發(fā)布時(shí)間：2018-05-31 14:33

  本文選題：miR-133 + 綿羊　； 參考：《東北林業(yè)大學(xué)》2015年碩士論文

【摘要】：已有研究證實(shí),miR-133在肌肉發(fā)育中起到非常重要的作用。間充質(zhì)干細(xì)胞是具有自我更新和多向分化潛能的細(xì)胞。為了探明利用miR-133誘導(dǎo)綿羊臍帶和骨髓間充質(zhì)干細(xì)胞為成肌細(xì)胞的方法,本研究采用脂質(zhì)體轉(zhuǎn)染法將肌肉特異表達(dá)的miR-133轉(zhuǎn)染到綿羊臍帶、骨髓間充質(zhì)干細(xì)胞后,檢測其誘導(dǎo)為成肌細(xì)胞的能力。其研究結(jié)果如下：1.綿羊臍帶、骨懿間充質(zhì)干細(xì)胞的轉(zhuǎn)染采用陽離子脂質(zhì)體轉(zhuǎn)染法將-miR-133a2表達(dá)載體或：niR-133mimics分別轉(zhuǎn)染綿羊臍帶間充質(zhì)和骨髓間充質(zhì)干細(xì)胞；2.轉(zhuǎn)染細(xì)胞培養(yǎng)與形態(tài)觀察將上述轉(zhuǎn)染細(xì)胞進(jìn)行續(xù)培養(yǎng),培養(yǎng)的第28d,在倒置熒光顯微鏡下細(xì)胞呈現(xiàn)梭形或紡錘形；當(dāng)細(xì)胞約達(dá)到80%匯合時(shí),細(xì)胞由渦旋狀生長變成定向有序的排列生長,并且鄰近細(xì)胞間出現(xiàn)融合傾向；3.轉(zhuǎn)染細(xì)胞的鑒定(1)成肌細(xì)胞特異表達(dá)基因的qRT-PCR檢測采用qRT-PCR方法檢測上述細(xì)胞骨骼肌特異基因MyoD、MyoG、Desmin、Tnni2、Myf5表達(dá)情況。在轉(zhuǎn)染前未檢測到MyoD和MyoG表達(dá),轉(zhuǎn)染后可以檢測到MyoD和MyoG的表達(dá)；Desmin、Tnni2、Myf5在轉(zhuǎn)染后相對(duì)表達(dá)量增加。此外,與綿羊骨髓間充質(zhì)干細(xì)胞相比,綿羊臍帶間充質(zhì)干細(xì)胞在誘導(dǎo)后Dismin、Tnni2、Myf5相對(duì)表達(dá)量增加更多；(2)成肌細(xì)胞特異表達(dá)基因的免疫熒光檢測 利用Desmin目應(yīng)α-actin抗體對(duì)轉(zhuǎn)染細(xì)胞進(jìn)行免疫熒光檢測,其結(jié)果,與對(duì)照組(未轉(zhuǎn)染細(xì)胞)在鏡下未檢測到相應(yīng)基因發(fā)出熒光相比,4組轉(zhuǎn)染細(xì)胞中,和MyoD蛋白檢測結(jié)果均呈現(xiàn)陽性,Desmin而呈現(xiàn)陰性；α-actin(3)成肌細(xì)胞特異表達(dá)基因的流式細(xì)胞檢測 當(dāng)將表達(dá)載體pcDNA3.1-miR-133a2分別轉(zhuǎn)染綿羊臍帶和骨髓間充質(zhì)干細(xì)胞后,采用流式細(xì)胞分析技術(shù)檢測成肌細(xì)胞特異蛋白和MyoD、Desmin其結(jié)果,在綿羊臍帶、骨髓間充質(zhì)干細(xì)胞中,表達(dá)α-actin,的細(xì)胞量分別為12.2%和29.6%,表達(dá)MyoD的細(xì)胞量分別為99.3%和99.5%,表達(dá)α-Desminact1n的細(xì)胞量分別為0.917%和9.24%；當(dāng)將分別轉(zhuǎn)染綿羊臍帶和骨髓miR-133mimics間充質(zhì)干細(xì)胞后,在這些細(xì)胞中檢測到表達(dá)的細(xì)胞量分別為99.3%和43.7%,表MyoD達(dá)的細(xì)胞量分別為99.0%和100%,表達(dá)Desmin的細(xì)胞量分別為1.07%和α-actin1.68%：上述結(jié)果表明,利用miR-133成功誘導(dǎo)綿羊臍帶間充質(zhì)干細(xì)胞和綿羊骨髓間充質(zhì)干細(xì)胞為成肌細(xì)胞,本研究結(jié)果為進(jìn)一步探討誘導(dǎo)間充質(zhì)細(xì)胞為成肌細(xì)胞和骨骼肌細(xì)胞分化機(jī)制提供了依據(jù)。
[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 偽 -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 偽 -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 偽 -actinin was 12.2% and 29.6%, the number of cells expressing MyoD was 99.3% and 99.5, the number of cells expressing 偽 -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 偽 -Desminact1n and 偽 -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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S826

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 田明;費(fèi)菁;胡曉湘;李寧;劉娣;孟慶勇;;microRNA-1,-133,-206在鼠肌肉中的表達(dá)譜[J];中國畜牧獸醫(yī);2009年08期

，

本文編號(hào)：1960107