【摘要】：肌肉衛(wèi)星細(xì)胞是一種肌源性的干細(xì)胞，在骨骼肌的再生和維持中發(fā)揮了重要作用，具有間充質(zhì)干細(xì)胞和多能性干細(xì)胞的部分生物學(xué)特性，可在體外向內(nèi)胚層、中胚層、外胚層和生殖細(xì)胞分化。生殖細(xì)胞是哺乳動(dòng)物體內(nèi)非常特殊的一類成體細(xì)胞，承擔(dān)著傳宗接代、種族延續(xù)的重任。國內(nèi)外已有多篇文獻(xiàn)報(bào)道表明間充質(zhì)干細(xì)胞、皮膚干細(xì)胞具有體外向雌性生殖細(xì)胞分化的潛力，但其誘導(dǎo)效率低、周期長。Reversine是一種類嘌呤小分子化合物，有資料顯示，其能使多種細(xì)胞去分化而呈多能性狀態(tài)，并且在合適的誘導(dǎo)條件下，能分化成多種細(xì)胞類型。目前，關(guān)于在體外如何誘導(dǎo)中胚層細(xì)胞向生殖細(xì)胞分化的資料還很缺乏。 本研究旨在分離純化出胎豬肌肉衛(wèi)星細(xì)胞，并對(duì)純化的肌肉衛(wèi)星細(xì)胞與早期貼壁的細(xì)胞進(jìn)行比較，分析其生物學(xué)特性；將分離純化的肌肉衛(wèi)星細(xì)胞用小分子化合物Reversine處理，并分析處理后細(xì)胞的變化；觀察純化的肌肉衛(wèi)星細(xì)胞用卵泡液處理后是否能向生殖細(xì)胞分化，進(jìn)一步用Reversine處理后再向生殖細(xì)胞誘導(dǎo)，檢測(cè)其誘導(dǎo)效率能否有所提高。 1.胎豬肌肉衛(wèi)星細(xì)胞的分離培養(yǎng)與生物學(xué)鑒定 本試驗(yàn)采用膠原酶和胰酶消化，結(jié)合差速貼壁的方法從60日胎齡的胎豬獲得肌肉衛(wèi)星細(xì)胞，通過細(xì)胞生長曲線的繪制、RT-PCR、細(xì)胞周期分析，發(fā)現(xiàn)純化后的肌肉衛(wèi)星細(xì)胞比早期貼壁的細(xì)胞具有較強(qiáng)的增殖能力，可以傳10代以上仍然保持較強(qiáng)的增殖能力，而早期貼壁的細(xì)胞傳到第4代就會(huì)出現(xiàn)老化；并用流式細(xì)胞儀分析表面抗原、免疫熒光染色，以及體外定向誘導(dǎo)分化等方法，對(duì)該細(xì)胞的生物學(xué)特性進(jìn)行了系統(tǒng)鑒定，證明其具有肌肉干細(xì)胞、經(jīng)典間充質(zhì)干細(xì)胞和部分胚胎干細(xì)胞的生物學(xué)特性，并具有定向分化為肌管和類神經(jīng)細(xì)胞的潛力。 2. Reversine對(duì)肌肉衛(wèi)星細(xì)胞的作用 體外培養(yǎng)的胎豬肌肉衛(wèi)星細(xì)胞經(jīng)Reversine后，通過免疫熒光染色、QRT-PCR檢測(cè)、Western-blotting、BrdU增殖分析，發(fā)現(xiàn)處理后胎豬肌肉衛(wèi)星細(xì)胞的多能性增強(qiáng)，但增殖能力減弱，細(xì)胞去分化呈多能性狀態(tài)；將Reversine處理后的細(xì)胞向脂肪細(xì)胞誘導(dǎo)，通過油紅O染色統(tǒng)計(jì)脂肪滴數(shù)量，QRT-PCR檢測(cè)，發(fā)現(xiàn)其能明顯提升胎豬肌肉衛(wèi)星細(xì)胞向脂肪細(xì)胞誘導(dǎo)效率。 3.胎豬肌肉衛(wèi)星細(xì)胞向生殖細(xì)胞誘導(dǎo) 優(yōu)化誘導(dǎo)條件，選定向培養(yǎng)液中添加20%的牛卵泡液為最佳誘導(dǎo)濃度，成功將胎豬肌肉衛(wèi)星細(xì)胞誘導(dǎo)成類卵母樣大圓細(xì)胞，這些大圓細(xì)胞呈Vasa、Dazl、Scp3、Stra8、Zp2和Zp3陽性；進(jìn)一步用Reversine處理后，再向生殖誘導(dǎo)，發(fā)現(xiàn)處理相同時(shí)間，Reversine處理組能明顯提高向生殖細(xì)胞分化的效率（能夠在短期內(nèi)誘導(dǎo)出大圓細(xì)胞，每個(gè)3.5cm皿中類生殖細(xì)胞的數(shù)量增加了6~7倍），生殖相關(guān)標(biāo)記基因的表達(dá)都明顯上調(diào)；體外轉(zhuǎn)染Figla基因和啟動(dòng)子，發(fā)現(xiàn)有類卵母樣大圓細(xì)胞出現(xiàn)，并且基因和啟動(dòng)子控制下的報(bào)告基因同時(shí)表達(dá)；將胎豬肌肉衛(wèi)星細(xì)胞誘導(dǎo)后移植到生殖缺陷的小鼠腎被膜下，30d后H.E.染色，發(fā)現(xiàn)移植側(cè)的卵巢功能較未移植側(cè)有明顯改善；并且移植塊中有類花環(huán)狀結(jié)構(gòu)出現(xiàn)，通過對(duì)移植塊切片染色發(fā)現(xiàn)，胎豬肌肉衛(wèi)星細(xì)胞在體內(nèi)可向生殖細(xì)胞分化。 綜上所述，，胎豬肌肉衛(wèi)星細(xì)胞具有很強(qiáng)的體外增殖能力，體外能夠向其他類型細(xì)胞進(jìn)行分化。Reversine處理后可以明顯提高向脂肪、生殖細(xì)胞分化的效率，可以為組織工程和再生醫(yī)學(xué)的研究提供足夠的細(xì)胞資源。
[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,
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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