色谱

色谱 ›› 2016, Vol. 34 ›› Issue (4): 414-421.DOI: 10.3724/SP.J.1123.2016.01006

• 研究论文 • 上一篇    下一篇

多层面验证共培养微环境诱导法诱导骨髓间充质干细胞心肌样分化

何林静, 邓伏雪, 胡云凤, 朱卫文, 伍家燕, 常静   

  1. 重庆医科大学附属第一医院心血管内科, 重庆 400016
  • 收稿日期:2016-01-06 出版日期:2016-04-08 发布日期:2012-11-16
  • 通讯作者: 常静
  • 基金资助:

    国家临床重点专科建设项目(2011170).

Feasibility of cardiac differentiation of bone marrow mesenchymal stem cells induced by co-culture indirectly with cardiomyocytes in several dimensions

HE Linjing, DENG Fuxue, HU Yunfeng, ZHU Weiwen, WU Jiayan, CHANG Jing   

  1. Department of Cardiology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
  • Received:2016-01-06 Online:2016-04-08 Published:2012-11-16
  • Supported by:

    National Key Clinical Specialties Construction Program of China (No. 2011170).

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摘要：

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。

关键词: 代谢组学, 共培养微环境, 骨髓间充质干细胞, 心肌细胞, 心肌样分化

Abstract:

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 and α-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 myocardium morphology of MSCs was observed under microscope. The positive immunofluorescence 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, obvious 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. Cardiac differentiation of MSCs can be induced by co-cultured with CMs indirectly in vitro. However, metabolism difference still existed between induced MSCs and CMs.

Key words: bone marrow mesenchymal stem cells (MSCs), cardiomyocytes (CMs), co-culture microenvironment, metabonomics, myocardium-like differentiation

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