TGF-β1对大鼠脂肪干细胞体外软骨形成能力影响的研究

发布时间：2018-06-21 06:26

  本文选题：脂肪干细胞 + TGF-β1　； 参考：《中南大学》2010年硕士论文

【摘要】： 目的本文研究大鼠脂肪干细胞(adipose-derived stem cells ADSCs)在体外分离培养、诱导分化成软骨细胞的能力,初步观察不同浓度的TGF-β1对其增殖、分化成软骨细胞的影响,为软骨组织工程及临床应用提供科学依据。 方法从3周龄SD大鼠腹股沟脂肪垫分离出脂肪组织,通过Ⅰ型胶原酶消化法分离干细胞,接种于含有胎牛血清的DMEM培养液中分离传代。每日以倒置显微镜观察细胞形态及生长情况。取第三代脂肪干细胞分成5组,包含1个对照组(培养液为人脂肪干细胞完全培养基：高糖DMEM、10%胎牛血清、50nmol/L抗坏血酸、6.25mg/L胰岛素、100U/ml青霉素、100μg/ml链霉素、100nmol/L地塞米松,不含TGF-β1)和4个TGF-β1浓度组(浓度分别是1、10、40、80μg/L加人脂肪干细胞完全培养基)。采用MTT比色法进行分化和增殖活性比较,Ⅱ型胶原免疫细胞化学染色和细胞甲苯胺蓝染色检测成软骨细胞分化情况。 结果原代细胞接种4-6小时后即可见沉降贴壁,7-8天左右可见大量增殖,按一定方向性排列呈漩涡状或束状,传代后细胞增殖迅速,生长稳定。成软骨诱导后行细胞甲苯胺蓝染色示：10μg/LTGF-β1浓度组阳性,40μg/LTGF-β1浓度组和80μg/LTGF-β1浓度染色较淡,染色细胞少,0μg/LTGF-β1浓度组和1μg/LTGF-β1浓度组为阴性。Ⅱ型胶原免疫组织化学染色显示：10μg/L TGF-β1浓度组阳性；40μg/LTGF-β1浓度组和80μg/LTGF-β1浓度组为弱阳性；0μg/LTGF-β1浓度组和1μg/LTGF-β1浓度组为阴性。MTT实验显示10μg/L的TGF-β1可以促进细胞增殖(P0.05)。 结论1.大鼠脂肪干细胞能向成软骨细胞诱导分化；2.10μg/L的TGF-β1是脂肪干细胞增殖、分化成软骨细胞的最佳浓度；3.0、1、40、80μg/L的TGF-β1均不利脂肪干细胞增殖及软骨细胞形成。
[Abstract]:Objective to study the ability of adipose-derived stem cells ADSCs isolated from rat adipose stem cells to induce the differentiation of chondrocytes in vitro, and to observe the effects of different concentrations of TGF- 尾 1 on the proliferation and differentiation of chondrocytes. To provide scientific basis for cartilage tissue engineering and clinical application. Methods Adipose tissue was isolated from 3 week-old SD rats with inguinal fat pad. Stem cells were isolated by type I collagenase digestion and cultured 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 5 groups, including one control group (human adipose stem cell complete medium: high glucose DMEM10% fetal bovine serum, 50nmol / L ascorbic acid 6.25mg / L insulin / 100U / ml penicillin 100 渭 g/ml streptomycin 100 nmol / L dexamethasone, 100 渭 mol / L ascorbic acid / L ascorbic acid, 100 渭 g/ml streptomycin 100 渭 mol / L dexamethasone. TGF- 尾 _ 1 and 4 TGF- 尾 _ 1 groups were added to human adipose stem cell culture medium with 40 渭 g / L TGF- 尾 _ (1) and 40 渭 g / L TGF- 尾 _ (1), respectively. The differentiation and proliferation activity of chondroblasts were compared by MTT colorimetry. The differentiation of chondroblasts was detected by type 鈪，

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