5-Aza诱导BMSC分化为心肌细胞的分子生物学检测

 【目的】利用分子生物学方法探讨5-氮杂胞苷（5-azacytidine，5-Aza）体外诱导牛骨髓间充质干细胞（bone marrow stromal cells，BMSC）向心肌细胞的分化，为转基因良种肉牛培育提供种子细胞，提高转基因效率。【方法】利用获取的纯化稳定的P4、P8、P12代BMSC各经5、10、15μmol?L-1的5-Aza进行体外诱导分化，统计并计算心肌细胞的转化率，对诱导出现的心肌样细胞进行形态学观察和RT-PCR 鉴定。【结果】诱导后细胞大多呈现梭形，排列方向渐趋一致，有肌管样结构形成。相同代数的BMSC在10μmol?L-1的5-Aza作用下诱导效果最佳;在相同浓度的5-Aza作用下，BMSC向心肌细胞的转化率随着传代次数的增加逐渐降低。RT-PCR显示诱导分化后的细胞明显表达心肌细胞的4种特异性基因，分别是α-心脏肌动蛋白(378bp)、结蛋白（601 bp）、心脏肌钙蛋白T (331 bp)、β-肌球蛋白重链（β-MHC）(273 bp)。【结论】5-Aza体外可诱导牛骨髓间充质干细胞分化为心肌细胞;利用P4代BMSC在10μmol?L-1的5-Aza的作用，诱导效果明显。

Molecular Biology Detection of 5-Aza Inducing BMSC into Cadiocyte

【Objective】 This research aimed to explore 5-Aza inducing BMSC into cadiocyte utilizing molecular biology technology so as to provide seed cells for genetically modified varieties of beef cattle and elevating transgenic efficiency. 【Method】 Test is divided into three groups，they were the P4, P8, P12 BMSC of purification and stability. Each group utilized L-DMEM containing 5, 10, 15 μmol?L-1 5-Aza to undertake respectively derivation，statistics and calculate transformation efficiency of cadiocyte, accredit cadiocyte by observation of morphology and RT-PCR.【Result】 Most cells after differentiation showed fusiform shape, orientating with one accord, forming myotubule-like structure. The inductivity of same passage BMSC was the highest in 10 μmol?L-1 5-Aza role.The conversion rate of BMSC to cadiocyte decreased with the increasing passage number at the same concentration of 5-Aza. RT-PCR results showed that cells after induction clearly expressed cardiac four specific genes, they are α-actin (378 bp), desmin (601 bp), cardiac troponin T (331 bp), β-myosin heavy chain (β -MHC) (273 bp). 【Conclusion】 5-Aza can induce bovine bone marrow mesenchymal stem cells into cadiocyte in vitro. Effect of derivation is obvious utilizing P4 BMSC with 10 μmol?L-1 5-Aza.