摘要
目的：研究体外不同诱导条件下大鼠骨髓间充质干细胞(MSCs)向心肌细胞分化的潜能。方法：取Wistar大鼠股骨和胫骨骨髓,分离培养MsCs,采用第二代或第三代MScs,以5-氮杂胞苷(5-aza)、碱性成纤维细胞生长因子(bFGF)及两者联合作用,作为分化诱导剂,连续观察三周,相差显微镜下观察其形态变化。免疫细胞化学方法鉴定心肌特异性蛋白T(Troponin T,cT- nT)、连接蛋白43(Connexin43)、α-横纹肌动蛋白(α-Sarcomeric Actin)的表达,应用半定量RT-PCR技术分析Nkx2．5、GATA-4、TGF-?等相关调控基因在分化过程中的表达。结果：免疫细胞化学显示cTnT、Connexin43、α-Sarcomcric Actin诱导前无表达,单纯bFGF诱导组及对照组未发现cTnT、Comexln43、α-Sarcomeric Actin染色阳性细胞,单纯5-aza诱导组诱导后上述三种蛋白阳性细胞表达比例分别为22%、28%、32%,5-aza与bFGF联合诱导组诱导后上述三者阳性细胞表达比例分别为28%、33%、40%,联合诱导组诱导后细胞阳性率明显高于单纯5-aza诱导组,两组相比较差异有显著性意义(P＜0．05)。RT-PCR检测结果显示,GATA-4、Nkx2．5、TGF-β在诱导前的MSCs有低表达,单纯5-aza诱导组及5-aza与bFGF联合诱导组诱导后3周,这三种基因有较强的表达,单纯5-aza诱导组诱导检测结果显示,GATA-4、Nkx2．5、TGF-β在诱导前的MSCs有低表达,单纯5-aza诱导组及5-aza与bFGF联合诱导组诱导后3周,这三种基因有较强的表达,单纯5-aza诱导组诱导前后相比较,差异有显著性意义(P＜0．05),5-aza与bFGF联合诱导组诱导前后相比较,差异也有显著性意义(P＜0．05),单纯5-aza诱导组与5-aza和bFGF联合诱导组诱导后两组之间相比较,差异亦有显著性意义(P＜0．05),表明单纯5-aza诱导及5-aza与bFGF联合诱导均可使MSCs向心肌细胞转化,联合诱导可以使更多的MSCs向心肌细胞方向转化。单纯bFGF诱导组诱导前后无显著性差异。结论：5-aza及bFGF联合诱导,可以作为更好的促进MSCs向心肌细胞分化的条件。
Objective： To evaluate, under the different inducing conditions, tire potentiality of bone marrow mesenchymal stem cells （MSCs） differentiating into cardiomyocytes in vitro. Methods： Wistar rat MSCs were isolated and cultured from rat bone marrow. MSCs derived from the second and the third passages were cultured for 21 days. During the inducing process, 5 - azacytidine, base fibroblast growth and 5 - aza ＋ bFGF were used as inducing factors. The MSCs differentiation was observed under phase - contrast microscope. Tile immunohistochemical technique was used for the expression of Troponin T（cTnT）, Counexin43, anda- Sarcomeric Actin. The expression of Nkx2.5, GATA- 4 and TGF - β were examined by semi - quantitative PCR. Results： MSCs showed spindle - hke morphology, but the shape and arrangement changed after induction. The 5 - aza group and 5 - aza ＋ bFGF group induced MSCs to change their morphology to that of cardiomyocyte type. The cTnT, Connexin43, and α - Sarcomeric Actin had no expression before induction and after the induction of bFGF. The rate of positive cells after 5 - aza ＋ bFGF induction was higher than the induction of 5 - aza（P 〈 0.05）. The expression levels of Nkx2.5, GATA - 4 and TGF - β genes after the induction of 5 - aza and 5 - aza＋ bFGF were significantly higher than that before induction and the induction of bFGF（P 〈 0.05）. The expression levels of these genes after induction of 5 - aza ＋ bFGF were higher than that of 5 - aza. Conclusion： The effect of 5 - aza plus bFGF is the better factor in inducing MSCs to differentiate into cardiomyocytes.
出处
《现代生物医学进展》
CAS
2006年第9期27-30,F0003,共5页
Progress in Modern Biomedicine
基金
黑龙江省科技计划项目(GC04C30103)
