兴义维蚋蚋卵(胚胎)原代细胞培养及鉴定

Culture of primary cells of eggs(embryos) in Simulium(Wilhelmia) xingyiense and its identification

【目的】筛选出最适宜兴义维蚋蚋卵（胚胎）原代细胞的培养条件和最佳培养基组合，并对其原代细胞进行鉴定，为蚋类细胞系的建立打下基础。【方法】采用组织块和单细胞法对兴义维蚋蚋卵（胚胎）进行原代细胞培养，分析培养基、温度、pH、CO2和培养器皿对细胞培养的影响；并测定蚋卵（胚胎）及原代细胞的rDNA-ITS区序列，通过构建基于ITS区序列的蚋类系统发育进化树鉴定其种属来源。【结果】兴义维蚋蚋卵（胚胎）原代细胞最适宜的培养方法为组织块法，培养基为改良型Shields and Sang M3，pH 6.0-6.5，培养温度28℃，恒温生化培养箱和5% CO2培养箱对原代细胞培养无明显差异，培养器皿为经多聚赖氨酸（PLL）处理的一次性塑料培养皿/板。由基于ITS区序列构建的蚋类系统发育进化树可知，蚋卵（胚胎）及其原代培养细胞均来源于兴义维蚋。【结论】成功建立的兴义维蚋蚋卵（胚胎）原代细胞培养方法可在科研生产中进一步推广应用。

Objective]The present study was conducted to select the most suitable culture conditions and optimal cul-ture media for culturing primary cells of eggs（embryos） in Simulium（Wilhelmia） xingyiense, and identify the primary cells in order to lay a foundation for establishment of blackfly cell lines. [Method]Primary cells of eggs（embryos） in S.（W.） xingyiense were cultured using tissue block method and single cell method. The influence factors for cell culture were ana-lyzed, including medium, temperature, pH value, CO2 and culture vessels. At the same time, the sequences of rDNA-ITS region of eggs（embryos） and primary cells were determined, and blackfly phylogenetic tree was established based on their ITS sequences to identify their species origin . [Result]The tissue block method was the optimal culture method for culturing the primary cells of eggs（embryos） in S.（W.） xingyiense. The appropriate culture medium was modified Shields and Sang M3 with pH 6.0-6.5 and 28℃culture temperature. Constant temperature biochemical incubator and 5%CO2 in-cubator made no significant differences on culturing primary cells. Poly-L-lysine（PLL） treated plastic petri dishes/plates could be used as culture vessels. The result of blackfly phylogenetic tree based on ITS sequences showed that blackfly eggs （embryos） and their primary cells were derived from S.（W.） xingyiense. [Conclusion]The culture method for primary cells of eggs（embryos） in S.（W.） xingyiense successfully established can be further applied in scientific research and production.