本文選題：BMP2 + 對(duì)人��； 參考：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：牙髓細(xì)胞(hDPCs)具有自我復(fù)制更新和多向分化克隆的潛能,可在生物因子的作用下可以向成牙本質(zhì)細(xì)胞分化。骨形態(tài)發(fā)生蛋-2(BMP-2)和地塞米松(DXM)是常見的成骨誘導(dǎo)因子,可以使牙髓細(xì)胞向成牙本質(zhì)樣細(xì)胞誘導(dǎo)分化。在成牙本質(zhì)細(xì)胞形成過(guò)程中亦發(fā)揮著重要作用。本研究的目的為探討B(tài)MP-2和DXM在誘導(dǎo)牙髓細(xì)胞增殖和牙向分化的作用以及兩者聯(lián)合運(yùn)用是否能增強(qiáng)誘導(dǎo)效果,為牙髓細(xì)胞分化的實(shí)驗(yàn)提供生物因子優(yōu)化組合的實(shí)驗(yàn)依據(jù)。目的:1、建立hDPCs培養(yǎng)模型,體外分離并培養(yǎng)hDPCs,鑒定細(xì)胞來(lái)源。2、探討B(tài)MP-2和DXM以及兩者聯(lián)合作用礦化及誘導(dǎo)hDPCs體外增殖及向成牙本質(zhì)細(xì)胞分化的影響。方法:1、hDPCs的體外分離、培養(yǎng)及鑒定:收集我院外科門診15-30周歲患者無(wú)牙體牙髓牙周疾病的正畸牙或智齒,半小時(shí)之內(nèi)迅速至無(wú)菌條件下劈開牙體取出髓腔中牙髓組織,并將其剪成1mm3小塊,置于培養(yǎng)瓶中,加入含20%優(yōu)質(zhì)胎牛血清的高糖DMEM培養(yǎng)液,于恒溫培養(yǎng)箱中培養(yǎng),設(shè)置條件為37℃、5%CO2濃度。待細(xì)胞生長(zhǎng)融合至25cm3培養(yǎng)瓶瓶底80%后,用無(wú)菌巴氏吸管加入0.25%胰酶,消化傳代細(xì)胞。取生長(zhǎng)狀態(tài)良好的第3-5代細(xì)胞行波形蛋白和角蛋白免疫化學(xué)染色鑒定。凍存穩(wěn)定傳代的3-6代hDPCs待用。2、BMP-2和DXM分別及聯(lián)合作用對(duì)hDPCs增殖的影響:用濃度為BMP-2(100ng/ml)、DXM(1×10-8mol/l)、BMP-2(100ng/ml)+DXM(1×10-8mol/l)誘導(dǎo)穩(wěn)定傳代的hDPCs,與對(duì)照組比較(僅含10%FBS的高糖DMEM培養(yǎng)液),在1day、3day、5day、7day消化細(xì)胞,制成細(xì)胞懸濁液,計(jì)數(shù),繪制各組細(xì)胞生長(zhǎng)曲線。3、BMP-2和DXM分別及聯(lián)合作用對(duì)h DPC向成牙本質(zhì)細(xì)胞分化的影響:誘導(dǎo)步驟同上,利用RT-PCR檢測(cè)BMP-2和DXM分別及聯(lián)合作用5day、7day后成牙本質(zhì)標(biāo)記基因DSPP、ALP、DMP-1的表達(dá)。作用14day后進(jìn)行堿性磷酸酶染色檢測(cè)ALP活性。作用21day,進(jìn)行茜素紅礦化結(jié)節(jié)染色,定量檢測(cè)礦化結(jié)節(jié)分析成牙本質(zhì)分化情況。結(jié)果:1、hDPCs的體外分離、培養(yǎng)及鑒定:(1)培養(yǎng)3-5天后,可見有梭型纖維狀細(xì)胞爬出,7天左右細(xì)胞密集生長(zhǎng)融合至瓶底的80%,可穩(wěn)定傳代,保持一定生長(zhǎng)能力。(3)經(jīng)波形蛋白、角蛋白免疫化學(xué)染色可見,細(xì)胞波形蛋白陽(yáng)性表達(dá),角蛋白陰性表達(dá),說(shuō)明培養(yǎng)的細(xì)胞來(lái)源于間葉組織中胚層,符合牙髓細(xì)胞的生物學(xué)特征。2、BMP-2和DXM分別及聯(lián)合作用對(duì)hDPCs增殖的影響:隨培養(yǎng)時(shí)間延長(zhǎng),各組細(xì)胞數(shù)均逐漸增加,5 d時(shí)達(dá)峰值,后逐漸下降,7 d降至3 d時(shí)水平。培養(yǎng)1d時(shí),各組細(xì)胞數(shù)無(wú)明顯差異(P0.05);培養(yǎng)3 d時(shí)BMP-2組、BMP-2+DXM組細(xì)胞數(shù)顯著高于DXM、對(duì)照組(P0.05)BMP-2組、BMP-2+DXM組間無(wú)明顯差異,DXM組、對(duì)照組間無(wú)明顯差異(P0.05);5 d時(shí)BMP-2、DXM、BMP-2+DXM組細(xì)胞數(shù)顯著高于對(duì)照組,BMP-2+DXM組BMP-2組DXM組對(duì)照組(P0.05);7d時(shí)細(xì)胞數(shù)下降,BMP-2+DXM組BMP-2組對(duì)照組DXM組(P0.05)。