Reversine对胎猪肌肉卫星细胞去分化及向雌性生殖细胞分化的作用

发布时间：2019-06-01 12:04

【摘要】：肌肉卫星细胞是一种肌源性的干细胞，在骨骼肌的再生和维持中发挥了重要作用，具有间充质干细胞和多能性干细胞的部分生物学特性，可在体外向内胚层、中胚层、外胚层和生殖细胞分化。生殖细胞是哺乳动物体内非常特殊的一类成体细胞，承担着传宗接代、种族延续的重任。国内外已有多篇文献报道表明间充质干细胞、皮肤干细胞具有体外向雌性生殖细胞分化的潜力，但其诱导效率低、周期长。Reversine是一种类嘌呤小分子化合物，有资料显示，其能使多种细胞去分化而呈多能性状态，并且在合适的诱导条件下，能分化成多种细胞类型。目前，关于在体外如何诱导中胚层细胞向生殖细胞分化的资料还很缺乏。本研究旨在分离纯化出胎猪肌肉卫星细胞，并对纯化的肌肉卫星细胞与早期贴壁的细胞进行比较，分析其生物学特性；将分离纯化的肌肉卫星细胞用小分子化合物Reversine处理，并分析处理后细胞的变化；观察纯化的肌肉卫星细胞用卵泡液处理后是否能向生殖细胞分化，进一步用Reversine处理后再向生殖细胞诱导，检测其诱导效率能否有所提高。 1.胎猪肌肉卫星细胞的分离培养与生物学鉴定本试验采用胶原酶和胰酶消化，结合差速贴壁的方法从60日胎龄的胎猪获得肌肉卫星细胞，通过细胞生长曲线的绘制、RT-PCR、细胞周期分析，发现纯化后的肌肉卫星细胞比早期贴壁的细胞具有较强的增殖能力，可以传10代以上仍然保持较强的增殖能力，而早期贴壁的细胞传到第4代就会出现老化；并用流式细胞仪分析表面抗原、免疫荧光染色，以及体外定向诱导分化等方法，对该细胞的生物学特性进行了系统鉴定，证明其具有肌肉干细胞、经典间充质干细胞和部分胚胎干细胞的生物学特性，并具有定向分化为肌管和类神经细胞的潜力。 2. Reversine对肌肉卫星细胞的作用体外培养的胎猪肌肉卫星细胞经Reversine后，通过免疫荧光染色、QRT-PCR检测、Western-blotting、BrdU增殖分析，发现处理后胎猪肌肉卫星细胞的多能性增强，但增殖能力减弱，细胞去分化呈多能性状态；将Reversine处理后的细胞向脂肪细胞诱导，通过油红O染色统计脂肪滴数量，QRT-PCR检测，发现其能明显提升胎猪肌肉卫星细胞向脂肪细胞诱导效率。 3.胎猪肌肉卫星细胞向生殖细胞诱导优化诱导条件，选定向培养液中添加20%的牛卵泡液为最佳诱导浓度，成功将胎猪肌肉卫星细胞诱导成类卵母样大圆细胞，这些大圆细胞呈Vasa、Dazl、Scp3、Stra8、Zp2和Zp3阳性；进一步用Reversine处理后，再向生殖诱导，发现处理相同时间，Reversine处理组能明显提高向生殖细胞分化的效率（能够在短期内诱导出大圆细胞，每个3.5cm皿中类生殖细胞的数量增加了6~7倍），生殖相关标记基因的表达都明显上调；体外转染Figla基因和启动子，发现有类卵母样大圆细胞出现，并且基因和启动子控制下的报告基因同时表达；将胎猪肌肉卫星细胞诱导后移植到生殖缺陷的小鼠肾被膜下，30d后H.E.染色，发现移植侧的卵巢功能较未移植侧有明显改善；并且移植块中有类花环状结构出现，通过对移植块切片染色发现，胎猪肌肉卫星细胞在体内可向生殖细胞分化。综上所述，，胎猪肌肉卫星细胞具有很强的体外增殖能力，体外能够向其他类型细胞进行分化。Reversine处理后可以明显提高向脂肪、生殖细胞分化的效率，可以为组织工程和再生医学的研究提供足够的细胞资源。
[Abstract]:Muscular satellite cells are a kind of myogenic stem cell, which plays an important role in the regeneration and maintenance of the skeletal muscle, and has the partial biological characteristics of the mesenchymal stem cells and the pluripotent stem cells, and can be differentiated into the endoderm, the mesoderm, the ectoderm and the germ cells in vitro. The germ cell is a very special kind of somatic cell in the mammalian body, which bears the important task of the generation and the extension of the race. There are many literature reports at home and abroad that the mesenchymal stem cells and the skin stem cells have the potential to differentiate into the female germ cells in vitro, but the induction efficiency is low and the period is long. Reversine is a kind of low-molecular compound of the type of methotrexate, and has the data to show that it can dedifferentiate multiple cells to be in a multi-functional state, and can be differentiated into multiple cell types under appropriate induction conditions. There is also a lack of information on how to induce the differentiation of mesoderm cells into germ cells in vitro. The purpose of this study was to separate and purify the porcine muscle satellite cells and to compare the purified muscle satellite cells with the early adherent cells to analyze their biological characteristics. The isolated and purified muscle satellite cells were treated with a small molecular compound, Reversine, and the changes of the treated cells were analyzed. To study whether the purified muscle satellite cells can differentiate to the germ cells after treatment with the follicular fluid, and then to induce the germ cells after the treatment with Reversine, and whether the induction efficiency of the purified muscle satellite cells can be improved. High.1. Isolation and culture of porcine muscle satellite cells In the