BMP2与DXM对人牙髓细胞增殖和分化的影响

发布时间：2018-04-26 14:10

本文选题：BMP2 + 对人　；参考：《广西医科大学》2017年硕士论文

【摘要】：牙髓细胞(hDPCs)具有自我复制更新和多向分化克隆的潜能,可在生物因子的作用下可以向成牙本质细胞分化。骨形态发生蛋-2(BMP-2)和地塞米松(DXM)是常见的成骨诱导因子,可以使牙髓细胞向成牙本质样细胞诱导分化。在成牙本质细胞形成过程中亦发挥着重要作用。本研究的目的为探讨BMP-2和DXM在诱导牙髓细胞增殖和牙向分化的作用以及两者联合运用是否能增强诱导效果,为牙髓细胞分化的实验提供生物因子优化组合的实验依据。目的:1、建立hDPCs培养模型,体外分离并培养hDPCs,鉴定细胞来源。2、探讨BMP-2和DXM以及两者联合作用矿化及诱导hDPCs体外增殖及向成牙本质细胞分化的影响。方法:1、hDPCs的体外分离、培养及鉴定:收集我院外科门诊15-30周岁患者无牙体牙髓牙周疾病的正畸牙或智齿,半小时之内迅速至无菌条件下劈开牙体取出髓腔中牙髓组织,并将其剪成1mm3小块,置于培养瓶中,加入含20%优质胎牛血清的高糖DMEM培养液,于恒温培养箱中培养,设置条件为37℃、5%CO2浓度。待细胞生长融合至25cm3培养瓶瓶底80%后,用无菌巴氏吸管加入0.25%胰酶,消化传代细胞。取生长状态良好的第3-5代细胞行波形蛋白和角蛋白免疫化学染色鉴定。冻存稳定传代的3-6代hDPCs待用。2、BMP-2和DXM分别及联合作用对hDPCs增殖的影响:用浓度为BMP-2(100ng/ml)、DXM(1×10-8mol/l)、BMP-2(100ng/ml)+DXM(1×10-8mol/l)诱导稳定传代的hDPCs,与对照组比较(仅含10%FBS的高糖DMEM培养液),在1day、3day、5day、7day消化细胞,制成细胞悬浊液,计数,绘制各组细胞生长曲线。3、BMP-2和DXM分别及联合作用对h DPC向成牙本质细胞分化的影响:诱导步骤同上,利用RT-PCR检测BMP-2和DXM分别及联合作用5day、7day后成牙本质标记基因DSPP、ALP、DMP-1的表达。作用14day后进行碱性磷酸酶染色检测ALP活性。作用21day,进行茜素红矿化结节染色,定量检测矿化结节分析成牙本质分化情况。结果:1、hDPCs的体外分离、培养及鉴定:(1)培养3-5天后,可见有梭型纤维状细胞爬出,7天左右细胞密集生长融合至瓶底的80%,可稳定传代,保持一定生长能力。(3)经波形蛋白、角蛋白免疫化学染色可见,细胞波形蛋白阳性表达,角蛋白阴性表达,说明培养的细胞来源于间叶组织中胚层,符合牙髓细胞的生物学特征。2、BMP-2和DXM分别及联合作用对hDPCs增殖的影响:随培养时间延长,各组细胞数均逐渐增加,5 d时达峰值,后逐渐下降,7 d降至3 d时水平。培养1d时,各组细胞数无明显差异(P0.05);培养3 d时BMP-2组、BMP-2+DXM组细胞数显著高于DXM、对照组(P0.05)BMP-2组、BMP-2+DXM组间无明显差异,DXM组、对照组间无明显差异(P0.05);5 d时BMP-2、DXM、BMP-2+DXM组细胞数显著高于对照组,BMP-2+DXM组BMP-2组DXM组对照组(P0.05);7d时细胞数下降,BMP-2+DXM组BMP-2组对照组DXM组(P0.05)。3、BMP-2和DXM分别及联合作用对hDPCs分化的影响:经诱导后各处组的成牙本质形成标记基因DSPP、DMP-1、ALP均有表达,碱性磷酸酶染色染色阳性,矿化结节茜素红染色阳性,说明牙髓细胞可以在BMP-2、DXM的诱导下分化为成牙本质细胞,RT-PCR检测示,经诱导5d,BMP-2、DXM、BMP-2+DXM组细胞的ALP、DSPP、DMP-1表达较对照组均上调,其中BMP-2+DXM组表达最高。BMP-2组与DXM组无统计学差异(P0.05),与BMP-2+DXM组、对两组有差异(P0.05);DXM组与BMP-2组无统计学差异(P0.05),与BMP-2+DXM组、对两组有差异(P0.05);BMP-2+DXM组、对照组与其他组间均存在差异(P0.05);提示经BMP-2和DXM诱导后各组hDPCs的分化标志基因强烈表达。经诱导7d各处理组基因表达量趋于同化平稳。DSPP基因表达量BMP-2组、DXM组、BMP-2+DXM组大于对照组(P0.05),但BMP-2组、DXM组、BMP-2+DXM组的组间比较无明显差异(P0.05);同时,ALP、DMP-1基因表达量四个组间无明显差异(P0.05)。培养14 d,经碱性磷酸酶染色检测,BMP-2组、DXM组、BMP-2+DXM组、对照组可见不同着色程度的紫黑色沉淀,其中BMP-2+DXM组着色程度最深,表现为强阳性;经半定量测定阳性染色率:BMP-2组、DXM组、BMP-2+DXM组、对照组依次为0.368±0.004,0.337±0.022,0.849±0.013,0.181±0.008,BMP-2+DXM组BMP-2组≈DXM组D组,BMP-2+DXM组、对照组与其他各组均有差异,差异有统计学意义(P0.05),BMP-2组、DXM组差异无统计学意义(P0.05),与BMP-2+DXM组、对照组均有差异,差异有统计学意义(P0.05)。培养21d,茜素红染色示,BMP-2组、DXM组、BMP-2+DXM组均出现大小及着色程度不一的橘红色矿化结节,其中BMP-2+DXM组矿化结节呈片状大面积分布,BMP-2组、DXM组呈散在分布;对照组无矿化结节形成。BMP-2组、DXM组、BMP-2+DXM组、对照组A值比较为BMP-2+DXM组BMP-2组DXM组对照组,差异有统计学意义(P0.05)。结论:1、利用组织块培养法分离培养hDPCs,可以成功获得稳定传代的牙髓细胞系,为后续各部分实验提供了基础。2、探究并比较了BMP2和DXM两者联合和单独作用对牙髓细胞增殖和分化的影响,丰富了对牙髓细胞分化机制作用的认识,为研究促进牙髓细胞增殖作用和分化作用的细胞因子组合及作用时间提供了依据。
[Abstract]:Dental pulp cells (hDPCs), which have the potential of self replicating and multidirectional differentiation, can differentiate into odontoblast cells under the action of biological factors. Bone morphogenetic eggs -2 (BMP-2) and dexamethasone (DXM) are common osteogenic inducible factors that can induce dental pulp cells to differentiate into odontoblast like cells. The purpose of this study is to explore the role of BMP-2 and DXM in inducing the proliferation and tooth differentiation of dental pulp cells and whether the combination of the two can enhance the induction effect and provide an experimental basis for the optimization of biological factors for the experiment of dental pulp cell differentiation. Objective: 1, to establish a hDPCs culture model and isolate in vitro And culture