3、BMP-2和DXM分別及聯(lián)合作用對(duì)hDPCs分化的影響:經(jīng)誘導(dǎo)后各處組的成牙本質(zhì)形成標(biāo)記基因DSPP、DMP-1、ALP均有表達(dá),堿性磷酸酶染色染色陽(yáng)性,礦化結(jié)節(jié)茜素紅染色陽(yáng)性,說(shuō)明牙髓細(xì)胞可以在BMP-2、DXM的誘導(dǎo)下分化為成牙本質(zhì)細(xì)胞,RT-PCR檢測(cè)示,經(jīng)誘導(dǎo)5d,BMP-2、DXM、BMP-2+DXM組細(xì)胞的ALP、DSPP、DMP-1表達(dá)較對(duì)照組均上調(diào),其中BMP-2+DXM組表達(dá)最高。BMP-2組與DXM組無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),與BMP-2+DXM組、對(duì)兩組有差異(P0.05);DXM組與BMP-2組無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),與BMP-2+DXM組、對(duì)兩組有差異(P0.05);BMP-2+DXM組、對(duì)照組與其他組間均存在差異(P0.05);提示經(jīng)BMP-2和DXM誘導(dǎo)后各組hDPCs的分化標(biāo)志基因強(qiáng)烈表達(dá)。經(jīng)誘導(dǎo)7d各處理組基因表達(dá)量趨于同化平穩(wěn)。DSPP基因表達(dá)量BMP-2組、DXM組、BMP-2+DXM組大于對(duì)照組(P0.05),但BMP-2組、DXM組、BMP-2+DXM組的組間比較無(wú)明顯差異(P0.05);同時(shí),ALP、DMP-1基因表達(dá)量四個(gè)組間無(wú)明顯差異(P0.05)。培養(yǎng)14 d,經(jīng)堿性磷酸酶染色檢測(cè),BMP-2組、DXM組、BMP-2+DXM組、對(duì)照組可見不同著色程度的紫黑色沉淀,其中BMP-2+DXM組著色程度最深,表現(xiàn)為強(qiáng)陽(yáng)性;經(jīng)半定量測(cè)定陽(yáng)性染色率:BMP-2組、DXM組、BMP-2+DXM組、對(duì)照組依次為0.368±0.004,0.337±0.022,0.849±0.013,0.181±0.008,BMP-2+DXM組BMP-2組≈DXM組D組,BMP-2+DXM組、對(duì)照組與其他各組均有差異,差異有統(tǒng)計(jì)學(xué)意義(P0.05),BMP-2組、DXM組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),與BMP-2+DXM組、對(duì)照組均有差異,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。培養(yǎng)21d,茜素紅染色示,BMP-2組、DXM組、BMP-2+DXM組均出現(xiàn)大小及著色程度不一的橘紅色礦化結(jié)節(jié),其中BMP-2+DXM組礦化結(jié)節(jié)呈片狀大面積分布,BMP-2組、DXM組呈散在分布;對(duì)照組無(wú)礦化結(jié)節(jié)形成。BMP-2組、DXM組、BMP-2+DXM組、對(duì)照組A值比較為BMP-2+DXM組BMP-2組DXM組對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1、利用組織塊培養(yǎng)法分離培養(yǎng)hDPCs,可以成功獲得穩(wěn)定傳代的牙髓細(xì)胞系,為后續(xù)各部分實(shí)驗(yàn)提供了基礎(chǔ)。2、探究并比較了BMP2和DXM兩者聯(lián)合和單獨(dú)作用對(duì)牙髓細(xì)胞增殖和分化的影響,豐富了對(duì)牙髓細(xì)胞分化機(jī)制作用的認(rèn)識(shí),為研究促進(jìn)牙髓細(xì)胞增殖作用和分化作用的細(xì)胞因子組合及作用時(shí)間提供了依據(jù)。
[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.

【學(xué)位授予單位】：廣西醫(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)】：R781

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