低血清培养基培养人胚肺二倍体细胞(2BS株)及其增殖甲型肝炎病毒的初步研究  

Preliminary study on human embryonic lung diploid fibroblast cell line (2BS) cultured in low serum medium and its proliferation of hepatitis A virus

作　　者：张宏丽 任琦 夏青娟 李春雷 杨利民 徐艳玲 ZHANG Hong-li;REN Qi;XIA Qing-juan;LI Chun-lei;YANG Li-min;XU Yan-ling(Second Department,of Vaccine,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司疫苗二室,吉林长春130012

出　　处：《微生物学免疫学进展》2021年第6期13-17,共5页Progress In Microbiology and Immunology

基　　金：甲型肝炎疫苗规格变更、质量提升与扩大产业化(20190404001YY)。

摘　　要：目的研究2种低血清培养基对人胚肺二倍体细胞(2BS株)的培养效果及甲型肝炎病毒(hepatitis A virus,HAV)在低血清培养基中的增殖情况。方法使用低血清培养基A+2%新生牛血清、B+5%新生牛血清和E-MEM培养基+10%新生牛血清培养细胞,显微镜下观察细胞形态、贴壁情况,细胞计数并绘制生长曲线;优化低血清培养基B的血清浓度;使用5层细胞工厂连续传代培养,放大低血清培养基细胞培养工艺;使用低血清培养基连续进行3次细胞传代后接种HAV,检测病毒滴度。结果使用低血清培养基A+2%新生牛血清、B+5%新生牛血清和E-MEM培养基+10%新生牛血清,细胞生长密度峰值分别为1.55×10^(5)、2.07×10^(5)和1.30×10^(5)个/cm^(2),统计学分析表明,低血清培养基B+5%新生牛血清达到细胞生长密度峰值高于低血清培养基A+2%新生牛血清和E-MEM+10%新生牛血清,差异具有统计学意义(P均<0.05);低血清培养基B+5%新生牛血清同B+3%新生牛血清连续培养3代细胞生长密度差异均无统计学意义(P=0.47、0.07、0.12);使用5层细胞工厂培养细胞,低血清培养基A+2%新生牛血清、B+5%新生牛血清连续培养3代,细胞生长密度均高于E-MEM+10%新生牛血清;低血清培养基B+3%新生牛血清与低血清培养基B+5%新生牛血清的病毒滴度分别为9.50和9.33 lgCCID_(50)/mL,低血清培养基A+2%新生牛血清和E-MEM+10%新生牛血清的病毒滴度分别为8.50和9.00 lgCCID_(50)/mL。结论低血清培养基适用于培养2BS细胞增殖HAV,为低血清培养基在甲肝疫苗生产中的应用奠定了基础。Objective To evaluate culture effect of two low serum media on human embryonic lung diploid fibroblast cell line(2 BS) and proliferation of hepatitis A virus(HAV) in low serum medium. Methods The 2 BS cells were cultured in low serum medium A containing 2% newborn calf serum, low serum medium B containing 5% newborn calf serum or E-MEM containing 10% newborn calf serum, respectively. Cell morphology and adhesion were observed under microscope, and cell numbers were counted using cell counter, then cell growth curves were drawn. The serum concentration of low serum medium B was optimized, and 5-layer cell factory was used for continuous subculture to amplify the cell culture process under the condition of low serum medium, then HAV was inoculated after 3 consecutive cell passages in low serum medium, and the cells were harvested for the virus titer detection. Results The peak cell densities of 2 BS cultured in low serum medium A, B and E-MEM were 1.55×10^(5), 2.07×10^(5)and 1.30×10^(5)cells/cm^(2), respectively, of which the peak cell density in low serum medium B was higher than that in low serum medium A and controlled E-MEM,and there were significant differences(all P < 0.05). There was no significant difference of cell density between low serum medium B and optimized B plus 3% newborn calf serum for three consecutive generations(P = 0.47, 0.07, 0.12). The cell densities cultured in low serum medium A and B were higher than that in E-MEM for three consecutive generations in 5-layer cell factory. The virus titers of HAV cultured in 2 BS in low serum medium A, B, optimized B and E-MEM were 8.50, 9.33, 9.50 and 9.00 lgCCID;/mL, respectively. Conclusion These findings indicate that HAV preparation by culture of 2 BS cells in low serum medium was feasible, which lays a foundation for the application of low serum medium in the production of hepatitis A vaccine.

关 键 词：人胚肺二倍体细胞(2BS株) 低血清培养基 甲型肝炎病毒 增殖 

分 类 号：R392[医药卫生—免疫学]