CHO细胞法检测百日咳毒素毒性的影响因素
|
摘要
目的考察不同培养基、不同牛血清、血清灭活与否及生产过程中添加的各外源物质对百日咳毒素(pertussis toxin, PT)在中华仓鼠卵巢细胞(chinese hamster ovary cell, CHO)簇集试验中的影响。方法分别使用3种培养基F-12K、DMEM/F12和1640培养CHO细胞,并进行CHO细胞簇集试验,观察细胞生长状态及PT引起细胞簇集的敏感性;分别选取2个厂家的牛血清(对2种血清进行灭活和不灭活处理)培养CHO细胞,观察4种牛血清对细胞生长及簇集的影响;选用生产过程中添加的物质进行CHO细胞簇集试验,观察细胞生长状态及是否出现簇集,确定不影响细胞生长的最高浓度,同时使用不影响细胞生长的各添加物质最高浓度进行小鼠组胺致敏试验,观察与CHO细胞簇集试验结果是否一致。结果 3种培养基对CHO细胞生长及CHO细胞簇集存在明显差异,F-12K培养基培养的细胞形态规则、典型,其他2种培养基培养的细胞生长缓慢,且对PT的敏感性均低于F-12K培养基;4种牛血清中胎牛血清培养的细胞生长最快且形态规则,簇集试验敏感性优于其他3组血清;添加的各外源物质均会导致细胞生长缓慢或死亡,在稀释至一定浓度后可以排除添加物质对CHO细胞簇集试验的影响,同时在小鼠组胺致敏试验中不会引起动物死亡。结论 F-12K培养基最适宜实验室CHO细胞生长,不同血清对细胞生长和簇集的敏感度有一定差异,添加的外源物质残留量应进行控制以保证试验结果的稳定可靠。
Objective To investigate the effects of different culture media, and bovine serum, with or without serum inactivation, added exogenous substances during detoxification on pertussis toxin(PT) by Chinese hamster ovary cell(CHO)clustering test. Methods CHO cells were cultured in F-12K, DMEM/F12 and 1640 medium, respectively, and CHO clustering tests were carried out to observe the cell growth state and the sensitivity of PT-induced cell clustering. Two kinds of bovine serums were used in culture of CHO cells, which were from different manufacturers. Comparison of the effects with or without inactivation of two kinds of serums on cell growth and clustering state were performed. The substances added in the production process were used for CHO cell clustering test in observation the cell growth and clustering state, and to make sure their's highest concentrations not to affect cells growth.At the same time, the highest concentration of each added substance that does not affect cell growth was used in mouse histamine sensitization test(HIST), to examine whether it was consistent with the CHO cell clustering test results. Results The three culture media showed significant differences in CHO cell growth and CHO cell clustering test. The cell morphology was in a regular and typical mode in F-12K medium, but cell grew slowly in the other two media and the sensitivity to PT was lower than that of F-12K; the cultured cells grew fastest in fetal bovine serum than in other bovine serums, with a regular morphology. The sensitivity of the clustering test was also better than other three serums. Each exogenous substance could cause cells growth slowly or death, the effect of the added substances on the CHO clustering test can be excluded after a certain dilution. HIST results were in consistent with cell test results. Conclusion F-12K was the most suitable medium for the growth of CHO cells preserved in our laboratory. Different serums had different sensitivity to cell growth and clustering test, and screening test should be carried out before the formal test. The concentration of exogenous substances should be controlled to ensure the stablity and reliablity for the test results.
Objective To investigate the effects of different culture media, and bovine serum, with or without serum inactivation, added exogenous substances during detoxification on pertussis toxin(PT) by Chinese hamster ovary cell(CHO)clustering test. Methods CHO cells were cultured in F-12K, DMEM/F12 and 1640 medium, respectively, and CHO clustering tests were carried out to observe the cell growth state and the sensitivity of PT-induced cell clustering. Two kinds of bovine serums were used in culture of CHO cells, which were from different manufacturers. Comparison of the effects with or without inactivation of two kinds of serums on cell growth and clustering state were performed. The substances added in the production process were used for CHO cell clustering test in observation the cell growth and clustering state, and to make sure their's highest concentrations not to affect cells growth.At the same time, the highest concentration of each added substance that does not affect cell growth was used in mouse histamine sensitization test(HIST), to examine whether it was consistent with the CHO cell clustering test results. Results The three culture media showed significant differences in CHO cell growth and CHO cell clustering test. The cell morphology was in a regular and typical mode in F-12K medium, but cell grew slowly in the other two media and the sensitivity to PT was lower than that of F-12K; the cultured cells grew fastest in fetal bovine serum than in other bovine serums, with a regular morphology. The sensitivity of the clustering test was also better than other three serums. Each exogenous substance could cause cells growth slowly or death, the effect of the added substances on the CHO clustering test can be excluded after a certain dilution. HIST results were in consistent with cell test results. Conclusion F-12K was the most suitable medium for the growth of CHO cells preserved in our laboratory. Different serums had different sensitivity to cell growth and clustering test, and screening test should be carried out before the formal test. The concentration of exogenous substances should be controlled to ensure the stablity and reliablity for the test results.
