發(fā)布時(shí)間：2018-04-24 09:50

  本文選題：脂肪間充質(zhì)干細(xì)胞 + 誘導(dǎo)分化　； 參考：《大連醫(yī)科大學(xué)》2011年碩士論文

【摘要】：背景:臨床上常見(jiàn)的慢性潰瘍、燒傷、外傷等造成的皮膚損傷,需要及時(shí)覆蓋創(chuàng)面,以促進(jìn)創(chuàng)面愈合及功能重塑。近年來(lái),采用自體干細(xì)胞和生長(zhǎng)因子的再生醫(yī)學(xué),為組織損傷的修復(fù)提供了新的思路,同時(shí)也為以細(xì)胞為基礎(chǔ)的治療提供了新的途徑。研究表明,成體干細(xì)胞治療,為組織損傷的修復(fù)提供了一種安全、有效的臨床治療方法。 目的:采用人脂肪間充質(zhì)干細(xì)胞(ADSCs)與EGF、bFGF、IGF-1、TGFβ1等生長(zhǎng)因子進(jìn)行體外誘導(dǎo)培養(yǎng),從ADSCs的分離培養(yǎng),到對(duì)其增殖能力和表面分子表達(dá)的檢測(cè),并從轉(zhuǎn)錄水平和蛋白水平進(jìn)行系統(tǒng)分析。擬建立ADSCs向表皮細(xì)胞和成纖維細(xì)胞誘導(dǎo)分化的規(guī)范實(shí)驗(yàn)體系。 方法:從脂肪組織中分離ADSCs,并檢測(cè)第3代和第10代ADSCs的表面分子表達(dá)及第3、5、10代ADSCs的細(xì)胞增殖情況。將ADSCs在含有EGF、bFGF、IGF-1等生長(zhǎng)因子的誘導(dǎo)培養(yǎng)基中進(jìn)行體外誘導(dǎo)培養(yǎng),蛋白水平,免疫熒光檢測(cè)CK 19的抗原表達(dá),同時(shí)透射電鏡下觀察細(xì)胞超微結(jié)構(gòu)。將ADSCs在含有TGFβ1、bFGF等生長(zhǎng)因子的誘導(dǎo)培養(yǎng)基中進(jìn)行體外誘導(dǎo)培養(yǎng),轉(zhuǎn)錄水平,檢測(cè)I型膠原的mRNA含量;蛋白水平,免疫熒光檢測(cè)I型膠原的抗原表達(dá),同時(shí)透射電鏡下觀察細(xì)胞超微結(jié)構(gòu)。 結(jié)果:(1)第3、10代ADSCs的CD90表達(dá)強(qiáng)陽(yáng)性,CD34表達(dá)弱陽(yáng)性,CD106表達(dá)陰性,第3、10代細(xì)胞均一性分別為94.2%,97.5%。第3代和第10代的ADSCs在細(xì)胞形態(tài)學(xué)、增殖能力和表面分子的表達(dá)方面無(wú)明顯改變。(2)ADSCs在含有EGF、bFGF、IGF-1等生長(zhǎng)因子的誘導(dǎo)培養(yǎng)基中進(jìn)行體外誘導(dǎo)培養(yǎng),第3天,部分細(xì)胞由梭形變成不規(guī)則形,第5天開(kāi)始,細(xì)胞逐漸融合成鋪石路樣改變。免疫熒光結(jié)果顯示,CK 19表達(dá)陽(yáng)性,陽(yáng)性率為15.6%。誘導(dǎo)15天后,透射電鏡下,可見(jiàn)黑素小體、透明角質(zhì)顆粒。(3)ADSCs在含有TGFβ1、bFGF等生長(zhǎng)因子的誘導(dǎo)培養(yǎng)基中進(jìn)行體外誘導(dǎo)培養(yǎng),ADSCs誘導(dǎo)7天后,轉(zhuǎn)錄水平上,I型膠原的mRNA含量為對(duì)照組的兩倍多;免疫熒光結(jié)果顯示,與陰性對(duì)照組比,誘導(dǎo)組I型膠原蛋白的表達(dá)增加。誘導(dǎo)15天后,透射電鏡下,可見(jiàn)膠原微纖維。 結(jié)論:聯(lián)合人脂肪間充質(zhì)干細(xì)胞與EGF、bFGF、IGF-1、TGFβ1等生長(zhǎng)因子進(jìn)行體外誘導(dǎo)培養(yǎng), ADSCs在一定條件下,可以向表皮細(xì)胞和成纖維細(xì)胞分化。從而,為皮膚組織再生提供了種子細(xì)胞的來(lái)源,同時(shí)也為皮膚損傷修復(fù)的臨床應(yīng)用提供了理論基礎(chǔ)。
[Abstract]:Background: skin injury caused by chronic ulcer, burn and trauma in clinic needs to cover the wound in time to promote wound healing and functional remodeling. In recent years, regenerative medicine with autologous stem cells and growth factors has provided new ideas for the repair of tissue injury and a new approach to cell-based therapy. Studies have shown that adult stem cell therapy provides a safe and effective method for the repair of tissue injury. Objective: to study the effects of human adipose mesenchymal stem cells (ADSCs) and EGFFGFGFIGF-1TGF- 尾 1 on the proliferation and surface molecular expression of human adipose mesenchymal stem cells (ADSCs) in vitro. To establish a standard experimental system for the differentiation of ADSCs into epidermal cells and fibroblasts. Methods: ADSCs