發(fā)布時(shí)間：2018-05-15 01:31

  本文選題：骨髓間充質(zhì)干細(xì)胞 + 誘導(dǎo)培養(yǎng)��； 參考：《遵義醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的建立體外分離、培養(yǎng)兔骨髓間充質(zhì)干細(xì)胞(BMSCs)的方法。初步探討B(tài)MSCs可定向分化為成骨細(xì)胞、軟骨細(xì)胞的途徑。 方法抽取家兔骨髓液,用淋巴細(xì)胞分離液梯度離心獲得單個(gè)核細(xì)胞,經(jīng)原代培養(yǎng)及傳代培養(yǎng),選擇狀態(tài)良好、經(jīng)過3次傳代的細(xì)胞與原代細(xì)胞一起用于下面實(shí)驗(yàn)。(1)誘導(dǎo)BMSCs向成骨細(xì)胞轉(zhuǎn)化：向培養(yǎng)液內(nèi)加入地塞米松1×10-7mol／L,B-磷酸甘油鈉10mmol/L,維生素C50μg/mL誘導(dǎo)培養(yǎng)后,行堿性磷酸酶活性檢查和Von kossa染色檢查。(2)向培養(yǎng)基中添加不同質(zhì)量濃度bFGF,行MTT檢測(3)誘導(dǎo)BMSCs向軟骨細(xì)胞轉(zhuǎn)化：向培養(yǎng)液內(nèi)加入地塞米松1×10-7mol／L、維生素C50μg/mL bFGF10ng/mL,培養(yǎng)1周后將bFGF換為TGF-β10ng/mL,繼續(xù)培養(yǎng)3周,行甲苯胺藍(lán)染色,RT-PCR技術(shù)檢測Ⅱ型膠原和聚集蛋白聚糖的表達(dá)。 結(jié)果(1)經(jīng)地塞米松、B-磷酸甘油鈉及維生素C誘導(dǎo)培養(yǎng)3-4周后,BMSCs轉(zhuǎn)化為成骨細(xì)胞,實(shí)驗(yàn)組ALP活性明顯高于對(duì)照組,Von kossa染色陽性。(2)10ng／mL的bFGF對(duì)BMSCs的促增值能力最強(qiáng)。(3)BMSCs經(jīng)軟骨誘導(dǎo)分化后骨髓間充質(zhì)干細(xì)胞呈軟骨細(xì)胞形態(tài),甲苯胺藍(lán)染色陽性,PCR證實(shí)轉(zhuǎn)化前的細(xì)胞主要表達(dá)Ⅰ型膠原mRNA.轉(zhuǎn)化后的細(xì)胞主要表達(dá)Ⅱ型膠原mRNA.Ⅱ型膠原和聚集蛋白聚糖的表達(dá)陽性。 結(jié)論兔骨髓間充質(zhì)干細(xì)胞可定向分化為成骨細(xì)胞、軟骨細(xì)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，

本文編號(hào)：1890393