本文選題：牙周膜干細(xì)胞　切入點(diǎn)：BMP9　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景與目的:慢性牙周炎、腫瘤、外傷、不恰當(dāng)?shù)恼委熅蓪?dǎo)致牙槽骨缺損。近年來,組織工程技術(shù)用于牙槽骨缺損的治療被證實(shí)具有一定效果,并且擁有巨大潛力。牙周膜干細(xì)胞(periodontal ligament stem cells,PDLSCs)因具有低免疫原性、自我更新能力和多向分化潛能等特點(diǎn)被證實(shí)為牙周組織工程中的首選種子細(xì)胞。如何更好的促進(jìn)種子細(xì)胞的增殖和成骨分化,并且發(fā)揮修復(fù)組織缺損的生物學(xué)功能,是目前亟需解決的問題。脈沖電磁場(Pulsed Electromagnetic Fields,PEMF)物理刺激和骨形態(tài)發(fā)生蛋白(bone morphogenetic proteins,BMPs)細(xì)胞因子均被證實(shí)具有促進(jìn)干細(xì)胞增殖和誘導(dǎo)成骨分化的能力。體外實(shí)驗(yàn)表明,適宜磁場參數(shù)的PEMF與其他生化因子(如分化因子和細(xì)胞生長因子)聯(lián)合作用時可明顯提高細(xì)胞增殖活性及成骨標(biāo)志分子的表達(dá)及礦化結(jié)節(jié)的生成。然而,目前尚沒有PEMF單獨(dú)刺激或聯(lián)合生長因子在牙槽骨修復(fù)方面的體內(nèi)外研究。因此,本課題探索性的研究不同磁場強(qiáng)度的PEMF單獨(dú)以及聯(lián)合骨形態(tài)發(fā)生蛋白9(bone morphogenetic protein 9,BMP9)對PDLSCs的增殖能力和成骨分化的影響,期望為牙周骨組織再生提供一種無創(chuàng)的輔助性療法。實(shí)驗(yàn)方法:1.在患者知情同意下,收集因正畸治療需要拔除的健康前磨牙,共20顆。采用組織塊貼壁法體外培養(yǎng)原代牙周膜細(xì)胞,采用CD146免疫磁珠分選獲得CD146陽性PDLSCs,然后通過流式細(xì)胞鑒定細(xì)胞表面抗原CD146和STRO-1的表達(dá)。通過免疫熒光染色鑒定細(xì)胞的組織來源,通過茜素紅S染色和油紅O染色鑒定細(xì)胞的成骨和成脂分化能力。2.構(gòu)建重組過表達(dá)BMP9腺病毒(adenoviruses overexpressing BMP9,Ad-BMP9),確定適宜的病毒感染濃度(multiplicities of infection,MOI)檢測感染PDLSCs的BMP9基因和蛋白的表達(dá)變化。然后通過細(xì)胞計數(shù)試劑盒(Cell counting kit-8,CCK8)檢測連續(xù)10天內(nèi)的細(xì)胞增殖活性,并繪制細(xì)胞增殖曲線。檢測Ad-BMP9對PDLSCs中早期和中期成骨標(biāo)志分子如堿性磷酸酶(alkaline phosphatase,ALP)、Runt相關(guān)轉(zhuǎn)錄因子2(Runt-related transcription factor 2,Runx2)、骨橋蛋白(osteopontin,OPN)的基因和蛋白表達(dá)影響,以及對成骨后期礦化結(jié)節(jié)形成的影響。3.設(shè)置15 HZ,5組不同場強(qiáng)的PEMF(0.6、1.2、1.8、2.4、3.0 m T)輻照體外培養(yǎng)的PDLSCs或者感染Ad-BMP9的PDLSCs,每天輻照1h,使用CCK8試劑盒連續(xù)10天檢測細(xì)胞的增殖活性,并繪制細(xì)胞增殖曲線。4.設(shè)置15 HZ,5組不同場強(qiáng)(0.6、1.2、1.8、2.4、3.0 m T,1h/d)的PEMF輻照PDLSCs,檢測ALP、Runx2、OPN基因表達(dá),篩選適宜的磁場強(qiáng)度。篩選出的PEMF(1.8 m T和2.4 m T)輻照轉(zhuǎn)染了Ad-BMP9的PDLSCs,檢測ALP、Runx2、OPN基因和蛋白表達(dá),采用茜素紅S染色檢測分化后期的鈣鹽沉淀情況。實(shí)驗(yàn)結(jié)果:1.流式細(xì)胞檢測顯示,CD146免疫磁珠分選所得的同時表達(dá)CD146陽性和STRO-1陽性的細(xì)胞比例約占32.2%。免疫熒光染色顯示角蛋白陰性、波形蛋白陽性。茜素紅S染色和油紅O檢測表明分選后的PDLSCs具有成骨和成脂的多向分化潛能。2.腺病毒感染PDLSCs的MOI確定為30-50時具有較高的轉(zhuǎn)染效率70%-80%;RT-PCR和Western blot結(jié)果顯示感染Ad-BMP9的PDLSCs明顯高表達(dá)BMP9基因和蛋白;CCK8檢測發(fā)現(xiàn)Ad-BMP9明顯促進(jìn)對數(shù)生長期的PDLSCs增殖活性;ALP定量檢測顯示Ad-BMP9明顯促進(jìn)了成骨分化早期細(xì)胞內(nèi)ALP蛋白的表達(dá),通過實(shí)時定量PCR(quantitative real-time polymerase chain reaction,q RT-PCR)檢測表明Ad-BMP9具有誘導(dǎo)PDLSCs表達(dá)ALP、Runx2、OPN等成骨標(biāo)記基因的能力。3.CCK8檢測發(fā)現(xiàn)PEMF對PDLSCs的細(xì)胞數(shù)量沒有明顯影響,而且PEMF沒有增強(qiáng)Ad-BMP9對PDLSCs增殖活性的影響。4.q RT-PCR結(jié)果顯示15Hz,1.8m T和2.4m T場強(qiáng)的PEMF明顯促進(jìn)了PDLSCs的早期和中期成骨基因的表達(dá)。q RT-PCR、Western blot、茜素紅S染色結(jié)果顯示PEMF聯(lián)合BMP9誘導(dǎo)PDLSCs表達(dá)ALP、Runx2、OPN的成骨分化能力以及成骨后期礦化結(jié)節(jié)的形成均高于單獨(dú)因素作用組。結(jié)論:本課題證實(shí)了BMP9具有促進(jìn)PDLSCs的增殖活性和誘導(dǎo)成骨分化的能力;單獨(dú)作用或在BMP9存在的前提下,PEMF刺激均沒有對細(xì)胞增殖活性產(chǎn)生顯著影響;適宜磁場參數(shù)下的PEMF具有誘導(dǎo)PDLSCs成骨分化的能力,而且聯(lián)合BMP9誘導(dǎo)PDLSCs成骨分化時存在交互作用。因此,適宜參數(shù)的PEMF可以增強(qiáng)BMP9誘導(dǎo)PDLSCs成骨分化的能力。
