【摘要】： 目的本文研究大鼠脂肪干細胞(adipose-derived stem cells ADSCs)在體外分離培養(yǎng)、誘導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Ⅰ型膠原酶消化法分離干細胞,型膠原酶消化法分離干細胞,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胞形態(tài)及生長情況。取第三代脂肪干細胞分五組,包含一個對照組(培養(yǎng)液為人脂肪干細胞完全培養(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)化生長因子β1,不含慶大霉素)和四個含慶大霉素濃度組(分別是10μg／ml、50μg／ml、100μg／ml、200μg／ml加人脂肪干細胞完全培養(yǎng)基)。采用MTT比色法進行增殖活性比較,Ⅱ型膠原免疫細胞化學染色和細胞甲苯胺藍染色檢測成軟骨分化情況。 結(jié)果1.體外培養(yǎng)的脂肪間充質(zhì)干細胞呈扁平的梭形成纖維樣細胞生長,且細胞形態(tài)均一,傳代穩(wěn)定。2.MTT實驗顯示慶大霉素在100μg／ml、200μg／ml抑制細胞增殖,其余各濃度組對細胞增殖無明顯影響3.誘導后細胞甲苯胺藍染色顯示100μg／ml組及200μg／ml組細胞細胞染色較淺,大部分細胞失染,余各組陽性表達：Ⅱ型膠原免疫組織化學染色顯示100μg／ml組及200μg／ml組細胞弱陽性表達,余各組陽性表達。 結(jié)論1大鼠脂肪干細胞易取材,培養(yǎng)傳代后增殖迅速,生長穩(wěn)定。2大鼠脂肪干細胞能向軟骨細胞誘導分化。3慶大霉素在一定濃度(10μg／ml、50μg／ml)下對脂肪干細胞的增殖和軟骨分化沒有影響,但高于此濃度(100μg／ml,200μg／ml)則抑制脂肪干細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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