全骨髓法培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞及其生物學(xué)特性

1、全骨髓法培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞及其生物學(xué)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(jiǎn)便有效的體外分離純化及培養(yǎng)擴(kuò)增大鼠骨髓間充質(zhì)干

2、細(xì)胞(MSC) 的方法，研究MSC的生物學(xué)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(xì)胞的形態(tài)變化；利用四唑鹽比色（MTT）法測(cè)定MSC的生長(zhǎng)曲線；流式細(xì)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(xì)胞形態(tài)為橢圓形、多角形及短梭形,12天時(shí)細(xì)胞呈長(zhǎng)梭形并達(dá)到90%單層融合。經(jīng)傳代擴(kuò)增,細(xì)胞進(jìn)一步純化。細(xì)胞傳代后2天內(nèi)處于潛伏期, 3天后進(jìn)入生長(zhǎng)期,7天后進(jìn)入平臺(tái)期。FCM檢測(cè)CD45、CD90陽(yáng)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(xì)胞生長(zhǎng)穩(wěn)定,增殖能力活躍,具有

3、MSC的一般生物學(xué)特性,為其成為組織工程理想的種子細(xì)胞提供了進(jìn)一步的支持。 【關(guān)鍵詞】  間充質(zhì)干細(xì)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(xì)胞(mesenchymal stem cells, MSC) 是骨髓內(nèi)除造血干細(xì)胞外另一種具有多向分化潛能的成體干細(xì)胞，在特定的條件下能分化為多種組織細(xì)胞，如成骨細(xì)胞、軟骨細(xì)胞、肌腱、脂肪細(xì)胞、成纖維細(xì)胞及神經(jīng)星狀細(xì)胞等，且具有極強(qiáng)的自我修復(fù)能力。加之其具有獲取簡(jiǎn)便、對(duì)供體損傷小、增殖力強(qiáng)、來(lái)源廣泛、免疫原性弱1、自體移植不發(fā)生排斥反應(yīng)2、不涉及倫理等特點(diǎn)，成為研

9、究的熱點(diǎn)。本實(shí)驗(yàn)通過(guò)分離培養(yǎng)、鑒定大鼠MSC，研究其生物學(xué)特性，從而為MSC的體外定向分化及作為種子細(xì)胞進(jìn)一步應(yīng)用于組織工程和再生醫(yī)學(xué)提供實(shí)驗(yàn)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(jí)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(xì)胞培養(yǎng)箱(Sanyo公司)。實(shí)驗(yàn)動(dòng)物為雄性SD大鼠, 3周齡, 100150 g, 清潔級(jí)(徐州醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供)。FITC 標(biāo)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(kuò)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?jī)?nèi)有大量懸浮細(xì)胞,主要為圓形紅細(xì)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?jī)?nèi)加培養(yǎng)液繼續(xù)培養(yǎng),均經(jīng)過(guò)23次換液后,漂浮的不貼壁細(xì)胞大部分隨換液除去,可見(jiàn)較多的貼壁細(xì)胞, 散在生長(zhǎng),分布不均,少部分呈集落狀或單個(gè)細(xì)胞存在,胞體呈長(zhǎng)梭形,兩端有長(zhǎng)突起,胞質(zhì)內(nèi)顆粒較多,折光性差,核不清楚。貼壁細(xì)胞前12天生長(zhǎng)緩慢,約3天后明顯增殖,細(xì)胞形態(tài)更加清晰,核明顯可見(jiàn),并有12個(gè)核仁（圖1A）。在第7天時(shí),形成明顯克隆，在1014天時(shí),血細(xì)胞隨換液全部清除,貼壁

11、細(xì)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(xì)胞變得扁平,胞體狹長(zhǎng),核呈橢圓形（圖1B）,進(jìn)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(xì)胞樣,無(wú)原代存有的圓形或類圓形細(xì)胞,增殖速度較原代細(xì)胞快,形態(tài)無(wú)明顯變化,每隔3天換液1次,57天傳代1次。部分細(xì)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(xué)特點(diǎn)（倒置相差顯微鏡,×100）（略）    A. 原代3天MSC貼壁生長(zhǎng);B. 原代14天MSC長(zhǎng)滿瓶底90%以上;C.第3代MSC2.2  大鼠骨髓MSC生長(zhǎng)規(guī)律  MSC傳代7代以前增殖速度