本文選題：骨髓間充質(zhì)干細胞 + 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)干細胞(BMSCs)的方法。初步探討B(tài)MSCs可定向分化為成骨細胞、軟骨細胞的途徑。 方法抽取家兔骨髓液,用淋巴細胞分離液梯度離心獲得單個核細胞,經(jīng)原代培養(yǎng)及傳代培養(yǎng),選擇狀態(tài)良好、經(jīng)過3次傳代的細胞與原代細胞一起用于下面實驗。(1)誘導(dǎo)BMSCs向成骨細胞轉(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,行甲苯胺藍染色,RT-PCR技術(shù)檢測Ⅱ型膠原和聚集蛋白聚糖的表達。 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)化為成骨細胞,實驗組ALP活性明顯高于對照組,Von kossa染色陽性。(2)10ng／mL的bFGF對BMSCs的促增值能力最強。(3)BMSCs經(jīng)軟骨誘導(dǎo)分化后骨髓間充質(zhì)干細胞呈軟骨細胞形態(tài),甲苯胺藍染色陽性,PCR證實轉(zhuǎn)化前的細胞主要表達Ⅰ型膠原mRNA.轉(zhuǎn)化后的細胞主要表達Ⅱ型膠原mRNA.Ⅱ型膠原和聚集蛋白聚糖的表達陽性。 結(jié)論兔骨髓間充質(zhì)干細胞可定向分化為成骨細胞、軟骨細胞。
[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，

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