本文關(guān)鍵詞：CD45，，CD90對(duì)分離得到大鼠骨髓間充質(zhì)干細(xì)胞純度的鑒定　出處：《中國(guó)醫(yī)科大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 密度梯度離心法 貼壁法 分離 鑒定 CD45 CD90

【摘要】： 目的 探討大鼠骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BW-MSCs)的分離方法;應(yīng)用流式細(xì)胞儀,用小鼠抗大鼠CD45, CD90檢測(cè)分離得到BW-MSCs的純度。 方法 在無(wú)菌條件下,采集雙側(cè)股骨,脛骨,用適量DMED／F12培養(yǎng)液沖出骨髓。用比重為1.073g／ml的Percol1分離液分離,取界面層,獲取單個(gè)核細(xì)胞,用PBS液以1 000r／min離心洗滌,每次10 min,共2次。分離的細(xì)胞以5×104／cm2個(gè)接種于培養(yǎng)瓶。培養(yǎng)體系為高糖EMDM/F12(1:1),20%胎牛血清。在37℃,體積分?jǐn)?shù)為5%CO2條件下培養(yǎng),首次24 h更換培養(yǎng)液,去掉未貼壁細(xì)胞。每3d換液1次。每天在倒置相差顯微鏡下觀察細(xì)胞形態(tài)。當(dāng)細(xì)胞達(dá)到80%融合時(shí),用1：1的0.1%胰蛋白酶和0.01%EDTA混合液消化,用含2%小牛血清的PBS洗滌后制成1.0×107／ml骨髓MSCs的單細(xì)胞懸液,加入EP管中。之后按說(shuō)明,取一定量CD90-FITC和CD45-PE加入EP管中。4℃避光孵育30min,流式細(xì)胞儀檢測(cè)。 結(jié)果 1、采用密度梯度離心法和貼壁法相結(jié)合可從大鼠骨髓中分離出骨髓間充質(zhì)干細(xì)胞。新鮮分離細(xì)胞在倒置顯微鏡下觀察呈圓形,大小不均一。24 h細(xì)胞貼壁,更換培養(yǎng)液后,見(jiàn)貼壁細(xì)胞呈圓形,橢圓形,多邊形。48 h見(jiàn)貼壁細(xì)胞體積變大,大小不均一,其漿核分界清楚,折光性強(qiáng)。絕大多數(shù)為單核,圓形,位于細(xì)胞中心位置。而后細(xì)胞形態(tài)仍然以圓形,橢圓形為主,長(zhǎng)梭形的細(xì)胞逐漸出現(xiàn)。10-12天后大部分細(xì)胞變?yōu)殚L(zhǎng)梭形,少部分呈多角形。12天左右可達(dá)80%融合。2、密度梯度離心法分離和貼壁法相結(jié)合得到的細(xì)胞,經(jīng)小鼠抗大鼠CD90-FITC和CD45-PE雙染后,應(yīng)用流式細(xì)胞儀檢測(cè)三次實(shí)驗(yàn)分離得到的細(xì)胞,CD9+CD45細(xì)胞的百分比40.02%,43.80%,42.63%。 結(jié)論 1、用比重為1.073g／ml的Percoll分離液離心之后貼壁培養(yǎng)可從大鼠股骨脛骨中分離得到骨髓間充質(zhì)干細(xì)胞。2、CD45, CD90雙染鑒定密度梯度離心法與貼壁法相結(jié)合分離得到骨髓間充質(zhì)干細(xì)胞的百分比約為42.15%。
[Abstract]:Purpose To investigate the isolation method of bone marrow mesenchymal stem cells (BW-MSCs) from rat bone marrow mesenchymal stem cells (BMSCs). The purity of BW-MSCs was determined by flow cytometry, anti-rat CD-45 and CD90. Method Under aseptic condition, bilateral femur and tibia were collected and the bone marrow was washed out with proper amount of DMED/F12 culture medium. The interfacial layer was separated with 1.073 g / ml Percol1 separation solution. Mononuclear cells were obtained and washed by centrifugation with PBS solution for 10 min each time. The isolated cells were inoculated in culture flask with 5 脳 10 4 / cm 2 cells for 2 times. The culture system was high sugar EMDM / F12: 1: 1% fetal bovine serum at 37 鈩

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