發(fā)布時間：2018-02-25 16:28

  本文關(guān)鍵詞： 骨髓 間質(zhì)干細胞 細胞 培養(yǎng)的 大鼠 Sprague-Dawley 干細胞 骨髓干細胞 貼壁分離篩選法 骨髓間充質(zhì)干細胞 生物學(xué)特性 SD大鼠 細胞形態(tài) 細胞周期 增殖　出處：《中國組織工程研究》2014年28期 　論文類型：期刊論文

【摘要】：背景:在正常情況下骨髓來源間充質(zhì)干細胞含量很少,且易與其他細胞相混雜,因此,建立一種簡便可行的體外培養(yǎng)擴增方法,獲得大量穩(wěn)定的骨髓間充質(zhì)干細胞具有重要的理論意義和應(yīng)用價值。目的:建立一種簡便可行的體外培養(yǎng)擴增方法,獲得大量穩(wěn)定的骨髓間充質(zhì)干細胞。方法:采用密度梯度離心法及貼壁分離篩選法相結(jié)合體外分離培養(yǎng)SD大鼠骨髓間充質(zhì)干細胞。倒置光學(xué)顯微鏡下觀察細胞形態(tài)變化,透射電鏡觀察細胞亞微結(jié)構(gòu),錐蟲藍拒染法計算活細胞數(shù),繪制細胞生長曲線,流式細胞儀分析細胞周期,免疫細胞化學(xué)檢測c-kit和CD45的表達,流式細胞儀分析CD45的表達情況。結(jié)果與結(jié)論:①接種后24 h倒置光學(xué)顯微鏡下可見有細胞貼壁并伸出偽足,4 d時可見有細胞集落形成,14 d時細胞可達到90%融合。經(jīng)傳代后細胞趨于一致,為纖維樣細胞,呈漩渦或火焰狀生長。②透射電鏡下可見第3代骨髓間充質(zhì)干細胞體積較小核大,核仁明顯。染色質(zhì)分布稀疏,電子密度低。細胞表面有微絨毛,胞質(zhì)稀松,內(nèi)有豐富核糖體,而內(nèi)質(zhì)網(wǎng)、線粒體、高爾基復(fù)合體等細胞器少見,提示細胞處于原始未分化狀態(tài)。③第3代骨髓間充質(zhì)干細胞接種后第1天細胞數(shù)量有所減少,第2天細胞開始增長,第3天細胞進入指數(shù)增生期,第7天進入平臺期,第9天細胞數(shù)量開始下降,繪制的生長曲線呈"S"形。④第3代骨髓間充質(zhì)干細胞經(jīng)DNA染色后采用流式細胞儀測定其DNA含量證明S期細胞比率為21.1%。⑤免疫細胞化學(xué)染色和流式細胞術(shù)證明c-kit陽性細胞比率為53.3%,CD45表達陽性率為1.68%。⑥骨髓間充質(zhì)干細胞向成骨細胞誘導(dǎo)分化16 d后,細胞的胞體呈橢圓形,有短突起彼此相連,胞漿較暗,提示可能富含粗面內(nèi)質(zhì)網(wǎng)和高爾基復(fù)合體,并具有分泌類骨質(zhì)的功能。結(jié)果證明該方法獲得的大鼠骨髓間充質(zhì)干細胞生長穩(wěn)定,增殖活躍。
[Abstract]:Background: under normal conditions, mesenchymal stem cells derived from bone marrow have little content and are easily mixed with other cells. Therefore, a simple and feasible method for culture and amplification in vitro is established. Obtaining a large number of stable bone marrow mesenchymal stem cells is of great theoretical significance and practical value. Objective: to establish a simple and feasible method for in vitro culture and amplification of bone marrow mesenchymal stem cells. A large number of stable bone marrow mesenchymal stem cells were obtained. Methods: bone marrow mesenchymal stem cells of SD rats were isolated and cultured in vitro by density gradient centrifugation and adherent separation screening. The ultrastructure of cells was observed by transmission electron microscope, the number of living cells was calculated by trypanosome blue exclusion method, the cell growth curve was drawn, the cell cycle was analyzed by flow cytometry, and the expression of c-kit and CD45 was detected by immunocytochemistry. Results and conclusion the adherent cells were observed under inverted optical microscope 24 h after inoculation and the cells could reach 90% fusion at 14 d after 14 d of colony formation. After passage, the cells tend to converge. The mesenchymal stem cells of the third generation were larger in size, obvious in nucleoli, sparse in chromatin distribution, low in electron density, and microvilli on the surface of the cells. There were abundant ribosomes in the cells, but few organelles such as endoplasmic reticulum, mitochondria and Golgi complex, which indicated that the number of cells in the third generation of bone marrow mesenchymal stem cells was decreased on the first day after inoculation. On the second day, the cells began to increase, on the third day the cells entered the exponential proliferative phase, on the 7th day into the plateau phase, and on the 9th day, the number of cells began to decrease. The growth curve of the third generation bone marrow mesenchymal stem cells was "S". 4. After DNA staining, flow cytometry was used to determine its DNA content. The ratio of S phase cells was 21.1.5 immunocytochemical staining and flow cytometry was used to prove c-kit positive fine cells. The positive rate of CD45 expression was 1.68.6 bone marrow mesenchymal stem cells differentiated into osteoblasts 16 days after induction. The cell body is elliptical, with short processes connected to each other, and the cytoplasm is dark, suggesting that the cells may be rich in rough endoplasmic reticulum and Golgi complex. The results showed that the bone marrow mesenchymal stem cells obtained by this method were stable in growth and active in proliferation.
【作者單位】： 武警后勤學(xué)院臨床醫(yī)學(xué)系實驗技術(shù)教研室;
【分類號】：R329

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