experiment, collagenase and pancreatin were used to digest, and the muscle satellite cells were obtained from the 60-day-old fetal pig by the method of differential adhesion, and the cell growth curve was drawn by RT-PCR. The cell cycle analysis revealed that the purified muscle satellite cells had a strong proliferative capacity than those of the early adherent cells, which could be transferred to the 4th generation for aging, and the surface antigen was analyzed by flow cytometry. The biological characteristics of the cells were systematically identified by the method of fluorescent staining of the phytophthora and the in vitro orientation-induced differentiation. The biological characteristics of the stem cells, the classical mesenchymal stem cells and the partial embryonic stem cells were demonstrated, and the directional differentiation was the myotube and the nerve-like nerve. Potential of the cell.2. Reversine vs. After reversine, the porcine muscle satellite cells, which were cultured in vitro, were analyzed by immunofluorescence staining, QRT-PCR, Western-blotting and BrdU. Strong, but the proliferation ability is weakened, the cell dedifferentiation is in a multi-functional state, the cells treated by the Reversine are induced to the fat cells, the number of fat drops is counted by the oil red O dyeing, and the QRT-PCR detection is found to be capable of obviously improving the porcine muscle satellite cells to the fat cell induction efficiency.3. The fetal pig's muscle in that condition of induction and optimization of the germ cell in the meat satellite cell,20% of the bovine follicular fluid is added to the culture solution to obtain the optimal induction concentration, and the porcine muscle satellite cell is successfully induced to form an egg-like large-circle cell, which is in the form of Vasa, Da, Zl, Scp3, Stra8, Zp2 and Zp3 were positive; after further treatment with Reversine, the same time was found to be treated with Reversine, and the Reversine treatment group was able to significantly improve the efficiency of differentiation to the germ cells (a large circle of cells can be induced in a short period of time, and in each 3.5 cm dish) The number of cells increased by 6-7 times, and the expression of the reproductive-related marker genes was up-regulated. Transfection of the hla gene and the promoter in vitro revealed the presence of a large-circle cell with an egg-like parent, and the gene and the start-up At the same time, the reporter gene under the subcontrol was expressed simultaneously, and the mouse kidney which was transplanted to the reproduction defect after induction of the porcine muscle satellite cells was stained with the membrane, and after 30 days, the H. E. staining showed that the ovarian function on the graft side was significantly improved than that of the non-grafted side, and a similar ring-like structure appeared in the graft block. by staining the graft block, the porcine muscle satellite in conclusion, that porcine muscle satellite cell has strong in-vitro proliferation ability, In vitro, the differentiation of other types of cells can be made. After the Reversine treatment, the efficiency of the differentiation of the fat and the germ cells can be obviously improved,
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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