小鼠MyoD基因诱导绵羊脐带间充质干细胞为成肌细胞的研究

doi:10.16431/j.cnki.1671-7236.2016.12.022

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (12): 3232-3238.doi: 10.16431/j.cnki.1671-7236.2016.12.022

小鼠MyoD基因诱导绵羊脐带间充质干细胞为成肌细胞的研究

张宝, 马丽媛, 王春生, 杜文敬, 朴善花, 安铁洙

东北林业大学生命科学学院, 哈尔滨 150040

收稿日期:2016-05-09 出版日期:2016-12-20 发布日期:2016-12-22
通讯作者: 安铁洙 E-mail:antiezhu@qq.com
作者简介:张宝(1992-),男,内蒙古呼伦贝尔人,硕士,研究方向:干细胞定向分化,E-mail:zhangbao182@163.com
基金资助:
黑龙江省自然科学基金面上项目（2016012）；国家自然科学基金（31000990）

Study on Differentiation of Sheep Umbilical Cord Mesenchymal Stem Cells into Muscle Cells Induced by Mouse MyoD Gene

ZHANG Bao, MA Li-yuan, WANG Chun-sheng, DU Wen-jing, PIAO Shan-hua, AN Tie-zhu

College of Life Science, Northeast Forestry University, Harbin 150040, China

Received:2016-05-09 Online:2016-12-20 Published:2016-12-22

摘要/Abstract

摘要：

为了探讨小鼠MyoD基因诱导绵羊脐带间充质干细胞（umbilical cord mesenchymal stem cells，UCMSCs）为成肌细胞的可能性，本研究用小鼠MyoD-pcDNA3.1真核表达载体质粒转染绵羊UCMSCs，在观察细胞形态变化的同时，检测成肌细胞标记蛋白表达、表达成肌细胞特异蛋白的细胞比率和成肌细胞特异基因mRNA相对表达量。结果显示，与对照组（未转染）相比，在转染MyoD-pcDNA3.1质粒后第21天，大部分细胞呈现似成肌细胞的细长管状；与对照组未表达相关蛋白相比，转染MyoD-pcDNA3.1后第8天，在荧光倒置显微镜下观察到细胞表达MyoD和Desmin蛋白荧光，转染后第16天，不仅观察到细胞表达MyoD和Desmin，而且观察到MyoG蛋白的表达；对于转染细胞后22 d的细胞进行流式细胞仪检测显示，表达MyoD、MyoG和Desmin的细胞比率分别达93.5%、97.4%和99.5%；此外，实时荧光定量PCR检测显示，转染MyoD-pcDNA3.1后第28天，其细胞中的MyoD、MyoG和Desmin mRNA相对表达量分别提高2.046、2.389和5.489倍。上述结果表明，利用小鼠MyoD构建真核表达载体具有诱导UCMSCs分化为成肌细胞的功效。

关键词: 小鼠; MyoD基因; 绵羊脐带间充质干细胞; 诱导; 成肌细胞

Abstract:

In order to investigate the differentiation of sheep umbilical cord mesenchymal stem cells (UCMSCs) into muscle cells induced by mouse MyoD gene. This study based on the previous work constructed the eukaryotic expression vector of MyoD-pcDNA3.1 in mice, and the vector was transfected into sheep UCMSCs. The morphological changes of cells were observed by fluorescent microsco, the expression of MyoD, Desmin and MyoG genes were detected by immunofluorescence, the percentage of cells expressing the cell specific factor (MyoD, Desmin and MyoG) was analyzed by flow cytometry and Real-time quantitative PCR to detect the relative expression of mRNA relative to muscle cell specific factor. Compared with the control group (no transfection), the vector was transfected into sheep UCMSCs, it was found that the cells were transformed into a long, slender, muscular cell state, and the cell spiral gradually disappeared at 21th day. It was found that MyoD and Desmin showed positive expression by immunofluorescence assay at 8th day, the expression of MyoG was also found after 16 d of induction, and the expression of MyoD decreased, the amount of Desmin expression was no change;By flow cytometry, the percentages of the expression of MyoD, MyoG and Desmin were 93.5%, 97.4% and 99.5%,respectively;Real-time quantitative PCR results showed that the relative expression of MyoD, MyoG and Desmin were increased and compared with the control group (non transfected cells), the cells were increased by 2.046, 2.389 and 5.489 times, respectively. The results showed that mouce MyoD gene could induce the differentiation of sheep UCMSCs into muscle cells.

Key words: mouse; MyoD gene; sheep umbilical cord mesenchymal stem cells; induction; myoblasts

中图分类号:

Q813

张宝, 马丽媛, 王春生, 杜文敬, 朴善花, 安铁洙. 小鼠MyoD基因诱导绵羊脐带间充质干细胞为成肌细胞的研究[J]. 《中国畜牧兽医》, 2016, 43(12): 3232-3238.

ZHANG Bao, MA Li-yuan, WANG Chun-sheng, DU Wen-jing, PIAO Shan-hua, AN Tie-zhu. Study on Differentiation of Sheep Umbilical Cord Mesenchymal Stem Cells into Muscle Cells Induced by Mouse MyoD Gene[J]. , 2016, 43(12): 3232-3238.

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小鼠MyoD基因诱导绵羊脐带间充质干细胞为成肌细胞的研究

Study on Differentiation of Sheep Umbilical Cord Mesenchymal Stem Cells into Muscle Cells Induced by Mouse MyoD Gene

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