为了多层面探讨共培养微环境诱导法定向诱导骨髓间充质干细胞（ MSCs）心肌样分化的可行性，取第3代MSCs与原代心肌细胞（ CMs）进行共培养。在显微镜下观察诱导1周后的 MSCs 形态学变化，用免疫荧光和实时荧光定量聚合酶链式反应（ RT-PCR）分别检测诱导的 MSCs 中心肌肌钙蛋白 I（ cTnI）、α-肌动蛋白（α-actin）、Nkx-2.5和 GATA-4的基因表达变化情况。采用超高效液相色谱-串联质谱（ UPLC-MS／MS）分别检测诱导组和对照组的代谢产物。诱导1周后的 MSCs形态呈心肌样改变，cTnI、α-actin、Nkx-2.5和 GATA-4的基因表达均明显升高，正交偏最小二乘法判别分析（ OPLS-DA）模型显示诱导的 MSCs 代谢物向 CMs 转变趋势明显。通过多元和单元统计分析筛选差异变量，根据一级质谱和二级质谱比对结果，最终确定12种差异代谢物。与未经诱导的MSCs 相比，经诱导的MSCs与CMs中变化趋势相同的差异代谢物有7种，变化趋势不同的差异代谢物有5种。实验结果表明，无论从形态、基因、蛋白质还是代谢层面看，MSCs 通过与 CMs 间接接触共培养后均发生了心肌样改变，但是与 CMs仍存在差异。
The objective of this research is to investigate the feasibility of cardiac differentiation of bone marrow mesenchymal stem cells( MSCs)by co-culture with cardiomyocytes( CMs)in vitro. The third generation of MSCs from bone marrow and CMs were co-cultured indirectly in a transwell. One week later,the expressions of muscle-specific markers( cardiac troponin I andα-actin) by immunofluorescence staining and the gene expressions of transcription factors ( Nkx-2. 5 and GATA-4 ) were measured by real-time polymerase chain reaction ( RT-PCR ). Then,orthogonal partial least squares discriminant analysis( OPLS-DA)models were employed to confirm the difference among MSCs,induced MSCs and CMs by ultra-performance liquid chromatography-tandem mass spectrometry( UPLC-MS/MS)analysis. The distinctive changes were identified by multivariate analysis and variate analysis,and the changed metabolites were identified by MS and MS/MS. One week after co-cultured with CMs indirectly,the specific myo-cardium morphology of MSCs was observed under microscope. The positive immunofluores-cence stainings against cTnI and α-actin were detected,and the positive expressions of the transcription factors Nkx-2. 5 and GATA-4 were measured by RT-PCR. In OPLS-DA mode,obvi-ous trend of cardiac differentiation of MSCs can be seen,and seven metabolites were tested both in induced MSCs and CMs,but five metabolites were tested in induced MSCs or CMs. Car-diac differentiation of MSCs can be induced by co-cultured with CMs indirectly in vitro. However,metabolism difference still existed between induced MSCs and CMs.
参考文献
| [1] | Tanaka,M.;Taketomi,K.;Yonemitsu,Y..Therapeutic angiogenesis: Recent and future prospects of gene therapy in peripheral artery disease[J].Current gene therapy,20144(4):300-308. |
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