茶黄素和转化生长因子-β1对兔骨髓基质干细胞向软骨细胞增殖及诱导分化的影响

发布时间：2018-07-01 08:24

  本文选题：骨髓基质干细胞 + 软骨细胞　； 参考：《安徽医科大学》2009年硕士论文

【摘要】： 目的:茶黄素是红茶中的主要成分,具有调节血脂、抗氧化、抗肿瘤、抗心脑血管疾病等多种药理活性。以往研究茶黄素对骨髓基质干细胞转化较少,本实验探讨茶黄素和转化生长因子β1(TGF-β1)对体外培养兔骨髓基质干细胞(BMSCs)增殖及向软骨细胞诱导分化的影响。 方法:实验于2008-05/8在安徽医科大学附属省立医院中心实验室完成。①实验动物:清洁级8周龄家兔12只,由安徽医科大学动物试验中心提供,实验过程中对动物的处置符合动物伦理学标准。②实验方法:兔全身肝素化后麻醉状态下处死,取双侧股骨和肱骨,去除软组织,切除包括骺板在内的两侧骺端,采用全骨髓法分离培养骨髓基质干细胞,按5×10~4/L的细胞密度接种,当细胞生长至80%～90%融合时消化传代。取传至第三代的细胞按1×10~4/ml的密度接种,加入含重组人胰岛素10mg/L、地塞米松10~(-8)mmol/L、TGF-β_15ng/L的DMBM成软骨诱导液培养2周。取第3代BMSC分为三组:A对照组,B组:完全培养基加地塞米松(10~(-8)mmol/L)、TGF-β1(5ng/L)、维生素C(10mmol/L)。C组:完全培养基加地塞米松(10~(-8)mmol/L)、TGF-β1(5ng/L)、维生素C(10mmol/L)、茶黄素(30 mg/L)。③实验评估:倒置显微镜下观察细胞生长状况,甲苯胺蓝染色、阿利新蓝比色法测定各板每孔中GAG含量、以及Ⅱ型胶原免疫组化鉴别。 结果:①细胞生长曲线:骨髓基质干细胞具有旺盛的增殖能力,培养1d为细胞的适应期,3d为对数增长期,8d时进入平台期,之后细胞增殖迅速减慢,细胞数下降。②观察细胞生长:骨髓中分离获得的BMSCs在体外增殖旺盛,TGF-β1和茶黄素诱导后细胞生长明显减缓。与对照组相比,经过诱导2周后,诱导组BMSCs均与对照组有显著的差异性(P＜0.05),诱导组之间也存在差异性(P＜0.05)。③甲苯胺蓝染色和免疫组化鉴定结果:经TGF-β1和茶黄素诱导2周后,细胞甲苯胺蓝异染明显,Ⅱ型胶原化学染色阳性,表现为软骨细胞生物学特性。 结论:茶黄素(30 mg/L)在TGF-β1存在的条件下能有效促进BMSCs在体外向软骨细胞分化。
[Abstract]:Objective: theaflavin is the main component of black tea. It has many pharmacological activities, such as regulating blood lipid, anti-oxidation, anti-tumor, anti-cardio-cerebrovascular disease and so on. The effect of theaflavine and transforming growth factor 尾 1 (TGF- 尾 1) on the proliferation and differentiation of rabbit bone marrow stromal cells (BMSCs) into chondrocytes in vitro was studied. Methods: the experiment was completed in the Central Laboratory of Provincial Hospital affiliated to Anhui Medical University in 2008-05 / 8. 1 Experimental animals: 12 rabbits of 8 weeks old of clean grade, provided by Animal Test Center of Anhui Medical University. In the course of the experiment, the disposal of animals was in accordance with the standard of animal ethics .2 Experimental methods: rabbits were killed under anesthesia after heparinization, bilateral femur and humerus were removed, soft tissue was removed, epiphysis including epiphyseal plate was removed. Bone marrow stromal cells were isolated and cultured by whole bone marrow method and inoculated with 5 脳 10 ~ (4) / L cell density. When the cells grew to 80% fusion, they were digested and subcultured. The third passage cells were inoculated at the density of 1 脳 10~4/ml, then added 10 mg / L of recombinant human insulin, 10 ~ (-8) mmol / L dexamethasone, TGF- 尾 to form cartilage inducer of 15 ng / L DMBM for 2 weeks. The third generation BMSC was divided into three groups: complete medium plus dexamethasone (10 ~ (-8) mmol / L) TGF- 尾 1 (5 ng / L), vitamin C (10 mmol / L) .C group: complete medium plus dexamethasone (10 ~ (-8) mmol / L) TGF- 尾 1 (5 ng / L), vitamin C (10 mmol / L), tea (30 mg / L) .3. Toluidine blue staining and Alisin blue colorimetry were used to determine the gag content in each hole of each plate, and the type 鈪，

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