MicroRNA-133诱导绵羊间充质干细胞分化为成肌细胞的研究

发布时间：2018-05-31 14:33

本文选题：miR-133 + 绵羊　；参考：《东北林业大学》2015年硕士论文

【摘要】：已有研究证实,miR-133在肌肉发育中起到非常重要的作用。间充质干细胞是具有自我更新和多向分化潜能的细胞。为了探明利用miR-133诱导绵羊脐带和骨髓间充质干细胞为成肌细胞的方法,本研究采用脂质体转染法将肌肉特异表达的miR-133转染到绵羊脐带、骨髓间充质干细胞后,检测其诱导为成肌细胞的能力。其研究结果如下：1.绵羊脐带、骨懿间充质干细胞的转染采用阳离子脂质体转染法将-miR-133a2表达载体或：niR-133mimics分别转染绵羊脐带间充质和骨髓间充质干细胞；2.转染细胞培养与形态观察将上述转染细胞进行续培养,培养的第28d,在倒置荧光显微镜下细胞呈现梭形或纺锤形；当细胞约达到80%汇合时,细胞由涡旋状生长变成定向有序的排列生长,并且邻近细胞间出现融合倾向；3.转染细胞的鉴定(1)成肌细胞特异表达基因的qRT-PCR检测采用qRT-PCR方法检测上述细胞骨骼肌特异基因MyoD、MyoG、Desmin、Tnni2、Myf5表达情况。在转染前未检测到MyoD和MyoG表达,转染后可以检测到MyoD和MyoG的表达；Desmin、Tnni2、Myf5在转染后相对表达量增加。此外,与绵羊骨髓间充质干细胞相比,绵羊脐带间充质干细胞在诱导后Dismin、Tnni2、Myf5相对表达量增加更多；(2)成肌细胞特异表达基因的免疫荧光检测利用Desmin目应α-actin抗体对转染细胞进行免疫荧光检测,其结果,与对照组(未转染细胞)在镜下未检测到相应基因发出荧光相比,4组转染细胞中,和MyoD蛋白检测结果均呈现阳性,Desmin而呈现阴性；α-actin(3)成肌细胞特异表达基因的流式细胞检测当将表达载体pcDNA3.1-miR-133a2分别转染绵羊脐带和骨髓间充质干细胞后,采用流式细胞分析技术检测成肌细胞特异蛋白和MyoD、Desmin其结果,在绵羊脐带、骨髓间充质干细胞中,表达α-actin,的细胞量分别为12.2%和29.6%,表达MyoD的细胞量分别为99.3%和99.5%,表达α-Desminact1n的细胞量分别为0.917%和9.24%；当将分别转染绵羊脐带和骨髓miR-133mimics间充质干细胞后,在这些细胞中检测到表达的细胞量分别为99.3%和43.7%,表MyoD达的细胞量分别为99.0%和100%,表达Desmin的细胞量分别为1.07%和α-actin1.68%：上述结果表明,利用miR-133成功诱导绵羊脐带间充质干细胞和绵羊骨髓间充质干细胞为成肌细胞,本研究结果为进一步探讨诱导间充质细胞为成肌细胞和骨骼肌细胞分化机制提供了依据。
[Abstract]:Studies have shown that miR-133 plays a very important role in muscle development. Mesenchymal stem cells are cells with self-renewal and multi-differentiation potential. In order to find out how to induce sheep umbilical cord and bone marrow mesenchymal stem cells to be myoblasts by miR-133, we used liposome transfection method to transfect muscle-specific miR-133 into sheep umbilical cord and bone marrow mesenchymal stem cells. The ability of inducing myoblast was tested. The results are as follows: 1. The transfection of sheep umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells was carried out by cationic liposome transfection of -miR-133a2 expression vector or the expression vector: 1 / niR-133mimics into sheep umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells respectively. The transfected cells were cultured with morphological observation. The cells were fusiform or fusiform under inverted fluorescence microscope on the 28th day of culture, and when the cells reached about 80% confluence, The cells changed from vortex growth to directional ordered growth, and fusion tendency was found between adjacent cells. The expression of myoblast specific gene MyoDnni2Tnni2mf5 was detected by qRT-PCR method. No expression of MyoD and MyoG was detected before transfection, and the expression of MyoD and MyoG was detected after transfection. The relative expression of MyoD and MyoG was increased after transfection. In addition, compared with sheep bone marrow mesenchymal stem cells, The expression of myoblast specific expression gene in sheep umbilical cord mesenchymal stem cells was increased after induction. Immunofluorescence assay was used to detect the specific expression of myoblast specific genes by using 伪 -actin antibody to Desmin. The results showed that the expression of myoblast specific expression gene was detected by immunofluorescence, and the expression of myoblast specific expression gene was detected by immunofluorescence assay with 伪 -actin antibody. Compared with the control group (untransfected cells), the corresponding gene emission fluorescence was not detected under microscope. The expression vector pcDNA3.1-miR-133a2 was transfected into sheep umbilical cord and bone marrow mesenchymal stem cells after transfection into sheep umbilical cord and bone marrow mesenchymal stem cells, and the expression vector pcDNA3.1-miR-133a2 was transfected into sheep umbilical cord and bone marrow mesenchymal stem cells by flow cytometry. Flow cytometry was used to detect myoblast specific protein and MyoDX Desmin in sheep umbilical cord and bone marrow mesenchymal stem cells. The number of cells expressing 伪 -actinin was 12.2% and 29.6%, the number of cells expressing MyoD was 99.3% and 99.5, the number of cells expressing 伪 -Desminact1n was 0.917% and 9.24%, respectively, when the mesenchymal stem cells of miR-133mimics were transfected into sheep umbilical cord and bone marrow, respectively, the expression rate of 伪 -Desminact1n and 伪 -Desminact1n were 0.917% and 9.24%, respectively. The number of cells detected in these cells was 99.3% and 43.7%, the number of cells expressed by MyoD was 99.0% and 100, and the number of cells expressing Desmin was 1.07% and 1.68%, respectively. Sheep umbilical cord mesenchymal stem cells and sheep bone marrow mesenchymal stem cells were successfully induced to be myoblasts by miR-133.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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