建立猪囊尾蚴细胞系的研究

Study on Establishing Cell Lines of Cysticercus cellulosae

为建立具有免疫原性的猪囊尾蚴细胞系,试验对猪囊尾蚴细胞进行了体外培养,并将建立的细胞系命名为猪囊尾蚴CC-97免疫细胞系。猪囊尾蚴细胞原代培养30 d后形成单层细胞,然后将单层细胞以1∶2分种率传代培养,相隔7 d传代一次,连传24代。结果表明:光镜下观察发现,细胞系由3种类型细胞组成,以椭圆形细胞占优势,梨形、球形细胞数量接近。放射自显影法测定细胞分裂情况,3H-TdR显示细胞生长指数,结果均表明,细胞对数增殖期长达7 d,细胞从12 h开始倍增分裂,第四、五天相继达到倍增分裂高峰,细胞增殖率达11倍,增量达6.32×109/L,后来达到1∶37的分种率,细胞数为3.7×1010/L;细胞周期约为32 h,细胞系以二倍体细胞为主,染色体有10对20条,细胞蛋白质组分有30个区带,酯酶同工酶谱显示一条区带,分子量约32 kDa;细胞系经荧光抗体检测表明,与猪囊尾蚴具有同源性,致肿瘤性与外源污染检验均为阴性。结果提示,建立的猪囊尾蚴细胞系是一个形态均一、生长旺盛、遗传性稳定、免疫原性高、无污染、无致肿瘤性、与猪囊尾蚴同源的生物学新品系。

CC-97 cell lines of Cysticercus cellulosae with immunogenicity were established through culturing Cysticercus cellulosae cells in vitro. After culan＇ing original generation cells for 30 days, primary monolayer cells were got. Then the monolayer cells were subcultured at 1 ： 2 split ratio, and the next generation was subcultured every 7 days, totally, the cell lines were subcultured suecesively for 24 generations. The cell line consisted of three cell types, the oval, the pear-shaped and the spherical cells under optical microscope. Among the cells, the oval cells were much more than others, while the number of pear-shaped cells and the number of spherical cells were nearly equal. Autoradiography method was used to detect cell division and 3H-TdR was used to analyze cell growth index, the two approaches demonstrated that the cell logarithmic proliferation phase was 7 days, the cell started to proliferate after culturing 12 hours and reached the proliferation peak at the 4th or 5th day during the culturing periods, and the proliferation rate reached 11 times, the amount of increment was 6.32 X 109/L, the increment eventually reached the speed of 1 ： 37 split ratio and the number of cells was 3.7 X 10^（10）/L. The total length of cell cycle was about 32 hours, the cell line mainly consisted of diploid cells, the number of chromosomes was 10 pairs and each pair has 2 chromatids. There were 30 fractions of protein components, the analysis with esterase lsozymes showed a fragment with molecular weight around 32 kDa. Fluorescent antibody test showed that the cell line was homologous with Cysticercus cellulosae, the test results of tumorigenicity and exogenous contamination were negative. These results indicate that Cysticercus cellulosae cell line established in this research is a new biological strain of the homolog of Cysticercus cellulosae with uniform morphology, vigorous growth, stable hereditary, high immunogenicity, non-contamination and non-tumorigeniciW.