本文選題：臍帶間充質(zhì)干細(xì)胞　切入點(diǎn)：豬　出處：《東北農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：臍帶間充質(zhì)干細(xì)胞(Umbilical cord mesnchymal stem cells, UCMSCs)是一種位于臍帶沃頓氏膠(Wharton's jelly)內(nèi)和血管周圍,來源于發(fā)育早期的中胚層和外胚層,具有自我更新能力和多分化潛能的一類成體干細(xì)胞。 在體外培養(yǎng)中發(fā)現(xiàn),臍帶間充質(zhì)干細(xì)胞具有分化為三個(gè)胚層細(xì)胞的能力,相對(duì)高量表達(dá)多能性基因Oct-4,sox-2和Nanog;同時(shí),較低的免疫排斥反應(yīng)使得臍帶間充質(zhì)干細(xì)胞在細(xì)胞替代治療和組織工程方面,成為替代有年齡和疾病限制的骨髓間充質(zhì)干細(xì)胞的種子細(xì)胞。 由于其在臨床應(yīng)用上的巨大潛力,關(guān)于人臍帶間充質(zhì)干細(xì)胞的研究很多,但多局限于體外實(shí)驗(yàn),而人的臨床實(shí)驗(yàn)受到限制,因此,在臨床應(yīng)用前尋找一種大動(dòng)物模型成為迫切的需要；但在人以外的其他物種相關(guān)研究報(bào)道匱乏。豬是除靈長類動(dòng)物外,在生理和解剖結(jié)構(gòu)上與人類最相似的動(dòng)物,是一種理想的大動(dòng)物模型,因此其用于基礎(chǔ)理論研究和臨床應(yīng)用研究的潛力巨大。 本實(shí)驗(yàn)以新生大白豬的臍帶為實(shí)驗(yàn)材料,比較不同的酶消化法和組織培養(yǎng)法對(duì)豬臍帶間充質(zhì)干細(xì)胞的分離效果,同時(shí),添加生長因子EGF和bFGF,觀察生長因子對(duì)臍帶間充質(zhì)干增殖速度和生長狀態(tài)的影響,在此基礎(chǔ)上,確定豬臍帶間充質(zhì)干細(xì)胞的培養(yǎng)體系,觀察細(xì)胞的生長特性。實(shí)驗(yàn)還通過免疫熒光和PCR方法,在蛋白水平和分子檢測(cè)了細(xì)胞多能性基因Oct-4,sox-2和Nanog的表達(dá)情況,同時(shí)跟蹤了其表達(dá)量的變化及趨勢(shì)。最后,為確定豬臍帶間充質(zhì)干細(xì)胞的分化能力,對(duì)體外培養(yǎng)的細(xì)胞進(jìn)行了成脂和成軟骨的誘導(dǎo)分化。 實(shí)驗(yàn)結(jié)果表明：在原代細(xì)胞的分離培養(yǎng)中,膠原酶Ⅰ和透明質(zhì)酸酶相結(jié)合的酶處理方法更適宜于豬臍帶間充質(zhì)干細(xì)胞的分離,而通過沃頓氏膠消化法得到的原代細(xì)胞純度更高,且生長能力更強(qiáng)；在觀察生長因子EGF和bFGF對(duì)豬臍帶間充質(zhì)干細(xì)胞增殖速度和生長狀態(tài)影響的實(shí)驗(yàn)中發(fā)現(xiàn),單獨(dú)添加EGF和bFGF的細(xì)胞與對(duì)照組相比,無明顯變化,而兩種因子同時(shí)添加的細(xì)胞在做生長曲線的檢測(cè)時(shí),明顯高于對(duì)照組和單獨(dú)添加組別,說明EGF和bFGF協(xié)同作用可以促進(jìn)維持細(xì)胞本身的自我更新能力；而后對(duì)細(xì)胞進(jìn)行傳代培養(yǎng),發(fā)現(xiàn)傳代培養(yǎng)細(xì)胞在接種6-12h后貼壁,細(xì)胞單層漩渦狀生長,呈現(xiàn)出典型的成纖維樣,但少量細(xì)胞,呈現(xiàn)出扁平,擴(kuò)張的形態(tài)；細(xì)胞具有一定的增殖能力,目前已有培養(yǎng)至13代的細(xì)胞,細(xì)胞形態(tài)維持成纖維樣,但增殖速度逐漸下降。 在多能性基因的檢測(cè)中發(fā)現(xiàn),通過免疫熒光的方法,可以看到多能性基因sox-2, nanog和nestin的表達(dá),通過分子水平的檢測(cè),可以看到Oct-4,sox-2和nanog的表達(dá),且其表達(dá)量隨傳代次數(shù)的增加而減少；最后對(duì)豬臍帶間充質(zhì)干細(xì)胞進(jìn)行了成脂和成軟骨的誘導(dǎo)分化,通過油紅0和阿辛藍(lán)的檢測(cè),證明其具有分化為成脂細(xì)胞和成軟骨細(xì)胞的能力。
[Abstract]:Umbilical cord mesenchymal cord mesnchymal stem cells (UCMSCs) are a kind of adult stem cells, which are located in and around the blood vessels of Whartonia jelly. they are derived from mesoderm and ectoderm in the early stage of development, and have the ability of self-renewal and multi-differentiation. It was found in vitro that umbilical cord mesenchymal stem cells had the ability to differentiate into three embryonic layer cells, and expressed the multipotent genes Oct-4, sox-2 and Nanog. at the same time, Due to low immune rejection, umbilical cord mesenchymal stem cells are used as seed cells to replace bone marrow mesenchymal stem cells with age and disease limitation in cell substitution therapy and tissue engineering. Because of its great potential in clinical application, there are many researches on human umbilical cord mesenchymal stem cells, but most of them are confined to in vitro experiments, but human clinical trials are limited. It is an urgent need to find a large animal model before clinical application, but there are few reports about other species other than human. Pigs are the most physiologically and anatomically similar animals except primates. It is an ideal large animal model, so it has great potential for basic theory research and clinical application research. In this experiment, the umbilical cord of newborn white pig was used as experimental material to compare the isolation effects of different enzyme digestion and tissue culture methods on porcine umbilical cord mesenchymal stem cells. The effects of EGF and bFGF on the growth rate and growth state of umbilical cord mesenchymal stem cells were observed, and the culture system of porcine umbilical cord mesenchymal stem cells was established. The cell growth characteristics were observed. The expression of the multipotent gene Oct-4Osox-2 and Nanog was detected at the protein level and molecular level by immunofluorescence and PCR methods, and the changes and trends of the expression of Oct-4Osox-2 and Nanog were followed. In order to determine the differentiation ability of porcine umbilical cord mesenchymal stem cells, adipogenic and chondrogenic differentiation of cultured cells was carried out. The results showed that collagenase I combined with hyaluronidase was more suitable for isolation of porcine umbilical cord mesenchymal stem cells. The primary cells obtained by Wharton's gum digestion method were of higher purity and stronger growth ability, and the effects of growth factors EGF and bFGF on the proliferation rate and growth state of porcine umbilical cord mesenchymal stem cells were observed. Compared with the control group, the cells supplemented with EGF and bFGF alone had no significant change, but the growth curves of the cells added with the two factors at the same time were significantly higher than those in the control group and the added group alone. The results showed that the synergistic effect of EGF and bFGF could promote the self-renewal ability of the cells, and then the cells were subcultured, and the cells adhered to the wall 6-12 hours after inoculation, and the monolayer whirlpool grew, showing a typical fibroblast. However, a small number of cells showed flat and dilated morphology, and the cells had the ability to proliferate to a certain extent. At present, the cells were cultured to the 13th passage, the morphology of the cells remained fibroid, but the proliferation rate decreased gradually. In the detection of multipotent genes, the expression of Sox-2, nanog and nestin could be seen by immunofluorescence, and Oct-4sox-2 and nanog could be detected by molecular level. And its expression decreased with the increase of passage times. Finally, adipogenic and chondrogenic differentiation of porcine umbilical cord mesenchymal stem cells was carried out, which was detected by oil red 0 and acinyl blue. It is proved that it has the ability to differentiate into adipocytes and chondroblasts.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 黃鵬;馬廉;;人臍帶間充質(zhì)干細(xì)胞的應(yīng)用研究現(xiàn)狀[J];中國輸血雜志;2009年03期

