本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：全骨髓培養(yǎng)法　出處：《新疆醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：研究大鼠骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cell, BMSCs)的不同培養(yǎng)方法的差異，及對(duì)培養(yǎng)出的不同代數(shù)的細(xì)胞做形態(tài)學(xué)觀察及表面標(biāo)記物鑒定。為今后向?qū)嶒?yàn)提供高純度的BMSCs提供方法及依據(jù)。方法：取150g~180g左右2只雄性SD大鼠后肢股骨、脛骨骨髓，分別行全骨髓培養(yǎng)法和密度密度梯度離心法培養(yǎng)。將2種方法分離出的細(xì)胞進(jìn)行貼壁細(xì)胞的篩選并培養(yǎng)，利用BMSCs的易于貼附于塑料制品的特性給予多次傳代純化。觀察兩種方法培養(yǎng)的不同代數(shù)的細(xì)胞形態(tài)，繪制貼壁的細(xì)胞生長曲線，流式細(xì)胞儀檢測P5代cd34、cd29、cd44、cd45細(xì)胞因子，鑒定是否為骨髓間充質(zhì)干細(xì)胞。結(jié)果：形態(tài)學(xué)學(xué)觀察上可見2種方法所獲取的細(xì)胞呈長梭形，呈現(xiàn)特征性的漩渦狀生長。繪制2種方法P0至P10代的細(xì)胞增殖生長曲線圖，以每一代單皿傳代數(shù)量為檢測指標(biāo)進(jìn)行單樣本配對(duì)T檢驗(yàn)分析結(jié)果為：P＜0.05。證明全骨髓培養(yǎng)法細(xì)胞增殖比較快。對(duì)P3代2組細(xì)胞進(jìn)行流式細(xì)胞檢測結(jié)果CD34、CD45陰性，CD29、CD44陽性。證明2種方法培養(yǎng)的細(xì)胞符合BMSCs的特性。結(jié)論：全骨髓培養(yǎng)方法相比于密度密度梯度離心法不僅可以成功分離骨髓間充質(zhì)干細(xì)胞而且對(duì)試劑要求少，更加經(jīng)濟(jì)，操作更簡單、易行，，培養(yǎng)所需周期較短。全骨髓培養(yǎng)法培養(yǎng)出的BMSCs并在5~10代時(shí)的細(xì)胞純度高，活性好且BMSCs純度較穩(wěn)定，可進(jìn)行BMSCs多向分化誘導(dǎo)及應(yīng)用BMSCs進(jìn)行動(dòng)物實(shí)驗(yàn)研究及臨床應(yīng)用。
[Abstract]:Objective: to study the different culture methods of bone marrow mesenchymal stem cells (BMSCs) in rat bone marrow mesenchymal stem cells (BMSCs). Morphological observation and surface marker identification of cultured cells from different generations were performed. Methods: the femur and tibial bone marrow of two male SD rats with 150g~180g were taken from the femur and tibial bone marrow of two male SD rats, and the method and basis of high purity BMSCs were provided for the future experiment. The whole bone marrow culture method and density gradient centrifugation method were used respectively. The cells isolated from the two methods were screened and cultured for adherent cells. The cell morphology of different generations was observed and the cell growth curve of adherent cells was plotted. Flow cytometry was used to detect the cytokines of cd34cd29cd4cd4cd45 in P5 passage. Results: according to the morphological observation, the cells obtained by the two methods were spindle-shaped, showing characteristic whirlpool growth. The cell proliferation and growth curves of P0 to P10 passages of the two methods were plotted. The result of single sample paired T test was: 1: P < 0.05, which proved that the whole bone marrow cell proliferation was faster. Flow cytometry was used to detect CD34, CD45 negative and CD29 CD44 positive in P3 passage 2 cells. Conclusion: compared with density density gradient centrifugation, the whole bone marrow culture method can not only successfully isolate bone marrow mesenchymal stem cells, but also require less reagent. BMSCs cultured by whole bone marrow culture has high purity, good activity and stable purity of BMSCs at 5 ~ 10 passages. It can be used to induce multidirectional differentiation of BMSCs and to use BMSCs for animal experimental study and clinical application.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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