發(fā)布時(shí)間：2018-02-11 11:28

  本文關(guān)鍵詞： 人骨髓間充質(zhì)干細(xì)胞 尿酸 成骨分化 骨形態(tài)形成蛋白-2　出處：《青島大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討人類骨髓間充質(zhì)干細(xì)胞(h BMSCs)向成骨細(xì)胞分化過(guò)程中,不同濃度尿酸對(duì)骨形態(tài)形成蛋白-2(BMP-2)表達(dá)的影響。方法:無(wú)菌取健康成人的骨髓樣本,利用全骨髓貼壁法分離、培養(yǎng)h BMSCs,選取生長(zhǎng)狀態(tài)良好的第3代h BMSCs為研究對(duì)象。將其分為5個(gè)組,分別為空白對(duì)照組(加入完全培養(yǎng)基)、成骨誘導(dǎo)組(加入成骨誘導(dǎo)液及含0、0.2、0.4、0.8 mmol/L尿酸的完全培養(yǎng)基)。倒置顯微鏡下觀察細(xì)胞形態(tài)的變化,各類培養(yǎng)基干預(yù)培養(yǎng)14天后,進(jìn)行茜素紅染色及堿性條件下抽磷酸酶(ALP)活性檢測(cè)來(lái)評(píng)估h BMSCs成骨分化情況。RT-PCR技術(shù)檢測(cè)各組BMP-2 m RNA的表達(dá)情況。結(jié)果:第3代的h BMSCs大多為形態(tài)單一的長(zhǎng)梭形,并逐漸形成旋渦狀生長(zhǎng);干預(yù)誘導(dǎo)后的細(xì)胞逐漸變?yōu)椴灰?guī)則的立方形,局部細(xì)胞重疊生長(zhǎng)形成團(tuán)塊狀結(jié)節(jié),以含尿酸濃度為0.8mmol/L的成骨誘導(dǎo)培養(yǎng)基組最為顯著。連續(xù)尿酸干預(yù)14天后,空白對(duì)照組未觀察到被茜素紅染色的橘紅色結(jié)節(jié),而各成骨誘導(dǎo)組細(xì)胞均有橘紅色結(jié)節(jié)形成。ALP活性隨尿酸濃度的增加和尿酸干預(yù)時(shí)間的延長(zhǎng)而增強(qiáng)(P0.05)。RT-PCR檢測(cè)結(jié)果顯示,空白對(duì)照組無(wú)BMP-2 m RNA的表達(dá)。成骨誘導(dǎo)組隨培養(yǎng)基中尿酸濃度的增加,BMP-2 m RNA表達(dá)水平逐漸升高,并呈尿酸濃度依賴性(P0.05)。結(jié)論:尿酸可以促進(jìn)h BMSCs的成骨分化過(guò)程,并可通過(guò)上調(diào)BMP-2 m RNA的表達(dá)促進(jìn)成骨,呈一定的濃度依賴性。
[Abstract]:Objective: to investigate the effect of different concentrations of uric acid on the expression of bone morphogenetic protein-2BMP-2 during the differentiation of human bone marrow mesenchymal stem cells (BMSCs) into osteoblasts. HBMSCs were cultured, and the third generation of h BMSCs was selected as the research object, which was divided into 5 groups. The cells were divided into blank control group (adding complete culture medium, osteogenic induction group and complete culture medium containing 0. 2 0. 4 0. 8 mmol/L uric acid) respectively. The changes of cell morphology were observed under inverted microscope. After 14 days of culture, all kinds of culture medium were interfered with each other for 14 days. Alizarin red staining and alkaline phosphatase (ALP) activity were used to evaluate the osteogenic differentiation of h BMSCs. RT-PCR was used to detect the expression of BMP-2 m RNA in all groups. The cells induced by the intervention gradually become irregular square, and the local cells overlap to form lumpy nodules. The osteogenic induction medium containing 0.8 mmol / L uric acid concentration was the most significant. After 14 days of continuous uric acid intervention, no orange nodules stained by alizarin red were observed in the blank control group. However, the activity of orange-red nodule formation and ALP increased with the increase of uric acid concentration and the time of uric acid intervention in all osteoblast induction groups. The results of RT-PCR showed that the activity of ALP was increased with the increase of uric acid concentration and the prolongation of uric acid intervention time. There was no expression of BMP-2 m RNA in the blank control group. In the osteogenic induction group, the expression of BMP-2 m RNA increased gradually with the increase of uric acid concentration in culture medium, and the expression of BMP-2 m RNA increased in a concentration dependent manner. Conclusion: uric acid can promote the osteogenic differentiation of h BMSCs. Osteogenesis could be promoted by up-regulating the expression of BMP-2 m RNA in a concentration dependent manner.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R580

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