骨形态发生蛋白2与P53抑制剂PFT-α联合诱导骨髓间充质干细胞分化为心肌样细胞的实验研究  

Bone morphogenetic protein 2 and the p53 inhibitor PFT-α induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells

作　　者：侯兆蕾[1] 吕安林[1] 达晶[1] 侯红[1] 艾世宜[1] 侯婧[1] 刘博武[1] 

机构地区：[1]第四军医大学西京医院心血管内科,西安710032

出　　处：《中华临床医师杂志（电子版）》2013年第6期121-123,共3页Chinese Journal of Clinicians(Electronic Edition)

摘　　要：目的观察骨形态发生蛋白2(BMP-2)与P53抑制剂PFT-α体外联合诱导骨髓间充质干细胞(BMMSCs)定向分化为心肌样细胞的作用。方法采用密度梯度离心法分离大鼠BMMSCs,取第4代BMMSCs分四组诱导:(1)空白对照组,仅用完全培养基诱导;(2)骨形态发生蛋白2组(BMP-2,终浓度10μg/L);(3)P53抑制剂组(PFT-α,终浓度20μmol/L);(4)BMP-2联合PFT-α组(BMP-2+PFT-α,终浓度分别为10μg/L与20μmol/L)。24h后换为普通培养液继续培养4周。用倒置相差显微镜观察细胞的形态学改变,免疫细胞化学法鉴定诱导后心肌样细胞中心肌特异标志物性肌钙蛋白I(cTnI)的表达,流式计数法计算诱导后心肌样细胞诱导率。结果原代BMMSCs10d形成集落,呈星形或梭形。传代后细胞体积变大,诱导4周后细胞呈长梭形,生长趋于一致性,出现肌岛样结构。免疫细胞化学结果显示诱导4周后BMMSCs表达心肌特异性蛋白cTnI。流式计数法结果显示:空白对照组、BMP-2组、PFT-α、BMP-2联合PFT-α组心肌样细胞诱导率分别为(1.4±0.3)%、(17.1±1.2)%、(28.2±1.0)%、(39.9±1.7)%,BMP-2联合PFT-α组心肌样细胞诱导分化率高于BMP-2组与PFT-α组(P<0.01)。结论 BMP-2联合PFT-α可在体外诱导大鼠BMMSCs定向分化为心肌样细胞,其诱导分化率明显高于单纯BMP-2或单纯PFT-α诱导。Objective To observe the effect of bone morphogenetic protein 2 （BMP-2） and the p53 inhibitor PFT-α on the differentiation of bone marrow mesenchymal stem cells （BMMSCs）into cardiomyocyte-like cells.Methods BMMSCs were isolated from bone marrow of SD rats and purified with a density gradient centrifugation method.The forth passage cells were divided into four groups：BMP-2 group（10 μg/L）,PFT-α group（20 μmol/L）,BMP-2（10 μg/L） ＋ PFT-α（20 μmol/L）group and an untreated group as control.After 24 h induction,the medium was changed to complete culture medium without any inductor and the cells cultured for 4 weeks.The morphological changes were observed under inverted phase contrast microscope.The cardiomyogenic cells were identified by immunofluorescence staining.The differentiation ratio was examined by flow cytometer.Results BMMSCs of primary culture formed cell colonies at 10 days.The passaged cells were larger than those of primary culture.After induction,the cells presented long spindle,aligned in parallel and formed＂ muscle island＂-like structure.The expression of specific protein of cardiac troponin Ⅰ （cTnⅠ） in induced BMMSCs was positive.Flow cytometer showed that the differentiation ratio in control group,BMP-2 group,PFT-α group and BMP-2 combined with PFT-α group were respectively（1.4 ± 0.3）％,（17.1 ± 1.2）％,（28.2 ± 1.0）％,（39.9 ± 1.7）％,demonstrating that the differentiation ratio in BMP-2 combined with PFT-α group was higher than that of in BMP-2 group or PFT-α group（P ＜0.01）.Conclusions BMP-2 and PFT-α can induce the differentiation of BMMSCs into cardiomyocyte-like cells,and the differentiation ratio was higher than that of in BMP-2 or PFT-α only.

关 键 词：间质干细胞 骨形态发生蛋白质类 肿瘤抑制蛋白质P53 细胞分化 心肌样细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]