本文選題：微環(huán)境 + 細(xì)胞培養(yǎng)�。� 參考：《鄭州大學(xué)》2012年碩士論文

【摘要】：骨髓間充質(zhì)干細(xì)胞(bone mesenchymal stem cells, BMSCs)也稱骨髓基質(zhì)來源干細(xì)胞,是存在于骨髓中除造血干細(xì)胞以外另一類具有多向分化潛能的干細(xì)胞。在一定誘導(dǎo)條件下,這類細(xì)胞可定向分化為多種造血以外的組織,特別是中胚層和神經(jīng)外胚層來源的組織細(xì)胞,例如成骨細(xì)胞、成軟骨細(xì)胞、脂肪細(xì)胞、腱細(xì)胞、肌肉細(xì)胞、神經(jīng)細(xì)胞等。另外間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)體外獲取容易,創(chuàng)傷微小,具有無限增殖和多向分化的潛能,再加上BMSCs具有貼壁生長的特性,在體外易分離和擴(kuò)增,還易于外源基因的轉(zhuǎn)入和表達(dá),所以BMSCs受到醫(yī)學(xué)界的青睞,它成為一種理想的細(xì)胞治療工具和基因治療的靶細(xì)胞。開創(chuàng)了再生醫(yī)學(xué)和組織工程的先河。然而間充質(zhì)干細(xì)胞在骨髓中的含量很低,需要進(jìn)行體外分離純化和大量增殖。這是目前面臨的主要問題。 目的 通過觀察不同血清微環(huán)境對大鼠骨髓間充質(zhì)干細(xì)胞的生長增殖狀態(tài)的影響得到一種更適合BMSCs體外生長的環(huán)境,以利于改良骨髓間充質(zhì)干細(xì)胞體外培養(yǎng)的方法,提高體外培養(yǎng)的獲得率,以便于其體外擴(kuò)增和純化,更好的應(yīng)用于臨床治療。 方法 自6-8W齡SD雄性大鼠股骨、脛骨和腓骨中分離提取骨髓間充質(zhì)干細(xì)胞。采用全骨髓貼壁法培養(yǎng)。分別給于以下三種血清微環(huán)境進(jìn)行培養(yǎng)：同種異體血清組：分離提取細(xì)胞后原代用含10%大鼠同種異體血清的培養(yǎng)基培養(yǎng),以后換液傳代時(shí)用含10%胎牛血清的培養(yǎng)基培養(yǎng)；胎牛血清組：分離提取細(xì)胞后原代用含10%胎牛血清的培養(yǎng)基培養(yǎng),以后換液傳代時(shí)仍用含10%胎牛血清的培養(yǎng)基培養(yǎng)；無血清組：分離提取細(xì)胞后原代用不含胎牛血清的DMEM培養(yǎng)基培養(yǎng),以后換液傳代時(shí)用含10%胎牛血清的培養(yǎng)基培養(yǎng)。在倒置顯微鏡下觀察不同血清微環(huán)境下骨髓間充質(zhì)干細(xì)胞的生長形態(tài)、細(xì)胞生長曲線,流式細(xì)胞儀檢測不同血清微環(huán)境培養(yǎng)條件下的細(xì)胞表面分子CD45和CD90的表達(dá)。應(yīng)用SPSS17.0軟件對數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果 大鼠同種異體血清微環(huán)境下培養(yǎng)細(xì)胞形態(tài)較小,呈紡錘狀,網(wǎng)狀生長。10%胎牛血清微環(huán)境下培養(yǎng)細(xì)胞呈梭形成纖維樣,集落式生長。無血清微環(huán)境下培養(yǎng)細(xì)胞稀少,只有稍許集落式生長。生長曲線中大鼠同種異體血清組倍增速度最快,其次為10%胎牛血清組,而無血清組未見明顯倍增速率。流式細(xì)胞儀檢測含血清培養(yǎng)基培養(yǎng)條件下各組第三代細(xì)胞的CD90陽性表達(dá)率均在80%以上,CD45陽性表達(dá)率在2%以下。而在無血清培養(yǎng)條件下CD90陽性表達(dá)率在74.09%,CD45陽性表達(dá)率卻在6.09%。這說明無血清微環(huán)境下培養(yǎng)的細(xì)胞純度明顯低于含血清微環(huán)境下培養(yǎng)的細(xì)胞。 結(jié)論 在含體積分?jǐn)?shù)為10%的大鼠同種異體血清微環(huán)境培養(yǎng)條件下所培養(yǎng)的細(xì)胞形態(tài)均一性好,融合度均高于其他組,且細(xì)胞首次傳代的時(shí)間明顯短于其他血清微環(huán)境。,且含體積分?jǐn)?shù)為10%的大鼠同種異體血清和10%胎牛血清均適合骨髓間充質(zhì)干細(xì)胞的生長,但在同種異體血清微環(huán)境下細(xì)胞增殖更快,而在無血清培養(yǎng)條件下未見明顯增殖現(xiàn)象。根據(jù)以上結(jié)果表明同種異體血清微環(huán)境下更適于骨髓間充質(zhì)干細(xì)胞的生長和純化,更有利于體外培養(yǎng)方法的改良。
[Abstract]:Bone marrow mesenchymal stem cells ( MSCs ) are also known as bone marrow stromal - derived stem cells , which are derived from bone marrow stromal - derived stem cells . These cells can be directionally differentiated into cells other than hematopoietic stem cells in the bone marrow . These cells can be directionally differentiated into various hematopoietic tissues , such as osteoblasts , chondrocytes , adipocytes , tendon cells , muscle cells , nerve cells , etc .

Purpose

by observing the effect of different serum microenvironment on the growth and proliferation state of bone marrow mesenchymal stem cells in rats , a method is more suitable for the in vitro growth of bone marrow mesenchymal stem cells , thereby being beneficial to improving the in vitro culture method of the bone marrow mesenchymal stem cells , improving the obtaining rate of in vitro culture so as to facilitate in vitro amplification and purification , and better application to clinical treatment .

method

Bone marrow mesenchymal stem cells were isolated from femur , tibia and fibula from 6 - 8 W - old SD male rats . All three kinds of serum micro - environments were cultured : allogeneic serum group : culture medium containing 10 % rat allogeneic serum after isolation and extraction of cells , and culture medium supplemented with 10 % fetal bovine serum at the later passage ;
Fetal bovine serum group : culture medium containing 10 % fetal bovine serum after isolation and extraction of cells was cultured in culture medium containing 10 % fetal bovine serum at the time of subculture ;
The growth form , cell growth curve and flow cytometry of bone marrow mesenchymal stem cells in different serum microenvironment were observed under reversed microscope .

Results

The results showed that the rate of CD90 positive expression of the third generation cells was above 80 % and the expression rate of CD90 was lower than 2 % under the condition of serum - free culture .

Conclusion

The results showed that the growth and purification of bone marrow mesenchymal stem cells were more suitable for the growth and purification of bone marrow mesenchymal stem cells .

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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相關(guān)期刊論文 前4條

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本文編號：1808937