本文關(guān)鍵詞：山羊脂肪間充質(zhì)干細(xì)胞的體外分離培養(yǎng)與定向分化　出處：《內(nèi)蒙古大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 脂肪間充質(zhì)干細(xì)胞 體外分離培養(yǎng) 細(xì)胞鑒定 細(xì)胞定向誘導(dǎo)培養(yǎng)

【摘要】：脂肪間充質(zhì)干細(xì)胞(adipose-derived stromal cells, ADSCs)在不同種類的動物細(xì)胞中越來越受到人們的關(guān)注。雖然從實驗動物(如大鼠)脂肪組織中已經(jīng)分離出大量脂肪間充質(zhì)干細(xì)胞并進(jìn)行了相關(guān)的研究,但對于家畜的脂肪間充質(zhì)干細(xì)胞(如山羊)卻研究的很少。 為了分離和培養(yǎng)山羊脂肪間充質(zhì)干細(xì)胞,本實驗借鑒分離大鼠脂肪間充質(zhì)干細(xì)胞的方法體外分離培養(yǎng)山羊脂肪間充質(zhì)干細(xì)胞、進(jìn)行定向誘導(dǎo)培養(yǎng)并與大鼠脂肪間充質(zhì)干細(xì)胞進(jìn)行比較。結(jié)果本實驗分離得到的山羊脂肪間充質(zhì)干細(xì)胞在體外增殖穩(wěn)定,經(jīng)過多次傳代后染色體仍保持正常的二倍性,但在相同接種密度和培養(yǎng)條件下,山羊脂肪間充質(zhì)干細(xì)胞比大鼠脂肪間充質(zhì)干細(xì)胞分裂增殖速度更快。山羊和大鼠脂肪間充質(zhì)干細(xì)胞經(jīng)CD49d和CD13染色呈陽性,CD34、CD106呈陰性,RT-PCR顯示CD44表達(dá)呈陽性；成骨誘導(dǎo)后經(jīng)茜素紅染色后均可見明顯的骨結(jié)節(jié)、ALP染色后細(xì)胞均呈陽性且表達(dá)Osteocalcin;不同的是山羊脂肪間充質(zhì)干細(xì)胞經(jīng)成骨誘導(dǎo)后形成的骨化結(jié)比大鼠脂肪間充質(zhì)干細(xì)胞經(jīng)誘導(dǎo)后形成的骨化結(jié)大且ALP染色更深。兩種細(xì)胞成脂誘導(dǎo)后經(jīng)O-油紅染色后均有紅色脂滴出現(xiàn)在細(xì)胞周圍且表達(dá)PPARy2,不同的是山羊脂肪間充質(zhì)干細(xì)胞誘導(dǎo)后形成的脂滴比大鼠脂肪間充質(zhì)干細(xì)胞誘導(dǎo)后形成的脂滴多且大。應(yīng)用貼壁培養(yǎng)法、三維培養(yǎng)法和懸滴培養(yǎng)法對兩種細(xì)胞進(jìn)行成軟骨誘導(dǎo),誘導(dǎo)后經(jīng)阿新藍(lán)染色均有軟骨陷窩形成且表達(dá)COL2A1,不同的是在大鼠脂肪間充質(zhì)干細(xì)胞軟骨分化誘導(dǎo)實驗中,懸滴培養(yǎng)法誘導(dǎo)后形成的軟骨陷窩數(shù)量較多、形態(tài)更好,在山羊脂肪間充質(zhì)干細(xì)胞軟骨分化誘導(dǎo)實驗中,貼壁誘導(dǎo)培養(yǎng)法和懸滴誘導(dǎo)培養(yǎng)法效果均好于三維誘導(dǎo)培養(yǎng)法,且形成的軟骨陷窩數(shù)量多、形態(tài)好,但貼壁誘導(dǎo)培養(yǎng)法和懸滴誘導(dǎo)培養(yǎng)法兩者相比較并無明顯差異。 研究結(jié)果表明本實驗中已經(jīng)得到純度較高的山羊和大鼠脂肪間充質(zhì)干細(xì)胞。
[Abstract]:Adipose-derived stromal cells. ADSCs have attracted more and more attention in different animal cells. Although from experimental animals (such as rats). A large number of adipose mesenchymal stem cells have been isolated and studied in adipose tissue. But little research has been done on adipose mesenchymal stem cells (such as goats) in domestic animals. In order to isolate and culture goat adipose mesenchymal stem cells, we used the method of rat adipose mesenchymal stem cells to isolate and culture goat adipose mesenchymal stem cells in vitro. Results the goat adipose mesenchymal stem cells isolated in this experiment proliferate stably in vitro. After several generations, the chromosomes remained normal diploidy, but under the same inoculation density and culture conditions. Goat adipose mesenchymal stem cells divide and proliferate faster than rat adipose mesenchymal stem cells. Goat and rat adipose mesenchymal stem cells are positive for CD34 by CD49d and CD13 staining. The expression of CD44 was positive by CD106 negative RT-PCR. After osteogenesis induced by alizarin red staining, all the cells were positive and expressed Osteocalcinin after ALP staining. The difference is that the ossification of goat adipose mesenchymal stem cells induced by osteogenesis is larger than that of rat adipose mesenchymal stem cells induced by ossification and ALP staining is deeper. After red staining, red lipid droplets appeared around the cells and expressed PPARy2. The difference was that the fat droplets of goat adipose mesenchymal stem cells were more and larger than that of rat adipose mesenchymal stem cells induced by adipose mesenchymal stem cells. Three dimensional culture method and suspension culture method were used to induce the cartilage of the two kinds of cells. After induction, cartilage lacunae were formed and COL2A1 was expressed. The difference is that in the differentiation of adipose mesenchymal stem cells, the number of cartilage lacunae induced by suspension culture is more and the shape is better. In the experiment of differentiation of goat adipose mesenchymal stem cells, the effect of adherent culture method and suspension culture method were better than that of three-dimensional culture method, and the number of cartilage lacunae was more and the shape was good. But there was no significant difference between the two methods. The results show that high purity goat and rat adipose mesenchymal stem cells have been obtained in this experiment.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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