北京生物醫(yī)學(xué)工程

豬骨髓間質(zhì)干細(xì)胞分離培養(yǎng)及表面抗原鑒定

Isolation, Expansion, and Antigen Expression of Porcine Bone Marrow Mesenchymal Stem Cells

作者：韓志軍任華

單位：中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院 (北京100730)

關(guān)鍵詞：豬；骨髓間充質(zhì)干細(xì)胞；細(xì)胞培養(yǎng)；抗原表達(dá)

分類號：

出版年·卷·期（頁碼）：2011·30·3（291-295）

摘要：

目的建立豬骨髓間質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMMSC)體外分離培養(yǎng)、純化的方法，觀察其生物學(xué)特性，為其作為組織工程學(xué)種子細(xì)胞提供實(shí)驗(yàn)基礎(chǔ)。方法取豬的胸骨骨髓6～8ml,用Ficoll淋巴細(xì)胞分離液提取單核細(xì)胞,采用貼壁法培養(yǎng)。接種后2h利用倒置顯微鏡觀察細(xì)胞的大體形態(tài)，并利用流式細(xì)胞儀檢測細(xì)胞抗原表達(dá)。結(jié)果原代培養(yǎng)接種3～4h后細(xì)胞開始貼壁，24h后貼壁細(xì)胞數(shù)量明顯增多，3~4d后細(xì)胞呈紡錘形且形成為單個或數(shù)個細(xì)胞克隆，7～9d后集落迅速增多，細(xì)胞融合，在10～12d細(xì)胞鋪滿全層。流式細(xì)胞儀監(jiān)測到BMMSC表達(dá)CD29、CD44、CD105、KDR，不表達(dá)HLA-DR、CD11a、CD14、CD31、CD34、CD45。結(jié)論采用密度梯度離心法培養(yǎng)豬的BMMSC生長穩(wěn)定,增殖能力強(qiáng)，具有間質(zhì)干細(xì)胞的表面抗原特征，該方法可作為培養(yǎng)豬BMMSC的常規(guī)方法。

Objective To establish the methods of isolation, expansion, and purification of porcine bone marrow mesenchymal stem cells (BMMSC) in vitro, and provide the experimental basis for BMMSC as tissue engineering seed cells by observing its biology characteristic. Method A volume of 6-8ml porcine bone marrow was aspirated from sternum. The mononuclear cells were harvested using Ficoll lymphocyte separating medium at a density of 1.077. the BMSC were cultured by the adherence method. Cellular morphology was observed by microscope, and cellular surface antigen expression was examined by the instrument flow cytometry. Result After seeded 3-4 hours, cells adhered to the bottom. Twenty-four hours later, the number of the adhered cells significantly increased, and 3-5 days later, the primary cultured cells were in uniformly long spindle-shaped form and formed cell-colony. After seeded 7-9 days, the cell-colonies rapidly increased, and 10-12 days later, cells spread to the bottom. The test of Flow cytometry showed that the BMMSC were positive for CD29,CD44,CD105 and KDR, while negative for HLA-DR, CD11a, CD14, CD31, CD34 or CD45. Conclusion By density gradient centrifugation and adherence method, BMMSC of porcine were obtained easily and proliferate rapidly, which showed the antigen characteristics of mesenchymal stem cell. The proposal method could be used as a routine method for BMMSC preparation.

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