DDM释放BMP-2相关性能研究

发布时间：2018-03-01 12:43

  本文关键词： 脱矿牙本质基质 BMP-2 Ⅰ型胶原蛋白 MC3T3-E1细胞 细胞趋化　出处：《昆明医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：[目的]:研究脱矿牙本质基质(decalcificated dentin matrix,DDM)释放BMP-2性能及其对细胞的趋化能力,为其作为骨移植材料提供理论支持。[方法]:1、收集牙齿,去除牙齿附着的软组织、牙釉质、牙骨质及牙髓,粉碎,筛选粒径100～400μm牙本质颗粒,脱脂、脱矿等一系列处理后制成DDM。同时使用Urist理化法提取5g DDM中的BMP-2,通过酶联免疫法检测检测其中BMP-2的含量。2、将自制DDM(A组)、成品DDM(B组)置于DMEM培养基中,设置单纯DMEM培养基为C组。A组、B组为实验组,C组为空白对照组,浸提1、2、3、5、7、9、11、13d,使用酶联免疫法检测检测浸提液中BMP-2及胶原蛋白的释放量。3、对小鼠成骨前体细胞MC3T3-E1进行传代培养,培养至第3代后,使用Transwell小室将细胞与下室的自制DDM(D组)、成品DDM(E组)、Bio-Oss(F组)和不含材料的培养基(G组)进行联合培养,D组、E组为实验组,F组为对照组,G组为空白对照组。无血清培养,培养至1、2、3天时,使用酶联免疫法检测检测下室培养液中BMP-2及胶原蛋白的释放量,与实验第二部分前3天BMP-2及胶原蛋白的释放量相比较,并对Transwell小室进行结晶紫染色,使用荧光显微镜观察穿过Transwell小室的细胞数量。所有数据用SPSS 19.0软件进行统计分析。[结果]:1、成功制备粒径为10O～400μm的DDM;成功从牙本质中提取出BMP-2,即约0.12ng/g。2、各实验组浸提液中BMP-2浓度随着时间的推移,呈现先上升、后下降至维持稳定浓度的趋势。分别在第1、2、3、5、7、9、11、13天检测浸提液中BMP-2浓度,结果采用多变量方差分析SNK-q进行两两比较,A、B两组与C组比较,P0.05,差异有统计学意义。A、B两组之间比较,P0.05,差异有统计学意义。分别在第1、2、3、5、7、9、11、13天检测浸提液中Ⅰ型胶原蛋白浓度,结果采用多变量方差分析SNK-q进行两两比较,A、B两组与C组比较,P0.05,差异有统计学意义。A、B两组之间比较,P0.05,差异有统计学意义。3、各组下室培养基中BMP-2浓度随着时间的推移,呈现逐步上升趋势。分别在第1、2、3天时测得各组下室培养基中BMP-2的浓度,结果采用多变量方差分析SNK-q进行两两比较,除F与G两组比较,P0.05,差异无统计学意义,其余各组两两比较均有统计学差异。将此组数据与第二部分1、2、3天数据相比较:两实验中,自制DDM组中BMP-2浓度均随着时间的推移呈现逐步上升趋势。在第1、2、3天三个时间点中,第1天和第三天实验二中的BMP-2浓度与实验三比较,P0.05,差异有统计学意义;第2天实验二比实验三相比较,P0.05,差异无统计学意义。分别在第1、2、3天时检测各组下室培养基中Ⅰ型胶原蛋白浓度,结果采用多变量方差分析SNK-q进行两两比较,除F组与G组比较,P0.05,差异无统计学意义,其余各组两两比较均有统计学差异。将此组数据与第二部分1、2、3天数据相比:两实验中,自制DDM组中Ⅰ型胶原蛋白浓度均随着时间的推移呈现逐步上升趋势。在第1、2、3天三个时间点实验二中Ⅰ型胶原蛋白浓度与实验三比较,P0.05,差异有统计学意义。分别在第1、2、3天时计数各组穿过Transwell小室细胞数目,统计结果采用多变量方差分析SNK-q进行两两比较,除F与G两组比较,P0.05,差异无统计学意义,其余各组两两比较均有统计学差异。[结论]:1、在本实验中,牙本质中存在BMP-2。DDM中的BMP-2含量约为 0.12ng/g。2、在本实验中,DDM可以缓释BMP-2。本实验中DDM释放BMP-2达近似稳平衡状态时,最终测得自制组为58.00±1.78pg/ml,成品组最后浓度为6.42±1.25pg/ml。3、在本实验中,与细胞共培养对DDM释放BMP-2有影响。4、在本实验中,对成骨源细胞具有一定趋化作用,可以作为骨移植材料。
[Abstract]:Objective: To study the demineralized dentin matrix (decalcificated dentin matrix, DDM BMP-2) release properties and cell chemotaxis ability, provide theoretical support for the method. As a bone graft material: 1, collect the teeth, remove soft tissue attachment, tooth enamel, cementum and pulp, crushing, screening the particle size of 100~400 mu m dentin particles, degreasing, demineralization and a series of processing made after extraction of 5g in DDM DDM. and BMP-2 Urist by using physicochemical method, enzyme-linked immunosorbent assay to detect the content of.2 and BMP-2, using DDM (A group), finished DDM (group B) on the DMEM medium in the setting of simple DMEM medium for C group,.A group, B group, C group was the control group, 1,2,3,5,7,9,11,13d extraction, detection using ELISA extraction.3 release BMP-2 and collagen in the liquid on mouse osteoblast precursor cells MC3T3-E1 were cultured and cultured After third generations, using Transwell cell cells and the lower chamber of the self-made DDM (D group), finished DDM (E group), Bio-Oss (group F) and medium containing materials (group G) were co cultured, D group, E group, F group, G group in the blank control group. Serum free culture, cultured for 1,2,3 days, detected by enzyme-linked immunosorbent assay and BMP-2 chamber culture under the collagen liquid release, release and the second part of the experiment, 3 days before BMP-2 and collagen compared to Transwell cells were stained with crystal violet, the number of cells using fluorescence microscope through the Transwell chamber. All data using SPSS 19 software for statistical