本文選題：間充質(zhì)干細胞 + 成骨細胞　； 參考：《福建醫(yī)科大學》2015年碩士論文

【摘要】：目的探討非甾體類抗炎藥(non-steroidal anti-inflammatory durgs,NSAIDs)在成骨細胞誘導分化過程中的作用,并探討經(jīng)典Wnt信號通路在其中的作用機制。方法(1)采用Ficoll密度梯度離心法體外分離培養(yǎng)大鼠骨髓間充質(zhì)干細胞(bone marrow mesenchymal stem cells,BMSCs),并進行傳代。(2)取傳至3-5代的大鼠骨髓間充質(zhì)干細胞,加入含地塞米松(10-8 mol/L)、β-甘油磷酸鈉(10-2 mol/L)、維生素C(0.05 g/L)等試劑的L-DMEM條件培養(yǎng)液誘導分化為成骨細胞,在細胞增殖和分化的過程中,分別加入不同濃度的雙氯芬酸鈉或塞來昔布(每組n=5)。(3)采用CCK-8試劑盒測定雙氯芬酸鈉或塞來昔布對BMSCs增殖活性的影響。(4)堿性磷酸酶(alkalinephosphatase,ALP)染色分析雙氯芬酸鈉對BMSCs向成骨細胞誘導分化過程中ALP表達的影響,鈣鹽染色分析塞來昔布對BMSCs向成骨細胞誘導分化過程中鈣鹽沉積的影響,使用Image Pro Plus 6.0軟件分析,計算IOD sum/Area sum值。(5)通過RT-PCR檢測塞來昔布對成骨細胞關鍵轉(zhuǎn)錄因子RUNX2 m RNA以及經(jīng)典Wnt信號通路中β-catenin m RNA表達的影響,使用Image J軟件分析RUNX2灰度值/GAPDH灰度值、β-catenin灰度值/GAPDH灰度值。結果(1)倒置相差顯微鏡下觀察BMSCs形態(tài)多樣,呈體積較大的圓形的單個核細胞,培養(yǎng)24小時后有細胞開始貼壁,貼壁的細胞有橢圓狀、淚滴狀、梭形及纖維狀等,分布不均勻。5天后首次換液,10天后所見細胞基本呈梭形或纖維狀,胞體大,胞核大,呈圓形或橢圓形,胞漿富含顆粒。當細胞融合至80%-90%時,可進行傳代。(2)CCK-8試劑盒測定結果顯示,1.25 umol/L、2.5 umol/L、5.0 umol/L、10.0 umol/L雙氯芬酸鈉對BMSCs增殖的抑制率分別為7.08%、12.42%、16.28%、21.10%(不同劑量組兩兩比較,P0.001),1.25 umol/L、2.5 umol/L、5.0 umol/L、10.0 umol/L塞來昔布的抑制率分別為7.27%、11.87%、12.78%、14.71%(不同劑量組兩兩比較,P0.001),表明雙氯芬酸鈉或塞來昔布對BMSCs增殖均呈現(xiàn)抑制作用,抑制作用呈劑量依賴。1.25 umol/L組雙氯芬酸鈉與塞來昔布對BMSCs增殖抑制率別為7.08%、7.27%,2.5 umol/L組雙氯芬酸鈉與塞來昔布對BMSCs增殖抑制率別為12.42%、11.87%(同一濃度組之間比較,P0.05),表明雙氯芬酸鈉組與塞來昔布對BMSCs增殖的抑制差別不大。(3)在條件培養(yǎng)液誘導7天后進行ALP染色,10 umol/L雙氯芬酸鈉組ALP的表達明顯低于對照組,IOD sum/Area sum值分別為0.270±0.256和0.412±0.113(P0.05);在誘導14天后,茜素紅S染色結果顯示,10 umol/L塞來昔布組鈣鹽沉積量明顯少于對照組,IOD sum/Area sum值分別為0.319±0.018和0.412±0.113(P0.05),表明雙氯芬酸鈉抑制了分化過程中的堿性磷酸酶,塞來昔布抑制了分化過程中的鈣鹽沉積。(4)RT-PCR結果顯示,0 umol/L、1.25 umol/L、2.5 umol/L、5.0 umol/L、10.0 umol/L塞來昔布組的RUNX2灰度值/GAPDH灰度值分別為0.7985±0.0356、0.6131±0.0244、0.3926±0.0189、0.2553±0.0294、0.1348±0.0071(不同濃度兩兩比較,P0.001),β-catenin灰度值/GAPDH灰度值分別為0.7959±0.03136、0.4002±0.0177、0.3388±0.0336、0.2209±0.0102、0.2162±0.0140(不同濃度兩兩比較,P0.001),表明塞來昔布抑制了RUNX2 m RNA和β-catenin m RNA的表達水平,并與劑量呈正相關。結論雙氯芬酸鈉和塞來昔布均能夠抑制BMSCs的增殖,并抑制其向成骨細胞的誘導分化。抑制作用可能與經(jīng)典Wnt信號通路的變化有關。
[Abstract]:Objective to investigate the role of non-steroidal anti-inflammatory durgs (NSAIDs) in the induction of osteoblast differentiation, and to explore the mechanism of the classical Wnt signaling pathway in it. Methods (1) Ficoll density gradient centrifugation was used to isolate and culture rat bone marrow mesenchymal stem cells (bone marrow mesenchymal stem) in vitro. Cells, BMSCs), and carry out the passage. (2) take the 3-5 generation of rat bone marrow mesenchymal stem cells, add dexamethasone (10-8 mol/L), beta glycerol phosphate (10-2 mol/L), vitamin C (0.05 g/L) and other reagent L-DMEM conditioned medium to induce differentiation into osteoblasts, in the process of cell proliferation and differentiation, adding different concentration of dichlorochloride, respectively. Sodium finate or celecoxib (each group of n=5). (3) the effect of diclofenac sodium or celecoxib on the proliferation of BMSCs by CCK-8 kit. (4) the effect of diclofenac sodium (alkalinephosphatase, ALP) on the expression of ALP in the process of induced differentiation of BMSCs into osteoblasts by alkaline phosphatase (alkalinephosphatase, ALP). Calcium salt staining analysis of celecoxib to BMSCs The effect of calcium salt deposition on osteoblast induced differentiation, Image Pro Plus 6 software analysis was used to calculate the IOD sum/Area sum value. (5) the effects of celecoxib on the expression of the