脂肪干细胞原代培养及诱导成肌细胞的实验研究

Primary culture of adipose-derived stem cells and differentiation induction into myoblasts

目的探讨大鼠脂肪干细胞(ADSC)的培养及诱导分化为成肌细胞的方法。方法取SD大鼠脂肪组织,酶消化法分离、培养ADSC,免疫荧光和流式细胞仪检测相关表面抗原CD90、CD105和CD34的表达。取培养至第2代处于对数生长期细胞,分别用含5-氮杂胞苷(5-aza)的诱导培养液(诱导组)和基础培养液(对照组)进行培养。诱导时间为7、14、21、28和35 d,倒置相差显微镜观察细胞的生长情况和形态变化,免疫荧光和流式细胞仪检测成肌细胞特异性抗原desmin和myosin的表达。结果成功从大鼠脂肪组织分离、培养出ADSC,相关表面抗原表达检测证实其干细胞特性。诱导组细胞诱导28 d后呈现成肌细胞特有的"漩涡"样生长形态,单个细胞表现出多核化;免疫荧光和流式细胞仪检测显示,desmin和myosin的表达率在诱导28 d时达最高,分别为52.57%和50.04%,而诱导前和对照组细胞均呈阴性表达。结论成功从大鼠脂肪组织分离、培养ADSC,含5-aza的诱导培养液可将其诱导分化为成肌细胞,诱导28 d成肌细胞特异性抗原表达率最高。

Objective To explore the method of culture of rat adipose-derived stem cells（ADSCs） and differentiation induction into myoblasts. Methods Adipose tissues were obtained from SD rats, and were isolated by enzyme digestion and cultured into ADSCs. The expression of surface antigen CD90, CD105 and CD34 was detected by immunofluorescence and flow cytometry. ADSCs of the second passage with logarithmic growth were obtained, and culture media containing 5-azacytidine （5-aza） and basic culture media were employed for cells in induction group and control group, respectively. The induction lasted for 7 d, 14 d, 21 d, 28 d and 35 d, respectively. Cell growth and cell morphology were observed by inverted phase contrast microscope, and immunofluorescence and flow cytometry were utilized to detect the expression of myoblast specific antigens desmin and myosin. Results ADSCs were successfully isolated and cultured, and were identified to be stem cells. On the 28th day of induction, cells in induction group displayed ＂swirl＂ morpholgy, and muhinucleation was observed. It was revealed by immunofluorescence and flow cytometry that the highest expression rates of desmin and myosin were 52.57% and 50.04%, respectively on the 28th day of induction, while there was no expression before induction and in control group. Conclusion ADSCs can be isolated and cultured from rat adipose tissues, and can further differentiate into myoblasts after induction by culture media with 5-aza. The expression of myoblast specific antigen is the highest on the 28th day of induction.