敲低PTEN表达对纺锤体正常结构的破坏作用及其相关机制研究

Disruption effect of PTEN knockdown on normal spindle structure and its relative mechanism

目的探讨PTEN干扰对有丝分裂纺锤体形成的影响及其可能机制。方法用慢病毒干扰载体及阴性对照载体转染HeLa细胞后采用qPCR及Westernblot技术检测PTEN mRNA及蛋白表达水平，有丝分裂测定实验镜下观察细胞有丝分裂指数，免疫荧光染色技术观察纺锤体的形态、结构，Westernblot实验检测Eg5蛋白表达变化。结果PTEN shRNA转染HeLa细胞后，PTEN的mRNA及蛋白表达水平均明显下调（P〈0．05）；与对照组相比，shPTEN组细胞的有丝分裂指数明显上调（P〈0．01）；免疫荧光结果显示shControl组中可见正常的纺锤体形成，而shPTEN组中可见形态及结构异常的纺锤体，且纺锤体的长度显著小于对照组（P〈0．01）；驱动蛋白Eg5在shPTEN组中表达明显低于对照组（P〈0．05）。结论PTEN干扰可能通过下调Eg5蛋白的表达水平导致有丝分裂及纺锤体形成异常。

Objective To investigate the effects of PTEN interference on spindle formation and explore the related mechanisms. Methods HeLa cells were transfected with PTEN - specific shRNA and control shRNA. The mRNA and protein level of PTEN were detected by qPCR and Western blot respectively. The mitotic index of different groups were e- valuated under microscope. The morphology and structure of spindle were analyzed by immunofluorescence. The expres- sion of Eg5 was detected by Western blot. Results After transfection of PTEN - specific shRNA, the mRNA and protein level of PTEN were suppressed markedly （P 〈 0.05）. Compared with control group, the mitotic index in shPTEN group was significantly increased （P 〈 0.01 ）. The immunofluorescence results showed normal spindle in shControl group, but in shPTEN group, there were many spindles with abnormal morphology and structure. The spindle length in shPTEN group was much shorter than that in shControl group （P 〈 0.01 ）. The protein expression of Eg5 was markedly decreased in shPTEN group compared with shControl group. Conclusions PTEN interference may lead to abnormal mitosis and spindle formation by downregulating EgS.