正交试验法筛选日本血吸虫细胞的培养条件

Studies on culture conditions of cells from Schistosoma japonicum by orthogonal tests

目的 筛选日本血吸虫成虫细胞的培养条件及评价细胞培养条件优劣的指标。方法 以琥珀酸脱氢酶(SDH)、乳酸脱氢酶(L DH)和葡萄糖- 6 -磷酸脱氢酶(G- 6 - PDH )为指标,运用正交试验法进一步研究合成培养基、细胞外基质和血清浓度3个因素在3个不同水平对虫龄为2 1d的日本血吸虫细胞的影响。培养第5天对日本血吸虫培养细胞进行SDH染色;培养第14天分别进行L DH、G- 6 - PDH染色,Olympus- BH2 显微镜观察并拍照,HPIAS- 2 0 0 0图像分析仪测量其含量,用SPSS10 .0作统计分析。结果 不论以SDH或L DH,还是G- 6 - PDH为指标,均显示RPMI- 16 4 0含2 0 %小牛血清组成的培养基培养接种于肝基质上的日本血吸虫细胞,培养细胞的SDH、L DH、G- 6 -PDH活性最强。血清浓度对培养细胞酶活性的影响最大,其次是细胞外基质(ECM) ,影响最小的是合成培养基。其中,血清浓度对3种酶活性的影响差异均显著;基质对SDH、L DH活性的影响差异显著,而对G- 6 - PDH活性的影响差异不显著;合成培养基的影响均无差异。结论 RPMI- 16 4 0含2 0 %小牛血清组成的培养基培养接种于肝基质上的日本血吸虫细胞,培养细胞的SDH、L DH、G- 6 -PDH活性最强。选择3种酶中任一,均可用来评价日本血吸虫细胞培养条件的优劣。

Objective To study the culture condition of primary cells from Schistosoma japonicum. Methods The effects of complex media, ex tracelluar matrix (ECM) and serum concentration at 3 different values on the act ivities of succinodehydrogenase (SDH), lactatede dehydrogenase (LDH) and glucos e-6-phosphate dehydrogenase (G-6-PDH) in the cells from S.j aponicum (21-day old) were studied by orthogonal tests. The cel ls from S.japonicum were stained to show the ac tivities of SDH after they were cultured for 5 days, and LDH and G-6-PDH after 14 days, respectively. Then they were observed and photographed by using the mi croscope (Olympus-BH 2). The concentrations of SDH, LDH and G-6-PDH were mea sured by using photometer-image analytical instrument and statistically analyze d. Results The highest activities of SDH, LDH a nd G-6-PDH in the cultured cells from S.japonicum were noted when the cells were inoculated on the glass coverslips which had smeared with the bio-matrixes from the liver of rabbits and cultured in the me dium RPMI-1640 containing 20% bovine serum. The effect of serum concentration o n the activities of the above enzymes in the cultured cells from S. japonicum was predominent, and the differences among the th ree enzymes were significant. The effect of ECM on the activities of the above e nzymes was the second, and there was a significant difference between SDH and LD H. The effect of complex media was the weakest, and the differences among the th ree enzymes were not significant. Conclu-sion The activities of SDH, LDH and G-6-PDH in the cultured c ells from S.japonicum are strongest when the cel ls are inoculated on the glass coverslips which has smeared with the bio-matrix es from the liver of rabbits and cultured by the medium RPMI-1640 containing 20 % bovine serum. Any of SDH, LDH and G-6-PDH could be used to estimate whether the culture condition of cells from S.japonicum is appropriate.