间接接触共培养下骨髓间充质干细胞分化为心肌样细胞及相关调控基因的时序表达
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摘要
目的应用新生大鼠心肌细胞(cardiomyocytes,CMs)与骨髓间充质干细胞(mesenchymal stem cells,MSCs)体外间接接触共培养的方法模拟心肌内环境,研究模拟环境下MSCs向CMs的分化情况及相关调控基因的时序表达,筛选MSCs定向分化为心肌细胞的重要调控基因。
方法MSCs及CMs按1:5的比例进行体外间接接触共培养,连续观察两周,相差显微镜下观察MSCs形态变化,免疫荧光方法鉴定心肌特征性肌动蛋白α(α-actin)和肌钙蛋白T(cTnT)的表达,应用半定量RT-PCR分析TGF-β、Nkx-2.5、GATA-4、MEF-2C、TEF-1等相关调控基因在分化过程中的时序表达。
结果共培养前MSCs呈成纤维细胞样,共培养后细胞形态发生变化,细胞体积增大,梭形形态逐渐变短变粗,近似棒状或椭圆形,细胞之间形成连接,排列方向趋于一致。MSCs共培养前及共培养1天、3天未见cTnT及α-actin阳性表达,共培养5天、7天、14天时cTnT阳性细胞百分率分别为7.14%、22.43%、28.77%,α-actin阳性细胞百分率分别为9.20%、22.88%、28.89%。TGF-β、Nkx-2.5、GATA-4和MEF-2C基因在共培养后一天表达开始增强,共培养后七天达高峰,以后虽有所下降但仍维持在较高水平;TEF-1基因在诱导过程中表达无明显变化。
结论间接接触共培养条件下MSCs可分化为心肌样细胞;且在此过程中,TGF-β、Nkx-2.5、GATA-4和MEF-2C基因可能是调控MSCs定向分化为心肌样细胞的重要调控基因。
Objective To investigate transdifferentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes and the temporal expression of regulative genes in the simulated myocardial micro-environment in which MSCs were co-cultured with cardiomyocytes by non– direct contact in vitro and screen some important genes in this differentiation.
Methods MSCs isolated from adult rats were cocultured with CMs obtained from neonatal rat ventricles at 1:5 ratio in a dual chamber dish separated by a semipermeable membrane for 2 weeks. During this non-direct contact coculture procedure,cardiomyogenic differentiation was confirmed by phase contrast microscope and immunostaining againstα-actin and cardiac troponin-T(cTnT), and reverse transcriptase-polymerase chain reaction was performed for the temporal expression of regulative genes TGF-β、Nkx-2.5、GATA-4、MEF-2C and TEF-1.
Results After coculture with CMs, MSCs showed a stick-like or elliptical morphology. Immunofluorescent stain results revealed thatα-actin and cardiac troponin T (cTnT) positive cells were found in MSCs at 5 days after coculture with CMs, and 9.20%、22.88%、28.89% of MSCs were positive forα-actin and 7.14%、22.43%、28.77% for cTnT at 5、7、14 days respectively. Results from RT-PCR demonstrated the expression level of TGF-β、Nkx-2.5、GATA-4 and MEF-2C genes began to increase at 1 day,reached the peak at 7 days after coculture and remained at relatively high afterwards, even though there was a slight drop; TEF-1 gene, on the other hand, had insignificant changes in expression level in our cocultures.
Conclusion Non-direct contact with CMs (1:5) is conducive for MSCs to differentiate into CMs in vitro, and TGF-β, Nkx-2.5, GATA-4 and MEF-2C genes may play a crucial role during the transdifferentiation.
方法MSCs及CMs按1:5的比例进行体外间接接触共培养,连续观察两周,相差显微镜下观察MSCs形态变化,免疫荧光方法鉴定心肌特征性肌动蛋白α(α-actin)和肌钙蛋白T(cTnT)的表达,应用半定量RT-PCR分析TGF-β、Nkx-2.5、GATA-4、MEF-2C、TEF-1等相关调控基因在分化过程中的时序表达。
结果共培养前MSCs呈成纤维细胞样,共培养后细胞形态发生变化,细胞体积增大,梭形形态逐渐变短变粗,近似棒状或椭圆形,细胞之间形成连接,排列方向趋于一致。MSCs共培养前及共培养1天、3天未见cTnT及α-actin阳性表达,共培养5天、7天、14天时cTnT阳性细胞百分率分别为7.14%、22.43%、28.77%,α-actin阳性细胞百分率分别为9.20%、22.88%、28.89%。TGF-β、Nkx-2.5、GATA-4和MEF-2C基因在共培养后一天表达开始增强,共培养后七天达高峰,以后虽有所下降但仍维持在较高水平;TEF-1基因在诱导过程中表达无明显变化。
结论间接接触共培养条件下MSCs可分化为心肌样细胞;且在此过程中,TGF-β、Nkx-2.5、GATA-4和MEF-2C基因可能是调控MSCs定向分化为心肌样细胞的重要调控基因。
Objective To investigate transdifferentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes and the temporal expression of regulative genes in the simulated myocardial micro-environment in which MSCs were co-cultured with cardiomyocytes by non– direct contact in vitro and screen some important genes in this differentiation.
Methods MSCs isolated from adult rats were cocultured with CMs obtained from neonatal rat ventricles at 1:5 ratio in a dual chamber dish separated by a semipermeable membrane for 2 weeks. During this non-direct contact coculture procedure,cardiomyogenic differentiation was confirmed by phase contrast microscope and immunostaining againstα-actin and cardiac troponin-T(cTnT), and reverse transcriptase-polymerase chain reaction was performed for the temporal expression of regulative genes TGF-β、Nkx-2.5、GATA-4、MEF-2C and TEF-1.
Results After coculture with CMs, MSCs showed a stick-like or elliptical morphology. Immunofluorescent stain results revealed thatα-actin and cardiac troponin T (cTnT) positive cells were found in MSCs at 5 days after coculture with CMs, and 9.20%、22.88%、28.89% of MSCs were positive forα-actin and 7.14%、22.43%、28.77% for cTnT at 5、7、14 days respectively. Results from RT-PCR demonstrated the expression level of TGF-β、Nkx-2.5、GATA-4 and MEF-2C genes began to increase at 1 day,reached the peak at 7 days after coculture and remained at relatively high afterwards, even though there was a slight drop; TEF-1 gene, on the other hand, had insignificant changes in expression level in our cocultures.
Conclusion Non-direct contact with CMs (1:5) is conducive for MSCs to differentiate into CMs in vitro, and TGF-β, Nkx-2.5, GATA-4 and MEF-2C genes may play a crucial role during the transdifferentiation.
引文
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