/LDH-C, cell cycle and cell apoptosiswere analyzed with flow cytometry, and mitochondrial membrane potential wasanalyzed with JC-1dye. Meanwhile, the expression of apoptosis related protein wereidentified by western blot.Results:(1) Human LDH-C gene was successfully cloned into eukaryoticexpression vector to make pcDNA3.0/LDH-C, and a stably transfection cell line withLDH-C overexpression was established. A clone#6named CHO/LDH-C#6wasselected for follow-up study through real-time PCR identification, fed-batch cultureexperiments and activity identification. Meanwhile, CHO/pcDNA3.0#4was selected asthe experimental control group.(2) Under the initial ondition, the cell growth profile of the two groups have noobvious difference, however, cell growth of the control was suppressed when40mM sodium pyruvate or40mm sodium lactate was added on the third day, the cell densityof CHO/LDH-Cwas significantly higher than control group, and more importantly,under the two conditions, the lactic acid concentration of the experiment group wasmuch lower than that of control group, especially in the later stages. Lactic acidconcentration can be reduced significantlly.(3) Cell cycle of CHO/LDH-C was analyzed with flow cytometry, the results showthat S phase increased while G0phase decreased, it suggested that DNA replication,proliferation activity increased in CHO/LDH-C cells. The results of cell cell apoptosisanalysis and mitochondrial membrane potential also demonstrated that CHO/LDH-Chad a much lower cell apoptosis rate, and the expression of apoptosis related proteinsprove that LDH-C can be advantage to inhibit apoptosis.Conclusion: In conclusion, this study has successfully established a stable LDH-Cgene transfection cell, and effect of the LDH-C gene on CHO cell growth and lactic acidmetabolism was examined through simulating high lactic acid environment by addingsodium lactate and sodium pyruvate. This Research has shown that in low lactic acidenvironment without adding lactic acid or sodium pyruvate, the cell growth andviability were not improved with LDH-C overexpression. And under the condition ofsodium lactate and pyruvate added, the CHO cells with LDH-C over-expressedachieved higher cell density and viability, and the lactic acid concentration decreasedsignificantly compared with the control group. In addition, the cells with LDH-Cover-expressed have faster cell proliferation and stronger ability to antagonizeapoptosis. |
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