hDPCs, identify cell source.2, explore the effect of BMP-2 and DXM as well as the combined effect of mineralization, hDPCs in vitro proliferation and differentiation to odontoblast. Methods: 1, hDPCs in vitro separation, culture and identification: the orthodontic teeth or wisdom teeth of the periodontal disease of the dental pulp of 15-30 years old patients in our hospital were collected for half an hour. The pulp tissue was removed from the pulp cavity under rapid and aseptic conditions, and the pulp was cut into 1mm3 small pieces and placed in a culture bottle. The high glucose DMEM culture containing 20% high quality fetal bovine serum was added to the incubator at the constant temperature. The conditions were 37 degrees C and 5%CO2 concentration. After the cell growth was fused to the bottle bottom 80% of the 25cm3 culture bottle, the aseptic pasteurized pipette was used. Adding 0.25% pancreatin and digesting the passage cells. The 3-5 generation cells with good growth state were identified by vimentin and keratin immunochemical staining. The effects of.2, BMP-2 and DXM on the proliferation of hDPCs for the 3-6 generation of frozen and stable passages: BMP-2 (100ng/ml), DXM (1 * 10-8mol/l), BMP-2 (100ng/ml) +DXM (1 x) Ol/l) induced the stable passages of hDPCs, compared with the control group (the high glucose DMEM medium with 10%FBS only), in 1day, 3day, 5day, 7day cells, to make cell suspension, count, and count the effects of.3, BMP-2 and DXM on the differentiation of the cell growth curve to the differentiation of the odontoblast cells. MP-2 and DXM respectively and combined action of 5day, 7day after 5day and 7day, the expression of DSPP, ALP, DMP-1. After 14day, the activity of ALP was detected by alkaline phosphatase staining. 21day, alizarin red mineralized nodule staining, and quantitative detection of mineralized nodules to analyze the odontoblast differentiation condition. Results: 1, hDPCs in vitro isolation, culture and identification: (1) 3-5 days after culture, there were fusiform fibroblast cells climbing out, 7 days or so, the cell dense growth fused to 80% of the bottom of the bottle, which could stabilize the passage and maintain a certain growth ability. (3) the positive expression of vimentin and negative keratin expressed by the vimentin and keratin immunochemistry staining, indicating that the cultured cells were derived from the embryo in the interleaf tissue. The biological characteristics of the dental pulp cells,.2, BMP-2 and DXM, and the effect of combined effect on the proliferation of hDPCs: with the prolongation of the culture time, the number of cells in each group increased gradually, the peak at 5 d, then decreased gradually, and decreased to the level of 3 D. The number of cells in each group was not distinct (P0.05) when the 1D was cultured, and the number of cells in the BMP-2+DXM group when 3 D was cultured. There was no significant difference between the control group and the control group (P0.05), and there was no significant difference between the DXM group and the control group (P0.05), and the number of BMP-2, DXM, BMP-2+DXM groups in the DXM group was significantly higher than that of the control group (P0.05) in the control group (P0.05). The number of cells in the BMP-2+DXM group was significantly higher than that of the control group (P0.05) at 5 d, and the number of fine cells