Hematology of Department,The Affiliated Hospital of Nantong University
China Postdoctoral Foundation
目的：用CLEC-2 缺陷小鼠检测CLEC-2在自噬过程中的作用,从而探究CLEC-2信号通路对巨核细胞发育的影响。方法：怀孕14.5 d的小鼠胚胎取出,分离并培养巨核细胞。体外雷帕霉素处理巨核细胞,检测巨核细胞CLEC-2和CD41的表达。小鼠经雷帕霉素处理后,检测小鼠骨髓自噬蛋白的表达。结果：自噬基因在胎肝来源的巨核细胞有表达,并且表达量很高。小鼠巨核细胞在雷帕霉素处理后,CLEC-2表达下调,提示CLEC-2参与调控自噬。雷帕霉素处理后,与野生型小鼠相比,敲除小鼠的巨核细胞CD41和CD61蛋白表达明显降低(P<0.001)。体内实验发现雷帕霉素能显著性的增强敲除小鼠LC3蛋白的表达。CLEC-2的缺陷能显著性的降低自噬紊乱导致的多倍体巨核细胞数。CLEC-2的缺陷降低雷帕霉素诱导胎肝来源的巨核细胞周期相关蛋白Cyclin D1,CyclinD2以及P21的表达。结论：血小板受体CLEC-2在巨核细胞自噬中具有重要的调控作用。
Objective: CLEC-2 signaling pathway is involved in the regulation of autophagy in megakaryocytes. However, the mechanism has not been fully elucidated. In this study, CLEC-2 deficient mice were used to detect whether CLEC-2 plays an important role in autophagy. Methods: Megakaryocytes were isolated and cultured from 14.5 day pregnant mouse embryos. The expression of CLEC-2 and CD41 in megakaryocytes were detected. After rapamycin treatment, the expression of autophagy protein was detected. Results: We found that autophagy gene was highly expressed in megakaryocytes derived from fetal liver. After rapamycin treatment, the expression of CLEC-2 was decreased, indicating that CLEC-2 was involved in the regulation of autophagy. Compared with wild-type mice, the expression of CD41 and CD61 in megakaryocytes of rapamycin knockout mice was significantly decreased. In vivo, rapamycin can significantly enhance the expression of LC3 protein in knockout mice. The defect in CLEC-2 can significantly reduce the number of polyploid megakaryocytes caused by autophagy disorder. And it also reduced the expression of cyclin D1, cyclind2 and p21 in megakaryocyte derived from fetal liver induced by rapamycin. Conclusion: CLEC-2 plays an important role in the regulation of autophagy of megakaryocytes.