本文選題：Oct-4　切入點：骨髓間充質(zhì)干細胞　出處：《石河子大學》2009年碩士論文　論文類型：學位論文

【摘要】：目的:體外分離、培養(yǎng)小鼠骨髓間充質(zhì)干細胞(mouse Bone marrow mesenchymal stem cells,mBMSCs),并誘導其向成骨細胞方向分化,檢測Oct-4在mBMSCs誘導分化前后的表達及甲基化狀態(tài)的改變,為進一步研究Oct-4在BMSCs增殖分化中的作用提供研究思路。 方法:采用全骨髓貼壁篩選法體外培養(yǎng)、擴增mBMSCs。用CD29、CD44、c-Kit以及CD34免疫細胞化學染色鑒定mBMSCs。取第3代小鼠BMSCs,用含10%FBS,0.1μmol/L地塞米松,50μg/ml維生素C和10mmol/Lβ?甘油磷酸鈉的高糖DMEM培養(yǎng)基定向誘導mBMSCs向成骨細胞分化,并用堿性磷酸酶(alkaline phosphatase, AKP)染色、茜素紅染色等方法進行鑒定,同時用體外分離培養(yǎng)的成骨細胞作陽性對照。采用RT-PCR及間接免疫熒光染色方法,從mRNA和蛋白水平檢測Oct-4在mBMSCs成骨誘導分化前后的表達;運用甲基化特異PCR檢測Oct-4在mBMSCs誘導分化前后的甲基化狀態(tài)。 結果:mBMSCs接種24h后已大量貼壁并伸展開;48h后貼壁細胞明顯增多并分裂增殖,貼壁細胞呈梭形或成纖維細胞樣形態(tài)。傳代培養(yǎng)后,細胞形態(tài)更加均一,增殖加快,呈渦旋樣生長。免疫細胞化學染色和間接免疫螢光染色結果顯示第3代mBMSCs CD29、CD44、c-Kit表達陽性,CD34表達陰性。第3代mBMSCs在成骨誘導誘導劑誘導4天后細胞形態(tài)變化,隨著誘導時間的延長,細胞逐漸增大,細胞形態(tài)由長梭形逐漸變?yōu)槎嘟切�。mBMSCs誘導10d后大多數(shù)細胞和體外培養(yǎng)成骨細胞形態(tài)相似,均呈AKP和茜素紅陽性反應。RT-PCR結果與間接免疫熒光染色結果同時顯示,第3代的mBMSCs有Oct-4表達,而向成骨方向誘導10d后的mBMSCs及小鼠成骨細胞均未見Oct-4表達;MSP結果顯示,mBMSCs在向成骨方向誘導10天后,Oct-4基因的啟動子區(qū)的CpG島出現(xiàn)甲基化,而未誘導的第3代mBMSCs為非甲基化狀態(tài)。 結論: 1.在本實驗室成功的建立了小鼠成骨細胞培養(yǎng)體系。 2.成功分離培養(yǎng)mBMSCs,并誘導其向成骨細胞分化,觀察到Oct-4在mBMSCs定向誘導分化中表達下調(diào)。 3.首次觀察到在mBMSCs定向誘導分化中Oct-4基因的甲基化狀態(tài)發(fā)生了改變,提示Oct-4基因在mBMSCs定向誘導分化中的表達下調(diào)可能與其啟動子區(qū)甲基化有關。
[Abstract]:Objective: to isolate and culture mouse Bone marrow mesenchymal stem stem mBMSCs, and to induce the differentiation of mouse Bone marrow mesenchymal stem cells into osteoblasts, and to detect the expression of Oct-4 before and after mBMSCs induction and the changes of its methylation state. To further study the role of Oct-4 in the proliferation and differentiation of BMSCs. Methods: mBMSCs were amplified by whole bone marrow adherent screening method in vitro. MBMSCs were identified by CD29 CD4C4c-Kit and CD34 immunocytochemical staining. BMSCs of the third generation of mice were obtained, and vitamin C and 10mmol/L 尾 were determined by 50 渭 g/ml vitamin C and 50 渭 g/ml of dexamethasone containing 10s and 0.1 渭 mol/L dexamethasone. The differentiation of mBMSCs into osteoblasts was induced by high glucose DMEM medium of sodium glycerophosphate. The differentiation of mBMSCs into osteoblasts was identified by alkaline phosphatase staining and alizarin red staining. At the same time, osteoblasts isolated and cultured in vitro were used as positive control. The expression of Oct-4 before and after osteogenic differentiation of mBMSCs was detected by RT-PCR and indirect immunofluorescence staining from the level of mRNA and protein. Methylation specific PCR was used to detect the methylation of Oct-4 before and after differentiation induced by mBMSCs. Results after 24 hours of inoculation, a large number of adherent cells were proliferated and proliferated, and the adherent cells were fusiform or fibroblast-like. After passage, the morphology of the cells was more uniform and the proliferation was accelerated. The results of immunocytochemical staining and indirect immunofluorescence staining showed that the third generation of mBMSCs CD29 + CD4c-Kit positive expression of CD34 was negative, the third generation of mBMSCs was induced by osteoblast inducer for 4 days, and the cell morphology changed with the prolongation of induction time. After 10 days of induction, most of the cells were similar to those of osteoblasts cultured in vitro. The results of AKP and alizarin red positive reaction. RT-PCR and indirect immunofluorescence staining showed that most of the cells were similar to those of osteoblasts cultured in vitro, and the results of RT-PCR and indirect immunofluorescence staining showed that most of the cells were similar to those of osteoblasts cultured in vitro. Oct-4 expression was found in the mBMSCs of the third generation, but no Oct-4 expression was found in the mBMSCs and mouse osteoblasts after 10 days of osteogenic induction. The results showed that there was methylation in the CpG island of the promoter region of Oct-4 gene 10 days after induction to the osteogenic direction. The uninduced third generation mBMSCs was demethylated. Conclusion:. 1. The mouse osteoblast culture system was successfully established in our laboratory. 2. MBMSCs were successfully isolated and cultured and induced to differentiate into osteoblasts. The down-regulated expression of Oct-4 was observed in mBMSCs induced differentiation. 3. It was observed for the first time that the methylation status of Oct-4 gene was changed during mBMSCs directed differentiation, suggesting that the down-regulation of Oct-4 gene expression in mBMSCs induced differentiation might be related to the promoter methylation.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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本文編號：1647102