2 劉忠;李素萍;楊宏友;王震;呂蓉;吳炳;方勤;李敏;吳學(xué)忠;;人臍帶間充質(zhì)干細(xì)胞體外分化成骨細(xì)胞的研究[J];中國輸血雜志;2011年05期

3 彭亮;高志良;;骨髓間充質(zhì)干細(xì)胞研究現(xiàn)狀和在肝臟疾病中的應(yīng)用[J];國外醫(yī)學(xué)(內(nèi)科學(xué)分冊(cè));2006年01期

4 何紅燕;崔冰琳;馮學(xué)永;肖平;林曉波;蔣學(xué)武;羅敏潔;馬廉;;人臍帶間充質(zhì)干細(xì)胞分化為神經(jīng)細(xì)胞的形態(tài)學(xué)改變[J];國際輸血及血液學(xué)雜志;2006年06期

5 楊曉娟;田衛(wèi)東;;成脂分化轉(zhuǎn)錄調(diào)控因子及其作用機(jī)制的研究進(jìn)展[J];國際口腔醫(yī)學(xué)雜志;2008年S1期

6 肖宏濤;牛希華;劉毅;;人臍帶間充質(zhì)干細(xì)胞的體外分離、培養(yǎng)及誘導(dǎo)分化[J];鄭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年02期

7 范存剛;周景儒;張慶俊;;人臍帶間充質(zhì)干細(xì)胞的生物學(xué)性質(zhì)研究進(jìn)展[J];基礎(chǔ)醫(yī)學(xué)與臨床;2010年02期

8 周永慶;胡圖強(qiáng);喻學(xué)洲;何俐;鄭鈞元;唐俊明;;表皮生長因子對(duì)間充質(zhì)干細(xì)胞遷移的影響及其信號(hào)機(jī)制[J];臨床口腔醫(yī)學(xué)雜志;2011年10期

9 莊淑波;劉毅;陳克明;肖斌;;不同方法分離的大鼠骨髓間充質(zhì)干細(xì)胞增殖活性和成脂分化能力的比較[J];中國美容醫(yī)學(xué);2007年01期

10 馬桂霞;何紅燕;羅敏潔;黃天華;肖升平;崔冰琳;馬廉;;人臍帶間充質(zhì)干細(xì)胞分化為胰島β細(xì)胞的實(shí)驗(yàn)研究[J];實(shí)用兒科臨床雜志;2008年08期

，

本文編號(hào)：1673275