critical transcription factor RUNX2 m RNA and the classical Wnt signal pathway were detected by RT-PCR. PDH gray value, the gray value of beta -catenin gray value /GAPDH gray value. Results (1) under the inverted phase contrast microscope, it was observed that the shape of BMSCs was a large round single nuclear cell. After 24 hours culture, the cells began to stick to the wall, and the cells attached to the wall were elliptical, tear, shuttle and fibrous, and were first changed in 10 days after the uneven distribution of.5. Cells were basically spindle or fibrous, large cells, large nuclei, round or oval, and rich in granules. When cells fused to 80%-90%, they could be passaged. (2) CCK-8 kit assay showed that 1.25 umol/L, 2.5 umol/L, 5 umol/L, 10 umol/L sodium diclofenac sodium inhibited the proliferation of BMSCs, 7.08%, 12.42%, 16.28%, 21.10%, respectively. Different dose group 22, P0.001), 1.25 umol/L, 2.5 umol/L, 5 umol/L, and 10 umol/L celecoxib were 7.27%, 11.87%, 12.78%, 14.71% (P0.001), which showed that diclofenac sodium or celecoxib inhibited the proliferation of BMSCs, and the inhibitory effect was dose dependent.1.25 umol/L group dichloro The inhibition rate of sodium finate and celecoxib on the proliferation inhibition of BMSCs was 7.08%, 7.27%, 2.5 umol/L, and the inhibition rate of diclofenac sodium and celecoxib on BMSCs proliferation was 12.42%, 11.87% (P0.05), indicating that diclofenac sodium and celecoxib had little difference in inhibition of BMSCs proliferation. (3) after induction of conditioned medium after 7 days The expression of ALP in the 10 umol/L diclofenac sodium group was significantly lower than that in the control group. The IOD sum/Area sum values were 0.270 + 0.256 and 0.412 + 0.113 (P0.05), and the alizarin red S staining results showed that the calcium salt deposition in the 10 umol/L celecoxib group was significantly less than the control group after the induction of the 14 days. The IOD sum/Area sum values were 0.319 + 0.018 and 0.412 + 0.412, respectively. 3 (P0.05) indicates that diclofenac sodium inhibits alkaline phosphatase in the differentiation process, and celecoxib inhibits the calcium salt deposition in the process of differentiation. (4) RT-PCR results show that 0 umol/L, 1.25 umol/L, 2.5 umol/L, 5 umol/L, and 10 umol/L celecoxib group RUNX2 gray value /GAPDH gray value is 0.7985 + 0.0356,0.6131 + 0.0244,0.3926 + 0.01, respectively 89,0.2553 + 0.0294,0.1348 + 0.0071 (different concentration 22, P0.001), the gray value of beta -catenin was 0.7959 + 0.03136,0.4002 + 0.0177,0.3388 + 0.0336,0.2209 + 0.0102,0.2162 + 0.0140 (22 comparison, P0.001), indicating that celecoxib inhibited the expression level of RUNX2 m and beta Conclusion the dose of diclofenac sodium and celecoxib can inhibit the proliferation of BMSCs and inhibit the induction of differentiation into osteoblasts. The inhibitory effect may be related to the changes of the classical Wnt signaling pathway.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R593.23

【參考文獻】

相關期刊論文 前5條

1 李建明;初同偉;周躍;;BMP-2和bFGF在強直性脊柱炎活動期骶髂關節(jié)滑膜組織中的表達[J];第三軍醫(yī)大學學報;2008年03期

2 柳約堅;李娟;;強直性脊柱炎骨病變特點及其機制的研究進展[J];熱帶醫(yī)學雜志;2010年06期

3 謝興文;李盛華;趙永利;李寧;宋敏;;Wnt信號通路在骨髓間充質(zhì)干細胞向成骨細胞分化機制的研究進展[J];中國骨質(zhì)疏松雜志;2011年12期

4 付姣;劉宏瀟;;BMP與強直性脊柱炎病理性成骨[J];中國免疫學雜志;2013年05期

5 潘京華;黃浩;查振剛;;間充質(zhì)干細胞向成骨細胞分化中的Wnt信號通路[J];中國組織工程研究;2013年40期

，

本文編號：1989647