were isolated from adipose tissue and the expression of surface molecules in the 3rd and 10th generation of ADSCs and the proliferation of ADSCs in the 3rd and 10th generation were detected. The expression of CK19 antigen was detected by immunofluorescence, and the expression of CK19 antigen was detected by immunofluorescence. The ultrastructure of the cells was observed by transmission electron microscope (TEM). ADSCs was cultured in culture medium containing TGF 尾 1bFGF in vitro, transcription level was measured to detect the mRNA content of type I collagen, protein level, antigen expression of type I collagen was detected by immunofluorescence. At the same time, the ultrastructure of cells was observed under transmission electron microscope. Results the strong positive expression of CD90 and the weak positive expression of CD106 in ADSCs in the 3rd and 10th generation of ADSCs were negative, and the homogeneity of the cells in the 10th generation of the 3rd generation were 94.2and 97.5g, respectively. The third and tenth passages of ADSCs had no obvious changes in cell morphology, proliferation ability and surface molecule expression. They were induced in vitro in the medium containing EGFFbFGFIGF-1, and cultured in vitro on the 3rd day. Some of the cells changed from fusiform to irregular, and gradually fused into paved road-like changes on the 5th day. Immunofluorescence showed positive expression of CK19, and the positive rate was 15.6. After 15 days of induction, melanosome and hyaline keratin granules were observed in the culture medium containing TGF 尾 1bFGF for 7 days. The mRNA content of type I collagen was more than twice as high as that of the control group at the transcriptional level, and the expression of type I collagen protein in the induced group was higher than that in the negative control group. After 15 days of induction, collagen microfibers were observed under transmission electron microscope. Conclusion: the combination of human adipose mesenchymal stem cells and EGFFGFF-1TGF- 尾 1 growth factors can differentiate ADSCs into epidermal cells and fibroblasts under certain conditions. Thus, it provides the source of seed cells for skin tissue regeneration, and also provides a theoretical basis for the clinical application of skin injury repair.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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2 艾國(guó)平,粟永萍,閆國(guó)和,王蒙,劉曉宏,徐輝,程天民;骨髓間充質(zhì)干細(xì)胞對(duì)合并局部放射損傷創(chuàng)面促愈作用及機(jī)理研究[J];中華醫(yī)學(xué)雜志;2002年23期

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本文編號(hào)：1796152