非甾体抗炎药抑制成骨细胞增生和分化研究

发布时间：2018-06-07 03:54

本文选题：间充质干细胞 + 成骨细胞　；参考：《福建医科大学》2015年硕士论文

【摘要】：目的探讨非甾体类抗炎药(non-steroidal anti-inflammatory durgs,NSAIDs)在成骨细胞诱导分化过程中的作用,并探讨经典Wnt信号通路在其中的作用机制。方法(1)采用Ficoll密度梯度离心法体外分离培养大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),并进行传代。(2)取传至3-5代的大鼠骨髓间充质干细胞,加入含地塞米松(10-8 mol/L)、β-甘油磷酸钠(10-2 mol/L)、维生素C(0.05 g/L)等试剂的L-DMEM条件培养液诱导分化为成骨细胞,在细胞增殖和分化的过程中,分别加入不同浓度的双氯芬酸钠或塞来昔布(每组n=5)。(3)采用CCK-8试剂盒测定双氯芬酸钠或塞来昔布对BMSCs增殖活性的影响。(4)碱性磷酸酶(alkalinephosphatase,ALP)染色分析双氯芬酸钠对BMSCs向成骨细胞诱导分化过程中ALP表达的影响,钙盐染色分析塞来昔布对BMSCs向成骨细胞诱导分化过程中钙盐沉积的影响,使用Image Pro Plus 6.0软件分析,计算IOD sum/Area sum值。(5)通过RT-PCR检测塞来昔布对成骨细胞关键转录因子RUNX2 m RNA以及经典Wnt信号通路中β-catenin m RNA表达的影响,使用Image J软件分析RUNX2灰度值/GAPDH灰度值、β-catenin灰度值/GAPDH灰度值。结果(1)倒置相差显微镜下观察BMSCs形态多样,呈体积较大的圆形的单个核细胞,培养24小时后有细胞开始贴壁,贴壁的细胞有椭圆状、泪滴状、梭形及纤维状等,分布不均匀。5天后首次换液,10天后所见细胞基本呈梭形或纤维状,胞体大,胞核大,呈圆形或椭圆形,胞浆富含颗粒。当细胞融合至80%-90%时,可进行传代。(2)CCK-8试剂盒测定结果显示,1.25 umol/L、2.5 umol/L、5.0 umol/L、10.0 umol/L双氯芬酸钠对BMSCs增殖的抑制率分别为7.08%、12.42%、16.28%、21.10%(不同剂量组两两比较,P0.001),1.25 umol/L、2.5 umol/L、5.0 umol/L、10.0 umol/L塞来昔布的抑制率分别为7.27%、11.87%、12.78%、14.71%(不同剂量组两两比较,P0.001),表明双氯芬酸钠或塞来昔布对BMSCs增殖均呈现抑制作用,抑制作用呈剂量依赖。1.25 umol/L组双氯芬酸钠与塞来昔布对BMSCs增殖抑制率别为7.08%、7.27%,2.5 umol/L组双氯芬酸钠与塞来昔布对BMSCs增殖抑制率别为12.42%、11.87%(同一浓度组之间比较,P0.05),表明双氯芬酸钠组与塞来昔布对BMSCs增殖的抑制差别不大。(3)在条件培养液诱导7天后进行ALP染色,10 umol/L双氯芬酸钠组ALP的表达明显低于对照组,IOD sum/Area sum值分别为0.270±0.256和0.412±0.113(P0.05);在诱导14天后,茜素红S染色结果显示,10 umol/L塞来昔布组钙盐沉积量明显少于对照组,IOD sum/Area sum值分别为0.319±0.018和0.412±0.113(P0.05),表明双氯芬酸钠抑制了分化过程中的碱性磷酸酶,塞来昔布抑制了分化过程中的钙盐沉积。(4)RT-PCR结果显示,0 umol/L、1.25 umol/L、2.5 umol/L、5.0 umol/L、10.0 umol/L塞来昔布组的RUNX2灰度值/GAPDH灰度值分别为0.7985±0.0356、0.6131±0.0244、0.3926±0.0189、0.2553±0.0294、0.1348±0.0071(不同浓度两两比较,P0.001),β-catenin灰度值/GAPDH灰度值分别为0.7959±0.03136、0.4002±0.0177、0.3388±0.0336、0.2209±0.0102、0.2162±0.0140(不同浓度两两比较,P0.001),表明塞来昔布抑制了RUNX2 m RNA和β-catenin m RNA的表达水平,并与剂量呈正相关。结论双氯芬酸钠和塞来昔布均能够抑制BMSCs的增殖,并抑制其向成骨细胞的诱导分化。抑制作用可能与经典Wnt信号通路的变化有关。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.23

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