MNNG诱导日本血吸虫成虫培养细胞的扫描电镜观察

SCANNING ELECTRON MICROSCOPICAL OBSERVATION ON THE CULTURED CELLS FROM ADULT SCHISTOSOMA JAPONICUM INDUCED BY MNNG

目的 观察甲基硝基亚硝基胍 (MNNG)对不同培养时间日本血吸虫成虫培养细胞作用的变化 ,确定培养细胞发生转化进而增殖的最佳诱导时间。 方法 将 2 3～ 2 8d虫龄的日本血吸虫成虫制成细胞悬液 ,接种于小盖玻片上 ,培养于含 2 0 %小牛血清及常量抗生素的RPMI 164 0常规培养基中。将接种培养第 4、5、6、7、8d的细胞分别设为 5个实验组及其相应的对照组。实验组用含终浓度为 3 μg/mlMNNG的常规培养基处理 48h ,对照组用不含MNNG的常规培养基处理相同时间 ,嗣后换用常规培养基培养 4周 ,再改用含 5 %小牛血清的培养基继续培养。MNNG诱导后每周取各组培养细胞固定、制样 ,每组随机取 3张小盖玻片进行电镜扫描观察 ,连续取样 11周。 结果 经MNNG诱导的日本血吸虫成虫培养细胞表面出现形态各异的结构 ,既有与对照组类似的表面较光滑和有乳突的培养细胞 ,也有表面光滑如玻璃珠状的细胞 ,还有具长纤突、微嵴、皱褶、微绒毛、蜂窝和仙人球状等形态的细胞 ;实验 1组培养至第 5周即出现大量分裂细胞。 结论 MNNG诱导培养第 4d的日本血吸虫成虫细胞培养至第 5周可发生转化而出现分裂、增殖。

Objective To observe the effects of N-Methy-N-Nitro-Nitrosoguanidine (MNNG) on the cultured cells from adult Schistosoma japonicum in different culture time by scanning electron microscopy (SEM), so as to determine the optimal period of time for MNNG to induce transformation and reproduction of the cultured cells. Methods The cells from the adult S. japonicum of 23～28-day old were inoculated on small coverslips and cultured in a routine medium RPMI-1640 containing 20% calf serum and a moderate amount of antibiotics. Cultures of 4, 5, 6, 7, 8 days were divided into 5 test and their corresponding control groups and treated with a routine medium containing MNNG in concentration of 3 μg/ml and 0 μg/ml for 48 hours, respectively. Afterwards, they were transferred into the routine medium. 4 weeks later, the percentage of calf serum in the medium was adjusted into to 5%. 3 of the coverslips with cultured cells were sampled and prepared for SEM once a group and week until the eleventh week after inducement. Results The cultured cells from adult S. japonicum treated by MNNG showed many different characteristics. On the surface of most cells, there were a few papilla-like tubercula or nothing, which were similar to the cells in the control groups. However, some cultured cells in the test groups showed different surface features, e.g. the surface were very slick, honeycomb-shaped or cactus-like, or with long filopodium, microridges, ruga, and many microvilli. In the test group 1, many divisive cells were observed in the fifth week. Among them, there were dichotomous and multipolar divisive cells. The multipolar divisive cells were equal or unequal. Conclusion Transformation and proliferation of the cultured cells from adult S. japonicum can be induced by MNNG. (For figures, to see inserts 13 and 14)