RSPO1基因转染间充质干细胞株的构建及其促进肠隐窝的生存和增殖作用研究

Establishment of RSPO1 gene transfected mesenchymal stem cell line and its role in promoting survival and proliferation of intestinal crypts

目的构建表达RSPO1的间充质干细胞株,并检测其在肠上皮生存和更新中作用。方法将表达人RSPO1的重组表达载体p EGZ-TermR/RSPO1及辅助病毒p HIT456和p HIT60共转染293T细胞,收取上清后感染小鼠细胞系C3H10 T1/2,而后经G418抗性筛选、细胞内细胞因子染色和流式细胞术鉴定获得稳定表达RSPO1基因的细胞株;通过体外肠隐窝培养体系,观察其对小鼠肠隐窝的生存和增殖的影响。结果成功建立了稳定表达RSPO1的间充质干细胞株,体外肠隐窝培养实验证实该细胞株可维持肠隐窝存活并促进其增殖。结论成功建立了稳定表达RSPO1的基因转染细胞株,为探讨修复肠上皮策略提供了有力工具。

Objective To establish a RSPO1 gene transfected mesenchymal stem cell line,and evaluate its biological effect on the survival and regeneration of intestinal epithelium. Methods The recombinant retroviral expressing vector p EGZ-TermR/RSPO1,which expressed human RSPO1,was co-transfected into the 293 T cells together with the helper virus vectors p HIT456 and p HIT60. The supernatant of the transfected 293 T cells was harvested to infect C3 H10 T1/2 cells. The G418 resistance screen,intracellular cytokine staining and flow cytometric assay were performed to identify the RSPO1 gene transfected cells. Its effect on the survival and proliferation of mouse intestinal crypt was observed by in vitro culture system. Results A stable RSPO1 gene transfected mesenchymal stem cell line was constructed. Its supernatant could promote mouse intestinal crypt survival and proliferation. Conclusion A stable RSPO1 gene transfected mesenchymal stem cell line was established. It provides a useful tool to rescue the intestinal epithelium injury.