【摘要】： 目的本文研究大鼠脂肪干細(xì)胞(adipose-derived stem cells ADSCs)在體外分離培養(yǎng)、誘導(dǎo)分化為成軟骨的能力,并初步觀察不同濃度(0μg／m、10μg／ml、50μg／ml、100μg／ml、200μg／ml)慶大霉素對(duì)其增殖、分化成軟骨的影響。 方法從3周齡SD大鼠腹股溝脂肪墊分離出脂肪組織,通過(guò)Ⅰ型膠原酶消化法分離干細(xì)胞,型膠原酶消化法分離干細(xì)胞,接種于含有胎牛血清的DMEM培養(yǎng)液中分離傳代。每日以倒置顯微鏡觀察細(xì)胞形態(tài)及生長(zhǎng)情況。取第三代脂肪干細(xì)胞分五組,包含一個(gè)對(duì)照組(培養(yǎng)液為人脂肪干細(xì)胞完全培養(yǎng)基：高糖DMEM、10%胎牛血清、50nmol／L抗壞血酸、6.25mg／L胰島素、100U／ml青霉素、100μg／ml鏈霉素、100nmol／L地塞米松,10μg／L轉(zhuǎn)化生長(zhǎng)因子β1,不含慶大霉素)和四個(gè)含慶大霉素濃度組(分別是10μg／ml、50μg／ml、100μg／ml、200μg／ml加人脂肪干細(xì)胞完全培養(yǎng)基)。采用MTT比色法進(jìn)行增殖活性比較,Ⅱ型膠原免疫細(xì)胞化學(xué)染色和細(xì)胞甲苯胺藍(lán)染色檢測(cè)成軟骨分化情況。 結(jié)果1.體外培養(yǎng)的脂肪間充質(zhì)干細(xì)胞呈扁平的梭形成纖維樣細(xì)胞生長(zhǎng),且細(xì)胞形態(tài)均一,傳代穩(wěn)定。2.MTT實(shí)驗(yàn)顯示慶大霉素在100μg／ml、200μg／ml抑制細(xì)胞增殖,其余各濃度組對(duì)細(xì)胞增殖無(wú)明顯影響3.誘導(dǎo)后細(xì)胞甲苯胺藍(lán)染色顯示100μg／ml組及200μg／ml組細(xì)胞細(xì)胞染色較淺,大部分細(xì)胞失染,余各組陽(yáng)性表達(dá)：Ⅱ型膠原免疫組織化學(xué)染色顯示100μg／ml組及200μg／ml組細(xì)胞弱陽(yáng)性表達(dá),余各組陽(yáng)性表達(dá)。 結(jié)論1大鼠脂肪干細(xì)胞易取材,培養(yǎng)傳代后增殖迅速,生長(zhǎng)穩(wěn)定。2大鼠脂肪干細(xì)胞能向軟骨細(xì)胞誘導(dǎo)分化。3慶大霉素在一定濃度(10μg／ml、50μg／ml)下對(duì)脂肪干細(xì)胞的增殖和軟骨分化沒(méi)有影響,但高于此濃度(100μg／ml,200μg／ml)則抑制脂肪干細(xì)胞的增殖和軟骨分化能力。
[Abstract]:Objective to study the ability of rat adipose stem cells (adipose-derived stem cells ADSCs) to differentiate into chondrocytes in vitro, and to observe the effects of different concentrations (0 渭 g / m ~ (10) 渭 g / ml ~ (10) 渭 g 路ml ~ (-1) of 50 渭 g / ml / ml ~ (100 渭 g / ml) on the differentiation of rat adipose stem cells into cartilage. Effects of gentamicin on proliferation and differentiation of cartilage. Methods Adipose tissue was isolated from the inguinal fat pad of 3-week-old SD rats. Stem cells were isolated by type I collagenase digestion. The stem cells were isolated and subcultured in DMEM medium containing fetal bovine serum. The morphology and growth of cells were observed by inverted microscope every day. The third generation of adipose stem cells were divided into five groups, which consisted of a control group (human adipose stem cell culture medium: high glucose DMEM,10% fetal bovine serum, 50nmol/L ascorbic acid, 6.25mg/L insulin, 100U/ml penicillin). 100 渭 g/ml streptomycin, 100nmol/L dexamethasone, 10 渭 g / L transforming growth factor 尾 1 (without gentamicin) and four gentamicin containing groups (100 渭 g / ml 100 渭 g 路ml / ml 100 渭 g / ml + 100 渭 g / ml 100 渭 g/ml) and 4 gentam The proliferative activity was compared by MTT colorimetry. The differentiation of chondrocytes was detected by type 鈪，

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