Comparison of mRNA expression of transcriptional factors and intercalated disk constituent proteins between in vivo and cultured cardiomyocytes
Comparison of mRNA expression of transcriptional factors and intercalated disk constituent proteins between in vivo and cultured cardiomyocytes
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DOI：
10.1007/s10047-008-0414-7
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发表时间：
2008-09-01
影响因子：
1.3
通讯作者：
Umezu, Mitsuo
中科院分区：
文献类型：
--
作者：
Nakamura, Takao;Feng, Zhonggang;Umezu, Mitsuo
尽管组织工程心肌在过去的十年里取得了很大的进展，但组织工程心肌的收缩力仍然很弱，这是组织工程心肌研究中的一个大问题。我们认为，收缩力弱的一个主要原因是调节心肌细胞分化和心脏组织合胞体的基因表达可能在体内和培养的细胞中不同。本研究通过比较体内和体外培养条件下心肌细胞mRNA表达的差异，为心肌组织构建过程中进一步的基因转移治疗寻找靶点。为此，四个主要的转录因子（SRF，p300，Nkx2.5，和myocardin）和两个闰盘组成蛋白（N-cadherin和connexin 43）在大鼠心肌细胞的mRNA表达测定通过比率逆转录聚合酶链反应。从18天（出生前约3天）的Wistar大鼠胚胎的心脏（胚胎细胞）、12天的新生大鼠心脏（新生细胞）或从18天胚胎收集的胚胎细胞（培养细胞）的14天连续培养皿培养物中收集心肌细胞。结果表明，除SRF外，其他mRNA在培养细胞中的表达均低于胚胎细胞和新生细胞，尤其是myocardin、Ncadherin和connexin 43的mRNA表达明显低于新生细胞。因此，myocardin是在构建工程化心脏组织过程中强制基因上调表达的候选者;此外，工程化心脏组织的弱收缩力的一个合理原因是闰盘的弱构成，因为已经阐明，与闰盘相关的蛋白质的mRNA表达在培养中较低。
The weak contractile force exerted by engineered cardiac muscle is a big problem in cardiac muscle tissue engineering, even though the field has made great progress over the past decade. We believe that one major reason for the weak contractile force is that the expression of genes regulating cardiomyocyte differentiation and cardiac tissue syncytium may be different for in vivo and cultured cells. In the present study, we investigated the difference of mRNA expression under in vivo and culture conditions in order to seek a target for further gene transfer treatment in the process of cardiac tissue construction. To this end, mRNA expression of four major transcriptional factors (SRF, p300, Nkx2.5, and myocardin) and two intercalated disk constituent proteins (N-cadherin and connexin43) in rat cardiomyocytes was measured by means of ratiometric reverse-transcription polymerase chain reaction. Cardiomyocytes were harvested from the hearts of 18-day (about 3 days before birth) Wistar-rat embryos (embryonic cells), 12-day neonatal rat hearts (neonatal cells), or 14-day successive dish culture of the embryonic cells harvested from 18-day embryos (cultured cells). The results indicated that, except for SRF, the mRNAs had a lower expression tendency in cultured cells than in embryonic and in neonatal cells; in particular, the mRNA expression of myocardin, Ncadherin, and connexin43 of cultured cardiomyocytes was significantly lower than that of neonatal cells. Therefore, myocardin is a candidate for forced gene up-expression during the construction of engineered cardiac tissue; in addition, a plausible reason for the weak contractile force of engineered cardiac tissue is the weak constitution of intercalated disk, because it was elucidated that mRNA expression of proteins related to intercalated disk were lower in culture.