in the BMP-2+DXM group was decreased. And the effect of combined effect on the differentiation of hDPCs: after induction, the odontogenic marker gene DSPP, DMP-1, ALP were expressed, alkaline phosphatase staining was positive, mineralized nodule alizarin red staining was positive, indicating that dental pulp cells could be differentiated into odontoblast cells under the guidance of BMP-2 and DXM, RT-PCR detection showed 5D, BMP-2, D. The expression of ALP, DSPP, DMP-1 in the XM, BMP-2+DXM group was up to that of the control group, and there was no statistical difference between the BMP-2+DXM group and the DXM group (P0.05). There was a difference between the two groups and the BMP-2+DXM group (P0.05); the DXM group and the group had no statistical difference. There was a difference between groups (P0.05), suggesting that the differentiation marker genes of hDPCs were strongly expressed in each group after BMP-2 and DXM induction. The gene expression levels of 7D treated groups tended to assimilate and smooth.DSPP gene expression in BMP-2 group, DXM group and BMP-2+DXM group were larger than those of control group (P0.05), but there was no significant difference in BMP-2 group, DXM group and group. At the same time, there was no significant difference between the four groups of ALP and DMP-1 gene expression (P0.05). The culture of 14 d was detected by alkaline phosphatase staining, group BMP-2, DXM, BMP-2+DXM, and the control group showed the black and black precipitation of different degrees of coloration. In the group BMP-2+DXM, the degree of coloring was the most deep, and the positive staining rate was determined by semi quantitative determination: BMP-2, DXM, BMP-. 2+DXM group, the control group was 0.368 + 0.004,0.337 + 0.022,0.849 + 0.013,0.181 + 0.008, BMP-2+DXM group BMP-2 group DXM group D group, BMP-2+DXM group, the control group were different with the other groups, the difference was statistically significant (P0.05), BMP-2 group, there was no statistical significance in the DXM group, and the control group had difference, the difference was statistically significant Significance (P0.05). Culture 21d, alizarin red staining showed that BMP-2 group, DXM group, BMP-2+DXM group were all size and coloring degree of orange red mineralized nodules, of which BMP-2+DXM group mineralized nodules were distributed in large area, BMP-2 group and DXM group were scattered, and no ore nodules in control group were formed in.BMP-2 group, DXM group, BMP-2+DXM group, and control group A value comparison. The difference between group BMP-2+DXM and group BMP-2 of group BMP-2+DXM was statistically significant (P0.05). Conclusion: 1, using tissue block culture method to isolate and culture hDPCs, a stable generation of dental pulp cell line can be successfully obtained, which provides a basic.2 for the subsequent parts of the experiment, and explores and compares the combination and separate action of both BMP2 and DXM to the proliferation and differentiation of dental pulp cells. It has enriched the understanding of the mechanism of the differentiation of dental pulp cells, and provides a basis for the study of the combination of cytokines and the time of action to promote the proliferation and differentiation of dental pulp cells.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781

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