本文關(guān)鍵詞： 臍帶間充質(zhì)干細胞 低氧誘導(dǎo)因子 培養(yǎng)環(huán)境 成軟骨分化　出處：《中南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的體外分離培養(yǎng)人臍帶間充質(zhì)干細胞并對其進行鑒定,探討體外低氧培養(yǎng)環(huán)境對其成軟骨分化能力的影響。 方法采用酶消化法分離培養(yǎng)人臍帶沃頓膠組織來源間充質(zhì)干細胞,加入含10%FBS的DMEM/F12培養(yǎng)基中進行原代培養(yǎng)；待細胞生長至80-90%融合時,1：3傳代培養(yǎng)。通過倒置顯微鏡觀察細胞形態(tài)變化,采用MTT法檢測細胞增殖能力,應(yīng)用流式細胞儀檢測細胞表面抗原。取傳3代細胞采用條件培養(yǎng)基誘導(dǎo)其向成軟骨方向分化,根據(jù)培養(yǎng)氧濃度分為實驗組(2%氧濃度)及對照組(20%氧濃度)。誘導(dǎo)2周后細胞爬片進行甲苯胺藍染色及II型膠原免疫組化染色,應(yīng)用RT-PCR技術(shù)檢測HIF-1α、Sox-9及CoL2al mRNA表達,研究低氧環(huán)境對人臍帶干細胞成軟骨分化的影響。 結(jié)果原代培養(yǎng)細胞貼壁生長,初始細胞形態(tài)呈短梭形、梭形或多角形；傳代后細胞多呈均一的成纖維樣細胞形態(tài),細胞貼壁和增殖速度明顯加快,呈平行排列生長或旋渦狀生長；MTT法檢測第3、5、7代臍帶干細胞均具有較強的增殖能力；流式細胞儀檢測傳3代臍帶干細胞95%表達CD29,96%表達CD44,僅有0.8%表達CD45、1.8%表達CD31,與間充質(zhì)干細胞表面抗原表達一致；誘導(dǎo)2周后低氧培養(yǎng)組細胞甲苯胺蘭染色及Ⅱ型膠原免疫組化染色均顯示強陽性反應(yīng),胞漿及胞膜異染明顯,而對照組染色顯示弱陽性反應(yīng)；RT-PCR檢測結(jié)果顯示實驗組HIF-1α、Sox-9及CoL2al mRNA表達均顯著強于對照組,差異有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論采用酶消化法可以成功分離培養(yǎng)人臍帶間充質(zhì)干細胞；低氧培養(yǎng)環(huán)境可促進人臍帶間充質(zhì)干細胞向成軟骨方向分化,其機制可能與HIF-1α基因表達上調(diào)有關(guān)。
[Abstract]:Objective to isolate and identify human umbilical cord mesenchymal stem cells (HMSCs) in vitro and to investigate the effect of hypoxia culture environment on the ability of chondrogenic differentiation of human umbilical cord mesenchymal stem cells. Methods mesenchymal stem cells derived from human umbilical cord Wharton gum tissue were isolated and cultured by enzyme digestion method. The mesenchymal stem cells were cultured in DMEM/F12 medium containing 10s. When the cells grew to 80-90% fusion, they were subcultured with 1: 3. The morphologic changes of cells were observed by inverted microscope, and the proliferative ability of cells was detected by MTT method. Flow cytometry was used to detect cell surface antigen. The third passage of cells was used to induce chondrogenic differentiation in conditioned medium. According to the culture oxygen concentration, the cells were divided into experimental group (2% oxygen concentration) and control group (20% oxygen concentration). After 2 weeks of induction, toluidine blue staining and type II collagen immunohistochemical staining were used. The expression of HIF-1 偽 -Sox-9 and CoL2al mRNA was detected by RT-PCR technique, and the effect of hypoxia on chondrogenic differentiation of human umbilical cord stem cells was studied. Results the primary cultured cells were adherent to the wall, and the initial cells were fusiform, fusiform or polygonal. After passage, most of the cells showed uniform fibroblast morphology, and the cell adhesion and proliferation were accelerated obviously, and the cells grew in parallel arrangement or swirl shape. All the umbilical cord stem cells of the 3rd and 5th generation were detected by MTT method, and all of the umbilical cord stem cells had strong proliferative ability. Flow cytometry was used to detect the expression of CD29996% in umbilical cord stem cells (95%) and CD31 in 0.8% (1.8%). The expression of surface antigen was consistent with that of mesenchymal stem cells. After 2 weeks of induction, toluidine blue staining and type 鈪，

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