人类心肌匀浆上清液对骨髓间充质干细胞诱导分化及相关基因时序表达的影响

Effects of human myocardial extracts on transdifferentiation of bone marrow mesenchymal stem cells and temporal expression of related genes

背景:大量有关动物的体内外研究表明,骨髓间充质干细胞在心肌微环境或模拟心肌微环境的作用下可分化为心肌细胞。然而,类似的人类研究报道较少,同时相关的分化机制也不甚清楚。目的:探讨人类心肌匀浆上清液对骨髓间充质干细胞诱导分化的影响及相关基因的时序表达。方法:体外分离培养人类骨髓间充质干细胞,传至第4代加入自体心肌匀浆上清液诱导骨髓间充质干细胞,持续作用2周。相差显微镜下观察骨髓间充质干细胞形态变化,免疫荧光方法鉴定心肌特征性肌动蛋白α和肌钙蛋白的表达,应用半定量RT-PCR技术分析Nkx-2.5、GATA-4、MEF-2C、α-MHC、肌钙蛋白等相关基因在分化过程中的动态时序表达。结果与结论:经心肌匀浆上清液诱导后,骨髓间充质干细胞体积变大,立体感增强,形态近似棒状。诱导2周时肌动蛋白α及肌钙蛋白阳性细胞比例分别为25.53%和23.48%。Nkx-2.5、GATA-4、MEF-2C基因在诱导后3d表达开始增强,其中GATA-4、MEF-2C诱导后5d达高峰,以后维持在较高水平,Nkx-2.5则随诱导时间逐渐增强;α-MHC、肌钙蛋白基因诱导前无表达,于诱导第3天出现表达,以后维持在高水平。结果提示,心肌匀浆上清液对骨髓间充质干细胞具有定向诱导作用,在此过程中,伴随着Nkx-2.5、GATA-4、MEF-2C、α-MHC和肌钙蛋白基因的时序表达变化。

BACKGROUND：Many studies on animals have suggested that bone marrow mesenchymal stem cells （BMSCs） have the potential to transdifferentiate into cardiomyocytes （CMs） within the myocardial environment both in vivo and in vitro.However,few studies in humans are reported in this field and the molecular signals that underlie this process are not fully understood.OBJECTIVE：To investigate the effects of human myocardial extracts on transdifferentiation of BMSCs and temporal expression of related genes.METHODS：Human BMSCs were harvested in vitro.Human BMSCs from passage 4 were induced by high level of autologous myocardial extracts for 2 weeks.Cardiomyogenic differentiation was confirmed by phase contrast microscope and immunostaining against α-actin and cardiac troponin-T （cTnT）,and reverse transcriptase-polymerase chain reaction （RT-PCR） was performed for temporal expression of related genes Nkx-2.5,GATA-4,MEF-2C,α-MHC and cTnT.RESULTS AND CONCLUSION：After induced by myocardial extracts,BMSCs showed a stick-like morphology,with a greater volume and stronger three-dimensional sense.25.53% of BMSCs were positive for α-actin and 23.48% for cTnT at 2 weeks.The expression level of Nkx-2.5,GATA-4 and MEF-2C genes began to increase at 3 days,the expression level of GATA-4 and MEF-2C genes reached a peak at 5 days after induction and remained at relatively high level afterwards,and the expression level of Nkx-2.5 was gradually increased with the induction time.The expression of α-MHC and cTnT genes,which were not detected before induction,began to emerge at 3 days after induction and remained at high level afterwards.Therefore,myocardial extracts are conducive for BMSCs to differentiate into CMs in vitro,along with the temporal expression changes of Nkx-2.5,GATA-4,MEF-2C,α-MHC and cTnT genes during the transdifferentiation.