心肌细胞裂解液对骨髓间充质干细胞向心肌细胞分化诱导作用的研究

目的通过向骨髓间充质干细胞(MSCs)培养体系中添加心肌细胞裂解液的方法,体外模拟心肌微环境,观察MSCs向心肌细胞分化的诱导作用,并与诱导分化剂5-氮杂胞苷(5-aza)比较。方法分离新生乳鼠的心肌细胞并制成心肌细胞裂解液,自成年大鼠骨髓中分离MSCs,用含有心肌细胞裂解液的培养基(A组)、含有5-aza的培养基(B组)、含有5-aza和心肌细胞裂解液的培养基(c组)以及普通培养基(对照组)培养。观察细胞形态的改变,并通过免疫组化分析分化后细胞表达α-肌动蛋白、心脏特异性肌钙蛋白T(cTnT)、连接蛋白43及CD31的情况。结果A、B组的MSCs在培养1周后均形成肌细胞形态,并且均表达α-肌动蛋白和cTnT;A组MSCs分化的肌样细胞所含的肌纤维较B组更丰实,细胞生长趋势也优于B组,并且可以表达CD31;B组MSCs分化的肌样细胞不表达CD31;对照组细胞仅表达α-肌动蛋白。结论心肌细胞裂解液是体外诱导MSCs分化为心肌样细胞的理想条件,优于传统的5-aza,在心肌细胞移植技术中可以用于体外模拟心肌细胞微环境。

Differentiation of mesenchymal stem cells into cardio myogenic cells under the induction of myocardial cell lysate

OBJECTIVE: In order to understand the effects of cardiac microenvironment on the differentiation of bone marrow mesenchymal stem cells (MSCs) into myocardial-like cells, we simulated the cardiac microenvironment in vitro by adding myocardial cell lysate into the culture system of MSCs, and compared the differentiation promoting effect of myocardial cell lysate with that of well-established inducer 5-azacytidine (5-aza). METHODS: Myocardial cells isolated from newly born rats were lysed by repeat freezing and defrosting. MSCs isolated from adult rat were cultured in four different systems. Medium A: medium with myocardial cell lysate; medium B: medium with 5-aza; medium C: medium with 5-aza and myocardial cell lysate; and control medium: ordinary medium without any addition reagent. The dynamic changes of MSCs morphology in different media were observed within 7 days after introduction of MSCs. Immunohistochemical staining against alpha-actin, cTnT, Connexin43 and CD31 were performed at the end of cultivation. RESULTS: MSCs in both medium A and B were differentiated into myocardial-like cells expressing alpha-actin and cTnT after 7-day cultivation. Cells in medium A developed more myofilaments than those in medium B, and expressed CD31, whereas cells in medium B did not. MSCs in control medium only expressed alpha-actin. CONCLUSIONS: Myocardial cell lysate is an ideal inducer to differentiate MSCs into myocardial-like cells in vitro. The differentiation promoting effect of myocardial cell lysate is more predominant than that of 5-aza.