引文
[1] 王祖森, 杨晓明, 何长民. 百日咳毒素及其应用[J]. 卫生研究, 1998, 27(增刊1): 160-167.
[2] AYALA V I, TEIJARO J R, FARBER D L, et al. Pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity[J]. PLoS One, 2011, 6(4): e19016-19019.
[3] 乔瑞洁, 杨晓明. 百日咳毒素S1亚单位的表达及其免疫原性研究[J].中国生物制品学杂志, 2003, 16(4): 205-207.
[4] 夏德菊, 潘殊男, 杨云凯, 等. 无细胞百日咳疫苗中百日咳毒素活性检测方法的建立[J]. 中国生物制品学杂志, 2014, 27(12): 1601-1606.
[5] 侯启明, 张路民, 梁雅文, 等. 应用CHO细胞法检测百日咳疫苗中的残余毒性[J]. 中国生物制品学杂志, 1997,10(2): 101-103.
[6] 曹方, 刘海昌, 王磊, 等. CHO-K1细胞簇集法百日咳毒素毒性的优化及验证[J]. 中国生物制品学杂志, 2015, 28(12): 1344-1346.
[7] 刘晓凤, 邓杰, 秦敏. 牛血清在百日咳毒素CHO细胞簇聚试验中的影响[J]. 微生物学免疫学进展, 2016, 44(2): 50-54.
[8] 徐颖华, 卫辰, 王丽婵, 等. CHO细胞簇集试验在无细胞百日咳疫苗质量控制中的应用[J]. 中国生物制品学杂志, 2014, 27(10): 1279-1282.
[9] WHO. Manual for Quality Control of Diphtheria, Tetanus and pertussis vaccines[S].WHO Technical Report Series, 1964, 293.
[10] TAN Y, FLECK R A, ASOKANATHAN C, et al. Microscopy study of pertussis toxin and toxoids on CHO-cells[J]. Hum Vaccin Immunother, 2014, 9(2): 332-338.
[2] AYALA V I, TEIJARO J R, FARBER D L, et al. Pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity[J]. PLoS One, 2011, 6(4): e19016-19019.
[3] 乔瑞洁, 杨晓明. 百日咳毒素S1亚单位的表达及其免疫原性研究[J].中国生物制品学杂志, 2003, 16(4): 205-207.
[4] 夏德菊, 潘殊男, 杨云凯, 等. 无细胞百日咳疫苗中百日咳毒素活性检测方法的建立[J]. 中国生物制品学杂志, 2014, 27(12): 1601-1606.
[5] 侯启明, 张路民, 梁雅文, 等. 应用CHO细胞法检测百日咳疫苗中的残余毒性[J]. 中国生物制品学杂志, 1997,10(2): 101-103.
[6] 曹方, 刘海昌, 王磊, 等. CHO-K1细胞簇集法百日咳毒素毒性的优化及验证[J]. 中国生物制品学杂志, 2015, 28(12): 1344-1346.
[7] 刘晓凤, 邓杰, 秦敏. 牛血清在百日咳毒素CHO细胞簇聚试验中的影响[J]. 微生物学免疫学进展, 2016, 44(2): 50-54.
[8] 徐颖华, 卫辰, 王丽婵, 等. CHO细胞簇集试验在无细胞百日咳疫苗质量控制中的应用[J]. 中国生物制品学杂志, 2014, 27(10): 1279-1282.
[9] WHO. Manual for Quality Control of Diphtheria, Tetanus and pertussis vaccines[S].WHO Technical Report Series, 1964, 293.
[10] TAN Y, FLECK R A, ASOKANATHAN C, et al. Microscopy study of pertussis toxin and toxoids on CHO-cells[J]. Hum Vaccin Immunother, 2014, 9(2): 332-338.