兔骨髓间质干细胞的成骨细胞、软骨细胞分化研究

发布时间：2018-05-15 01:31

  本文选题：骨髓间充质干细胞 + 诱导培养　； 参考：《遵义医学院》2012年硕士论文

【摘要】：目的建立体外分离、培养兔骨髓间充质干细胞(BMSCs)的方法。初步探讨BMSCs可定向分化为成骨细胞、软骨细胞的途径。 方法抽取家兔骨髓液,用淋巴细胞分离液梯度离心获得单个核细胞,经原代培养及传代培养,选择状态良好、经过3次传代的细胞与原代细胞一起用于下面实验。(1)诱导BMSCs向成骨细胞转化：向培养液内加入地塞米松1×10-7mol／L,B-磷酸甘油钠10mmol/L,维生素C50μg/mL诱导培养后,行碱性磷酸酶活性检查和Von kossa染色检查。(2)向培养基中添加不同质量浓度bFGF,行MTT检测(3)诱导BMSCs向软骨细胞转化：向培养液内加入地塞米松1×10-7mol／L、维生素C50μg/mL bFGF10ng/mL,培养1周后将bFGF换为TGF-β10ng/mL,继续培养3周,行甲苯胺蓝染色,RT-PCR技术检测Ⅱ型胶原和聚集蛋白聚糖的表达。 结果(1)经地塞米松、B-磷酸甘油钠及维生素C诱导培养3-4周后,BMSCs转化为成骨细胞,实验组ALP活性明显高于对照组,Von kossa染色阳性。(2)10ng／mL的bFGF对BMSCs的促增值能力最强。(3)BMSCs经软骨诱导分化后骨髓间充质干细胞呈软骨细胞形态,甲苯胺蓝染色阳性,PCR证实转化前的细胞主要表达Ⅰ型胶原mRNA.转化后的细胞主要表达Ⅱ型胶原mRNA.Ⅱ型胶原和聚集蛋白聚糖的表达阳性。 结论兔骨髓间充质干细胞可定向分化为成骨细胞、软骨细胞。
[Abstract]:Objective to establish a method for isolation and culture of rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro. To explore the pathway that BMSCs can differentiate into osteoblasts and chondrocytes. Methods the rabbit bone marrow fluid was extracted and mononuclear cells were obtained by gradient centrifugation with lymphocyte separator. After primary culture and passage culture, the selection of mononuclear cells was good. After three times of passage, the cells were used together with the primary cells to induce the transformation of BMSCs into osteoblasts: adding dexamethasone 1 脳 10 ~ (-7) mol 路L ~ (-1) sodium glycerine phosphate 10 mmol / L, vitamin C _ (50) 渭 g/mL into the culture medium. Alkaline phosphatase activity test and Von kossa staining. 2) adding different concentration bFGF3 to culture medium and MTT detecting 3) inducing BMSCs to transform into chondrocytes: adding dexamethasone 1 脳 10-7 mol / L, vitamin C 50 渭 g/mL bFGF10ngmL into culture medium for 1 week. Change bFGF to TGF- 尾 10ng / mL and continue to grow for 3 weeks. The expression of type 鈪，

本文编号：1890393