analysis. Results: 1, the successful preparation of the particle size of 10O ~ 400 m DDM; extract BMP-2 from the dentin, which is about 0.12ng/g.2, the experimental group in the extracts of BMP-2 concentration with time, showing first increased and then decreased to To maintain a stable concentration trend. The leaching concentration of BMP-2 solution respectively on day 1,2,3,5,7,9,11,13 detection results using multivariate analysis of variance and SNK-q were 22, A, B two group, P0.05 group and C group, there was significant difference between the two groups.A, B, P0.05, the difference was statistically significant. The extraction of liquid collagen concentration respectively on day 1,2,3,5,7,9,11,13 detection results using multivariate analysis of variance of SNK-q 22, A two, B group compared with C group P0.05, there were statistically significant differences between the two groups.A, B, P0.05, there was a significant difference between the.3 and BMP-2 in the medium concentration with time in each group the lower chamber culture, showed a gradual upward trend. The measured concentration of BMP-2 in the medium of each room under cultivation in the first 1,2,3 days, the results using multivariate analysis of variance and SNK-q 22 comparison, except F and G between the two groups, P0.05, no significant difference Meaning, 22 other groups were statistically significant. The second part of the 1,2,3 group data and the comparison of the data: two day experiment, self-made DDM group BMP-2 concentrations were over time showed a gradual upward trend. At the three time points in 1,2,3 days, first days and third days of Experiment 2 BMP-2 concentration and in experiment three, P0.05, the difference was statistically significant; second days of experiment two than experimental three-phase comparison, P0.05, the difference was not statistically significant. Each type respectively under chamber culture medium collagen concentration detection on day 1,2,3, multivariate analysis of variance results using SNK-q 22, except for the F group compared with G group P0.05, the difference was not statistically significant, 22 other groups were statistically significant. The data set and the second part 1,2,3: two days compared to the data in the experiment, self-made DDM type I collagen concentration group increased along with the time of pushing Shift showed a gradual upward trend. P0.05 in the first 1,2,3 days of three time points in Experiment II collagen concentration and three experimental comparison, the difference was statistically significant. After 1,2,3 days were counted through the cell number of Transwell, the statistical results by multivariate variance analysis and comparison of SNK-q 22, in addition to F and G two group P0.05, no significant difference between the 22 groups were statistically significant. Conclusion: 1. In this experiment, dentin in the presence of BMP-2 content in BMP-2.DDM is about 0.12ng/g.2, in this experiment, DDM can release the experiment of BMP-2. DDM release BMP-2 reached a quasi steady state of equilibrium. The final measured self-made group is 58 + 1.78pg/ml, finished the final group concentration was 6.42 + 1.25pg/ml.3, in this experiment, co cultured with DDM cells to release BMP-2 of.4, in this experiment, the osteoblast derived cells has certain tendency It can be used as a bone graft material.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.6

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