全骨髓法培養(yǎng)大鼠骨髓間充質(zhì)干細胞及其生物學特性

1、全骨髓法培養(yǎng)大鼠骨髓間充質(zhì)干細胞及其生物學特性         10-11-01 14:00:00     編輯：studa20                     作者：豐炳峰，董晨，關(guān)鳳軍【摘要】  目的 建立一種簡便有效的體外分離純化及培養(yǎng)擴增大鼠骨髓間充質(zhì)干

2、細胞(MSC) 的方法，研究MSC的生物學特性。方法 貼壁培養(yǎng)法分離純化大鼠MSC，體外培養(yǎng)和連續(xù)傳代, 在倒置顯微鏡下連續(xù)觀察細胞的形態(tài)變化；利用四唑鹽比色（MTT）法測定MSC的生長曲線；流式細胞儀（FCM）鑒定MSC膜抗原。結(jié)果 原代分離的MSC在接種后48 h貼壁,細胞形態(tài)為橢圓形、多角形及短梭形,12天時細胞呈長梭形并達到90%單層融合。經(jīng)傳代擴增,細胞進一步純化。細胞傳代后2天內(nèi)處于潛伏期, 3天后進入生長期,7天后進入平臺期。FCM檢測CD45、CD90陽性率分別為19.60%、95.38%。結(jié)論 貼壁培養(yǎng)法能有效分離純化大鼠MSC,用此方法培養(yǎng)的細胞生長穩(wěn)定,增殖能力活躍,具有

3、MSC的一般生物學特性,為其成為組織工程理想的種子細胞提供了進一步的支持。 【關(guān)鍵詞】  間充質(zhì)干細胞;骨髓;貼壁法分離培養(yǎng)    Abstract:Objective  To establish a simple and effective method of isolation, purification and cultivation of rat bone marrow-derived mesenchymal stem cells (MSC) in vitro, and explore their biological characte

4、ristics.Methods  MSC were isolated and purified for culture in vitro and serial passage by the attachment culture method. The morphological changes of the MSC were continuously observed under the inverted microscope. The cell growth curve was measured by MTT method, and membrane antigens were d

5、etected with flow cytometry (FCM).Results  The MSC in primary culture which were adhered to plastic surface within 48 h after displacement took the oval, asteroid, short and fusiform shapes. 12 days after culture in DMEM-F12 medium containing 10% FBS, the cells reached 90% confluence in single

6、layers, taking a long and fusiform shape. Further purification was achieved by expansion at serial passages. MSC were in latency for 2 days, converted into growth period on the 3rd day and entered the stationary phase on the 7 th day. FCM results indicated that the positive rate of CD45 and CD90 was

7、 19.60% and 95.38%, respectively.Conclusion  The attachment culture method can be effectively used to isolate and purify rat MSC. The cultured MSC are stable in growth with active proliferation and share the general biological characteristics of MSC, which will further make them ideal seed cell

8、s for tissue engineering.    Key words: mesenchymal stem cells; bone marrow; attachment culture骨髓間充質(zhì)干細胞(mesenchymal stem cells, MSC) 是骨髓內(nèi)除造血干細胞外另一種具有多向分化潛能的成體干細胞，在特定的條件下能分化為多種組織細胞，如成骨細胞、軟骨細胞、肌腱、脂肪細胞、成纖維細胞及神經(jīng)星狀細胞等，且具有極強的自我修復能力。加之其具有獲取簡便、對供體損傷小、增殖力強、來源廣泛、免疫原性弱1、自體移植不發(fā)生排斥反應2、不涉及倫理等特點，成為研

9、究的熱點。本實驗通過分離培養(yǎng)、鑒定大鼠MSC，研究其生物學特性，從而為MSC的體外定向分化及作為種子細胞進一步應用于組織工程和再生醫(yī)學提供實驗基礎(chǔ)。1  材料和方法1.1  材料  DMEM-F12培養(yǎng)基(Gibco公司),優(yōu)級胎牛血清(杭州四季青有限公司) ，蛋白酶(Sigma公司),倒置顯微鏡(Nikon公司)，細胞培養(yǎng)箱(Sanyo公司)。實驗動物為雄性SD大鼠, 3周齡, 100150 g, 清潔級(徐州醫(yī)學院實驗動物中心提供)。FITC 標記CD45、CD90抗體(北京中杉金橋生物技術(shù)有限公司)。1.2  方法2  結(jié)果2.1

10、0; MSC的分離培養(yǎng)、擴增結(jié)果  將分離的未離心的MSC懸液直接接種培養(yǎng)24 h后,發(fā)現(xiàn)培養(yǎng)瓶內(nèi)有大量懸浮細胞,主要為圓形紅細胞。72 h行首次半量換液后仍不易觀察MSC的貼壁情況,將首次換液的吸出液在新培養(yǎng)瓶內(nèi)加培養(yǎng)液繼續(xù)培養(yǎng),均經(jīng)過23次換液后,漂浮的不貼壁細胞大部分隨換液除去,可見較多的貼壁細胞, 散在生長,分布不均,少部分呈集落狀或單個細胞存在,胞體呈長梭形,兩端有長突起,胞質(zhì)內(nèi)顆粒較多,折光性差,核不清楚。貼壁細胞前12天生長緩慢,約3天后明顯增殖,細胞形態(tài)更加清晰,核明顯可見,并有12個核仁（圖1A）。在第7天時,形成明顯克隆，在1014天時,血細胞隨換液全部清除,貼壁

11、細胞形態(tài)比較一致,已基本貼滿培養(yǎng)孔板底部,呈漩渦狀或輻射狀排列,細胞變得扁平,胞體狹長,核呈橢圓形（圖1B）,進行第1次傳代培養(yǎng)。傳3代后培養(yǎng)的MSC(圖1C) 形態(tài)上更加趨于一致,為成纖維細胞樣,無原代存有的圓形或類圓形細胞,增殖速度較原代細胞快,形態(tài)無明顯變化,每隔3天換液1次,57天傳代1次。部分細胞傳代10次后, 就變得寬大扁平，增殖速度減慢。圖1  MSC的形態(tài)學特點（倒置相差顯微鏡,×100）（略）    A. 原代3天MSC貼壁生長;B. 原代14天MSC長滿瓶底90%以上;C.第3代MSC2.2  大鼠骨髓MSC生長規(guī)律  MSC傳代7代以前增殖速度