P19细胞心肌体外分化诱导条件的优化

Optimization for differentiation of P19 cells to cardiomyocytes in vitro

目的 分别观察P19细胞心肌分化诱导中的几种影响因素，优化P19细胞心肌分化的诱导条件。方法使用悬滴法或96孔板悬浮培养法制备细胞聚集体（aggregates），二甲基亚砜（DMSO）为诱导剂诱导P19细胞的心肌分化。普通光学显微镜下观察跳动的心肌细胞的产生来确定诱导是否成功，免疫荧光染色确认心肌特异性蛋白Troponin T的表达，RT-PCR方法检测分化诱导后心肌细胞标志基因的表达情况。结果使用96孔板悬浮培养法诱导细胞形成聚集体后转入贴壁培养阶段时细胞聚集体贴壁状态优于传统的悬滴法。DMSO的加入可以提高细胞形成聚集体后贴壁时细胞聚集体的完整性。1×10^7～2×10^8L^-1的接种浓度下心肌细胞分化诱导效率较高。在可观察到跳动的心肌细胞之前即可检测到Troponin T的表达。结论通过对细胞形成聚集体的诱导方法、诱导剂、接种浓度等因素的优化可以提高P19细胞的心肌分化效率，为开发高效心肌分化诱导法提供了条件。

Objective Evaluating important factors which have effects on cardiomyocyte differentiation and to optimize differentiation efficiency. Methods Hanging drop method or 96-well plate method were used to produce aggregates. DMSO was used to induce cardiomyocyte differentiation of P19 cells. Beating cells were detected with microscopy and cardiomyocyte specific protein Troponin T was examined by immunostaining. RT-PCR was applied to evaluate the expression of cardiomyocyte related genes. Results Compared with Hanging drop method, 96-well plate method facilitated aggregate formation and adhesion. Adding DMSO improved aggregates integrity. Seeding concentration of 1 ×10^7- 2 × 10^8L^-1 caused the most effective differentiation. Troponin T immunostaining was observed before cells started beating. Conclusion By optimizing the method for aggregates formation, differentiation inducing chemicals and cell seeding concentration, it is possible to improve cardiomyocyte differentiation effect of P19 cells. This might provide us a way for cardiomyocyte differentiation with high efficiency.