本文選題：間充質(zhì)干細(xì)胞 + 成骨細(xì)胞　； 參考：《福建醫(yī)科大學(xué)》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(xì)胞誘導(dǎo)分化過程中的作用,并探討經(jīng)典Wnt信號(hào)通路在其中的作用機(jī)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(xì)胞(bone marrow mesenchymal stem cells,BMSCs),并進(jìn)行傳代。(2)取傳至3-5代的大鼠骨髓間充質(zhì)干細(xì)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(dǎo)分化為成骨細(xì)胞,在細(xì)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(cè)定雙氯芬酸鈉或塞來昔布對(duì)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(duì)BMSCs向成骨細(xì)胞誘導(dǎo)分化過程中ALP表達(dá)的影響,鈣鹽染色分析塞來昔布對(duì)BMSCs向成骨細(xì)胞誘導(dǎo)分化過程中鈣鹽沉積的影響,使用Image Pro Plus 6.0軟件分析,計(jì)算IOD sum/Area sum值。(5)通過RT-PCR檢測(cè)塞來昔布對(duì)成骨細(xì)胞關(guān)鍵轉(zhuǎn)錄因子RUNX2 m RNA以及經(jīng)典Wnt信號(hào)通路中β-catenin m RNA表達(dá)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(jié)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(gè)核細(xì)胞,培養(yǎng)24小時(shí)后有細(xì)胞開始貼壁,貼壁的細(xì)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(xì)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(dāng)細(xì)胞融合至80%-90%時(shí),可進(jìn)行傳代。(2)CCK-8試劑盒測(cè)定結(jié)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(duì)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(duì)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(duì)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(duì)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(duì)BMSCs增殖的抑制差別不大。(3)在條件培養(yǎng)液誘導(dǎo)7天后進(jìn)行ALP染色,10 umol/L雙氯芬酸鈉組ALP的表達(dá)明顯低于對(duì)照組,IOD sum/Area sum值分別為0.270±0.256和0.412±0.113(P0.05);在誘導(dǎo)14天后,茜素紅S染色結(jié)果顯示,10 umol/L塞來昔布組鈣鹽沉積量明顯少于對(duì)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(jié)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(dá)水平,并與劑量呈正相關(guān)。結(jié)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(xì)胞的誘導(dǎo)分化。抑制作用可能與經(jīng)典Wnt信號(hào)通路的變化有關(guān)。
[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(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.23

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