本文關(guān)鍵詞： 脫礦牙本質(zhì)基質(zhì) BMP-2 Ⅰ型膠原蛋白 MC3T3-E1細(xì)胞 細(xì)胞趨化　出處：《昆明醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]:研究脫礦牙本質(zhì)基質(zhì)(decalcificated dentin matrix,DDM)釋放BMP-2性能及其對(duì)細(xì)胞的趨化能力,為其作為骨移植材料提供理論支持。[方法]:1、收集牙齒,去除牙齒附著的軟組織、牙釉質(zhì)、牙骨質(zhì)及牙髓,粉碎,篩選粒徑100～400μm牙本質(zhì)顆粒,脫脂、脫礦等一系列處理后制成DDM。同時(shí)使用Urist理化法提取5g DDM中的BMP-2,通過(guò)酶聯(lián)免疫法檢測(cè)檢測(cè)其中BMP-2的含量。2、將自制DDM(A組)、成品DDM(B組)置于DMEM培養(yǎng)基中,設(shè)置單純DMEM培養(yǎng)基為C組。A組、B組為實(shí)驗(yàn)組,C組為空白對(duì)照組,浸提1、2、3、5、7、9、11、13d,使用酶聯(lián)免疫法檢測(cè)檢測(cè)浸提液中BMP-2及膠原蛋白的釋放量。3、對(duì)小鼠成骨前體細(xì)胞MC3T3-E1進(jìn)行傳代培養(yǎng),培養(yǎng)至第3代后,使用Transwell小室將細(xì)胞與下室的自制DDM(D組)、成品DDM(E組)、Bio-Oss(F組)和不含材料的培養(yǎng)基(G組)進(jìn)行聯(lián)合培養(yǎng),D組、E組為實(shí)驗(yàn)組,F組為對(duì)照組,G組為空白對(duì)照組。無(wú)血清培養(yǎng),培養(yǎng)至1、2、3天時(shí),使用酶聯(lián)免疫法檢測(cè)檢測(cè)下室培養(yǎng)液中BMP-2及膠原蛋白的釋放量,與實(shí)驗(yàn)第二部分前3天BMP-2及膠原蛋白的釋放量相比較,并對(duì)Transwell小室進(jìn)行結(jié)晶紫染色,使用熒光顯微鏡觀察穿過(guò)Transwell小室的細(xì)胞數(shù)量。所有數(shù)據(jù)用SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)分析。[結(jié)果]:1、成功制備粒徑為10O～400μm的DDM;成功從牙本質(zhì)中提取出BMP-2,即約0.12ng/g。2、各實(shí)驗(yàn)組浸提液中BMP-2濃度隨著時(shí)間的推移,呈現(xiàn)先上升、后下降至維持穩(wěn)定濃度的趨勢(shì)。分別在第1、2、3、5、7、9、11、13天檢測(cè)浸提液中BMP-2濃度,結(jié)果采用多變量方差分析SNK-q進(jìn)行兩兩比較,A、B兩組與C組比較,P0.05,差異有統(tǒng)計(jì)學(xué)意義。A、B兩組之間比較,P0.05,差異有統(tǒng)計(jì)學(xué)意義。分別在第1、2、3、5、7、9、11、13天檢測(cè)浸提液中Ⅰ型膠原蛋白濃度,結(jié)果采用多變量方差分析SNK-q進(jìn)行兩兩比較,A、B兩組與C組比較,P0.05,差異有統(tǒng)計(jì)學(xué)意義。A、B兩組之間比較,P0.05,差異有統(tǒng)計(jì)學(xué)意義。3、各組下室培養(yǎng)基中BMP-2濃度隨著時(shí)間的推移,呈現(xiàn)逐步上升趨勢(shì)。分別在第1、2、3天時(shí)測(cè)得各組下室培養(yǎng)基中BMP-2的濃度,結(jié)果采用多變量方差分析SNK-q進(jìn)行兩兩比較,除F與G兩組比較,P0.05,差異無(wú)統(tǒng)計(jì)學(xué)意義,其余各組兩兩比較均有統(tǒng)計(jì)學(xué)差異。將此組數(shù)據(jù)與第二部分1、2、3天數(shù)據(jù)相比較:兩實(shí)驗(yàn)中,自制DDM組中BMP-2濃度均隨著時(shí)間的推移呈現(xiàn)逐步上升趨勢(shì)。在第1、2、3天三個(gè)時(shí)間點(diǎn)中,第1天和第三天實(shí)驗(yàn)二中的BMP-2濃度與實(shí)驗(yàn)三比較,P0.05,差異有統(tǒng)計(jì)學(xué)意義;第2天實(shí)驗(yàn)二比實(shí)驗(yàn)三相比較,P0.05,差異無(wú)統(tǒng)計(jì)學(xué)意義。分別在第1、2、3天時(shí)檢測(cè)各組下室培養(yǎng)基中Ⅰ型膠原蛋白濃度,結(jié)果采用多變量方差分析SNK-q進(jìn)行兩兩比較,除F組與G組比較,P0.05,差異無(wú)統(tǒng)計(jì)學(xué)意義,其余各組兩兩比較均有統(tǒng)計(jì)學(xué)差異。將此組數(shù)據(jù)與第二部分1、2、3天數(shù)據(jù)相比:兩實(shí)驗(yàn)中,自制DDM組中Ⅰ型膠原蛋白濃度均隨著時(shí)間的推移呈現(xiàn)逐步上升趨勢(shì)。在第1、2、3天三個(gè)時(shí)間點(diǎn)實(shí)驗(yàn)二中Ⅰ型膠原蛋白濃度與實(shí)驗(yàn)三比較,P0.05,差異有統(tǒng)計(jì)學(xué)意義。分別在第1、2、3天時(shí)計(jì)數(shù)各組穿過(guò)Transwell小室細(xì)胞數(shù)目,統(tǒng)計(jì)結(jié)果采用多變量方差分析SNK-q進(jìn)行兩兩比較,除F與G兩組比較,P0.05,差異無(wú)統(tǒng)計(jì)學(xué)意義,其余各組兩兩比較均有統(tǒng)計(jì)學(xué)差異。[結(jié)論]:1、在本實(shí)驗(yàn)中,牙本質(zhì)中存在BMP-2。DDM中的BMP-2含量約為 0.12ng/g。2、在本實(shí)驗(yàn)中,DDM可以緩釋BMP-2。本實(shí)驗(yàn)中DDM釋放BMP-2達(dá)近似穩(wěn)平衡狀態(tài)時(shí),最終測(cè)得自制組為58.00±1.78pg/ml,成品組最后濃度為6.42±1.25pg/ml。3、在本實(shí)驗(yàn)中,與細(xì)胞共培養(yǎng)對(duì)DDM釋放BMP-2有影響。4、在本實(shí)驗(yàn)中,對(duì)成骨源細(xì)胞具有一定趨化作用,可以作為骨移植材料。
[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.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.6

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本文編號(hào)：1552005