發(fā)布時間：2018-07-17 20:02

【摘要】：目的：分離培養(yǎng)兔骨髓間充質(zhì)干細胞(Bone Marrow-derived Mesenchymal Stem Cells, BMSCs)。體外立體誘導骨髓間充質(zhì)干細胞向成軟骨方向分化,觀察立體誘導過程中骨髓間充質(zhì)干細胞中Sox9及11型膠原mRNA的含量隨時間的表達關(guān)系,對組織工程化軟骨進行基因水平上的初步研究。 方法：新西蘭大白兔3只。股骨粗隆處備皮,用骨髓穿刺針抽取骨髓,采用全骨髓貼壁培養(yǎng)生長法培養(yǎng)骨髓間充質(zhì)干細胞,使之得到分離純化。傳代至第3代備用。MTT法測細胞增值率,描繪增殖曲線,觀察細胞生長狀態(tài)。取3代細胞,調(diào)整細胞數(shù)量為1.5×106/離心管,150g離心后分為實驗組(立體成軟骨誘導培養(yǎng)組)與對照組(立體非成軟骨誘導培養(yǎng)組)。實驗組采用含有l(wèi)Ong/mlrhTGF-p,的成軟骨誘導液培養(yǎng),對照組采用不含TGF-β1的誘導液進行培養(yǎng)。分別在第4天,8天,12天,16天提取總RNA,行RT-PCR,檢測Sox9及Ⅱ型膠原mRNA表達。 結(jié)果：全骨髓培養(yǎng)后骨髓間充質(zhì)干細胞呈貼壁生長,隨著換液骨髓中其他不貼壁生長的細胞逐漸被棄去,使得骨髓間充質(zhì)干細胞得到進一步純化。傳代后細胞生長速度明顯加快,并呈旋渦狀生長。3代骨髓間充質(zhì)干細胞生長良好,經(jīng)歷潛伏期、對數(shù)期、平臺期。實驗組細胞第2天形成小球,并逐漸長大。而對照組細胞無法形成小球,松散沉積于離心管底。RT-PCR檢測實驗組Sox9與Ⅱ型膠原表達含量均隨時間逐漸升高,兩者呈高度相關(guān)性。對照組Sox9表達隨時間變化無明顯統(tǒng)計學意義,Ⅱ型膠原表達未檢出。 結(jié)論：全骨髓貼壁生長法可使骨髓間充質(zhì)干細胞得到分離純化。含有TGF-β1的誘導培養(yǎng)液可使骨髓間充質(zhì)干細胞立體誘導成軟骨分化,而不含TGF-β1的誘導培養(yǎng)液無法誘導成軟骨分化。成軟骨分化中,Sox9與Ⅱ型膠原表達逐漸增高,且兩者呈相關(guān)性。TGF-β1可以上調(diào)Sox9的表達量,由于Sox9是Ⅱ型膠原轉(zhuǎn)錄因子,表明在本實驗中立體誘導過程是通過上調(diào)Sox9表達從而促使成軟骨分化的。
[Abstract]:Objective: to isolate and culture rabbit bone Marrow-derived mesenchymal stem cells (BMSCs). Bone marrow mesenchymal stem cells were induced to differentiate into chondrocytes in vitro. The expression of Sox9 and type 11 collagen mRNA in bone marrow mesenchymal stem cells was observed with time. A preliminary study on the gene level of tissue engineered cartilage was carried out. Methods: 3 New Zealand white rabbits. Bone marrow mesenchymal stem cells (BMSCs) were isolated and purified from femoral trochanter by bone marrow puncture needle. Bone marrow mesenchymal stem cells (BMSCs) were cultured by whole bone marrow adherent growth method. After passage to the third passage, the proliferation rate was measured by MTT method, the proliferation curve was depicted and the state of cell growth was observed. After centrifugation, the cells were divided into experimental group (stereotactic chondrogenic culture group) and control group (stereotactic non-chondrogenic culture group). The experimental group was cultured with chondrogenic medium containing lOng / ml rhTGF-pand the control group was cultured without TGF- 尾 1. Total RNAs were extracted on day 4, day 8, day 12, day 16, respectively. RT-PCR was performed to detect the expression of Sox9 and collagen 鈪，

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