本文選題：骨髓間充質(zhì)干細胞 + Osterix　； 參考：《浙江大學》2011年碩士論文

【摘要】：目的：檢測特異性的轉(zhuǎn)錄因子osterix對骨髓間充質(zhì)干細胞誘導分化成骨細胞以及軟骨細胞途徑的影響,且與外源性BMPs長期培養(yǎng)對骨髓間充質(zhì)干細胞誘導分化各途徑的效應相比較。同時,BMP-2能代表性的誘導種子細胞分化為成骨細胞或軟骨細胞。因此該實驗合成并構(gòu)建特異性的細胞系,可穩(wěn)定下調(diào)基因osterix的表達,且抑制骨髓間充質(zhì)干細胞誘導分化軟骨細胞系后期出現(xiàn)的肥大變性時基因指標的表達,從而促進軟骨組織工程臨床應用和推廣。 方法：構(gòu)建特異性的慢病毒載體,其帶有攻擊目的基因osterix的shRNA序列,通過轉(zhuǎn)染液介導后可轉(zhuǎn)染骨髓間充質(zhì)干細胞,干擾并致基因osterix沉默效應,穩(wěn)定下調(diào)基因osterix的表達。同時挑選已公開的不針對基因osterix的靶基因,構(gòu)建慢病毒載體行陰性對照。最后另一組為BMP-2為主要誘導劑的誘導液,促進骨髓間充質(zhì)干細胞誘導分化。分別于不同時間點觀察并行ELISA檢測各組堿性磷酸酶表達水平,組織染色(阿爾新藍染色以及茜素紅染色)檢測成骨細胞系以及軟骨細胞水平,行實時定量PCR檢測各組目的基因表達水平,行Western blot檢測各組目的蛋白表達水平。 結(jié)果：基因osterix下調(diào)組基因osterix表達降低,多數(shù)成骨型指標如Runx2,10型膠原蛋白下降,同時成軟骨性指標如2型膠原蛋白等基因以及蛋白表達量增加。BMP-2誘導組多數(shù)基因以及蛋白表達情況基本與osterix下調(diào)組相反。 結(jié)論：BMP-2對骨髓間充質(zhì)干細胞成骨分化誘導增強,效果明顯；對軟骨途徑誘導分化時,效果較osterix基因沉默組效果差。基因osterix下調(diào)二十一天內(nèi)可明顯抑制骨髓間充質(zhì)干細胞成骨細胞系誘導分化的途徑,且顯著增強骨髓間充質(zhì)干細胞向軟骨細胞誘導分化的途徑,且誘導骨髓間充質(zhì)干細胞分化成軟骨細胞時后期出現(xiàn)的類軟骨細胞肥大變性現(xiàn)象通過對成骨關(guān)鍵性基因osterix的下調(diào)可顯著抑制。穩(wěn)定下調(diào)基因osterix的表達水平可明確增強骨髓基質(zhì)干細胞表面軟骨細胞系顯型表達,將有效阻抑長期培養(yǎng)后軟骨細胞系肥大性顯型基因的表達水平,從而對解決廣泛大量培養(yǎng)的骨髓基質(zhì)干細胞誘導分化為軟骨細胞后期出現(xiàn)的肥大變性的問題提示了方向。
[Abstract]:Aim: to investigate the effects of specific transcription factor osterix on osteoblast differentiation and chondrocyte pathway induced by bone marrow mesenchymal stem cells (MSCs), and to compare the effects of long-term culture of exogenous BMPs on the differentiation pathways of bone marrow mesenchymal stem cells (BMSCs). BMP-2 can induce seed cells to differentiate into osteoblasts or chondrocytes. Therefore, the synthesis and construction of specific cell lines can stably down-regulate the expression of gene osterix and inhibit the expression of gene markers during hypertrophic degeneration in the later stage of differentiation of chondrocytes induced by bone marrow mesenchymal stem cells. So as to promote the clinical application and promotion of cartilage tissue engineering. Methods: a specific lentivirus vector containing shRNA sequence of target gene osterix was constructed and transfected into bone marrow mesenchymal stem cells mediated by transfection medium. The silencing effect of gene osterix was interfered with and gene osterix expression was steadily down-regulated. At the same time, the target genes which were not specific to gene osterix were selected, and the lentivirus vector was constructed for negative control. The last group was treated with BMP-2 as the main inducer to promote the differentiation of bone marrow mesenchymal stem cells. The expression of alkaline phosphatase was detected by Elisa at different time points, and the osteoblasts and chondrocytes were detected by tissue staining (Alxin blue staining and alizarin red staining). The expression level of target gene in each group was detected by real-time quantitative PCR, and the expression level of target protein was detected by Western blot. Results: the expression of gene osterix was decreased in the down-regulated group of gene osterix, and the expression of most osteogenic markers such as Runx2O10 collagen was decreased. At the same time, the expression of most genes and proteins in the chondrogenic index such as collagen type 2 and protein increased. The expression of most genes and proteins in BMP-2 induced group was basically opposite to that in osterix down-regulated group. Conclusion the osteogenic differentiation of bone marrow mesenchymal stem cells induced by bone morphogenetic protein (BMP-2) was significantly enhanced, and the effect on cartilage pathway was worse than that in osterix gene silencing group. The down-regulation of osterix could significantly inhibit the differentiation of bone marrow mesenchymal stem cells in osteoblasts within 21 days, and enhance the differentiation of bone marrow mesenchymal stem cells into chondrocytes. The hypertrophic denaturation of chondrocytes induced by bone marrow mesenchymal stem cells differentiation into chondrocytes was significantly inhibited by down-regulation of osteoblast key gene osterix. The stable down-regulation of the expression of osterix gene can obviously enhance the expression of chondrocytes on the surface of bone marrow stromal cells, and can effectively inhibit the expression of hypertrophic explicit genes in long-term cultured chondrocytes. Therefore, it suggests the direction to solve the problem of hypertrophic degeneration induced by a large number of cultured bone marrow stromal cells into chondrocytes.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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1 李彥林;韓睿;王福科;;骨髓間充質(zhì)干細胞在骨及軟骨組織工程中的應用[J];中國臨床康復;2006年05期

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