本文選題：干細(xì)胞 + 微團(tuán)培養(yǎng)��； 參考：《中國醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的 比較脂肪干細(xì)胞在BMP-7存在時(shí)平面培養(yǎng)和微團(tuán)培養(yǎng)兩種方式下誘導(dǎo)分化成軟骨細(xì)胞的情況,探討不同培養(yǎng)方式對脂肪干細(xì)胞成軟骨能力的影響。尋求一種更好的促進(jìn)脂肪干細(xì)胞成軟骨分化的培養(yǎng)方式。 方法 細(xì)胞的分離和擴(kuò)增,選用6周齡健康Wistar雄性大鼠,無菌條件下取大鼠雙側(cè)腹股溝脂肪組織,眼科剪剪碎,0.1%的膠原酶37度震蕩消化20分鐘,含10%胎牛血清的DMEM終止消化,1500g離心10min,含10%胎牛血清的DMEM重懸,接種于培養(yǎng)瓶中培養(yǎng)。每3天換液一次,待細(xì)胞生長至接近融合時(shí)胰酶/EDTA消化傳代。將培養(yǎng)至第3代細(xì)胞胰酶/EDTA消化后計(jì)數(shù),調(diào)整密度為1×10~5/ml,在BMP-7存在條件下進(jìn)行平面和微團(tuán)培養(yǎng)。指標(biāo)檢測(1)光鏡觀察原代脂肪干細(xì)胞及誘導(dǎo)7,21天后的生長情況,流式細(xì)胞學(xué)檢測脂肪間充質(zhì)干細(xì)胞的表面標(biāo)志;(2)MTT法檢測不同培養(yǎng)方式下脂肪干細(xì)胞的增殖能力;(3)免疫組織化學(xué)染色檢測培養(yǎng)三周后兩組Ⅱ型膠原表達(dá),RT-PCR檢測誘導(dǎo)14,21天后平面和微團(tuán)培養(yǎng)組軟骨特異蛋白聚糖和Ⅱ型膠原mRNA的表達(dá)及肥大軟骨細(xì)胞的標(biāo)志X型膠原mRNA的表達(dá)。 結(jié)果 脂肪干細(xì)胞的形態(tài)特征原代脂肪干細(xì)胞接種24小時(shí)后光鏡下可見有少量的細(xì)胞貼壁,形態(tài)為短梭形或多角形,48小時(shí)后貼壁細(xì)胞明顯增多,約9天左右細(xì)胞融合超過90%。誘導(dǎo)7天后平面培養(yǎng)組細(xì)胞呈長梭形,微團(tuán)培養(yǎng)組呈短梭形。誘導(dǎo)21天后兩組細(xì)胞形態(tài)均為圓形。流式細(xì)胞儀檢測表明,脂肪間充質(zhì)干細(xì)胞高表達(dá)CD29和CD44,而CD34,CD45的表達(dá)均不足2%。 MTT檢測結(jié)果表明平面培養(yǎng)組細(xì)胞增殖趨勢平緩,10天左右進(jìn)入平臺(tái)期。而微團(tuán)培養(yǎng)組各檢測點(diǎn)MTT值均顯著高于平面培養(yǎng)組,二者統(tǒng)計(jì)學(xué)有顯著性(P＜0.05),表示微團(tuán)組細(xì)胞的增殖要明顯快于平面培養(yǎng)組,且微團(tuán)培養(yǎng)組從生長曲線上看未見明顯的平臺(tái)期。 免疫組化結(jié)果顯示,培養(yǎng)三周后微團(tuán)組Ⅱ型膠原表達(dá)強(qiáng)陽性而平面組表達(dá)弱陽性,RT-PCR結(jié)果顯示培養(yǎng)14和21天后兩組均有軟骨特異蛋白聚糖和Ⅱ型膠原mRNA的表達(dá),但微團(tuán)組表達(dá)較強(qiáng)。而微團(tuán)組培養(yǎng)21天時(shí)其X型膠原mRNA的表達(dá)弱于平面組。 結(jié)論 在BMP-7誘導(dǎo)下脂肪干細(xì)胞可以向軟骨細(xì)胞分化,同時(shí)微團(tuán)培養(yǎng)比平面培養(yǎng)更能促進(jìn)脂肪干細(xì)胞的增殖、分化,并能更好的維持軟骨細(xì)胞的表型,可以作為組織工程構(gòu)建軟骨的理想選擇。
[Abstract]:Objective to compare the differentiation of adipose stem cells into chondrocytes in the presence of BMP-7 and to explore the effects of different culture methods on the chondrogenic ability of adipose stem cells. To seek a better way to promote the differentiation of adipose stem cells into cartilage. Methods the cells were isolated and expanded. Healthy male Wistar rats aged 6 weeks were selected and bilateral inguinal adipose tissue was harvested under aseptic condition. The tissue was digested by 37 degree shock of 0.1% collagenase in ophthalmic shears for 20 minutes. DMEM containing 10% fetal bovine serum was digested by 1500g and centrifuged for 10 min. The DMEM containing 10% fetal bovine serum was suspended and inoculated in culture flask. Once every 3 days, trypsin / EDTA digested and subcultured when the cells grew close to fusion. After digesting trypsin / EDTA at the third passage, the cell density was adjusted to 1 脳 10 ~ (5) / ml, and the flat and microclusters were cultured in the presence of BMP-7. The primary adipose stem cells were observed under light microscope and the growth of primary adipose stem cells was observed 21 days after induction. Proliferation of adipose Mesenchymal Stem cells detected by flow Cytometry and MTT method) Immunohistochemical staining was used to detect the expression of type 鈪，

本文編號(hào)：2049305