[Abstract]:Background and objective: chronic periodontitis, tumor, trauma, orthodontic treatment can lead to inappropriate alveolar bone defect. In recent years, tissue engineering technology for the treatment of alveolar bone defect was confirmed to have a certain effect, and has great potential. Periodontal ligament stem cells (periodontal ligament stem cells, PDLSCs) due to its low immunogenicity the characteristics of self-renewal and multilineage differentiation potential was confirmed for periodontal tissue engineering seed cells. The choice of how to better promote cell proliferation and osteogenic differentiation, and helps to repair tissue defect of biological function, is an urgent problem. The pulse electromagnetic field (Pulsed Electromagnetic Fields, PEMF) physics stimulation and bone morphogenetic protein (bone morphogenetic, proteins, BMPs) were identified with cytokines to promote stem cell proliferation and induce osteogenic differentiation capacity in vitro. Experimental results show that the suitable magnetic field parameters of PEMF and other biochemical factors (such as differentiation factor and fibroblast growth factor) when combined can generate expression and mineralized nodule increased cell proliferation and osteogenic markers. However, there is no PEMF alone or combined with stimulation of growth factor in alveolar bone repair in vivo. Therefore, this topic research of different magnetic field intensity PEMF alone and combined with bone morphogenetic protein 9 (bone morphogenetic 9 protein, BMP9) PDLSCs on the proliferation and osteogenic differentiation, is expected to provide a non-invasive adjuvant therapy for the regeneration of periodontal tissue. Methods: 1. informed consent of patients, because of the need to collect extracted healthy premolars for orthodontic treatment, a total of 20. Cultured by tissue adherent method in vitro cultured periodontal ligament cells by immunomagnetic separation, CD146 CD1 46 positive PDLSCs expression by flow cytometry, and identification of cell surface antigen CD146 and STRO-1. The cells were identified by immunofluorescence staining of tissue derived cells were identified by alizarin red staining, S staining and oil red O osteogenic and adipogenic differentiation ability of.2. to construct the recombinant adenovirus overexpressing BMP9 (adenoviruses overexpressing BMP9, Ad-BMP9) sure, the suitable concentration of virus infection (multiplicities of infection, MOI) of the BMP9 gene and protein expression of PDLSCs infection detection. Then through cell counting Kit (Cell counting, kit-8, CCK8) to detect the cell proliferation activity for 10 consecutive days, and cell proliferation curve. Detection of Ad-BMP9 of PDLSCs in the early and middle osteogenic markers such as alkaline phosphatase (alkaline phosphatase, ALP), Runt related transcription factor 2 (Runt-related transcription 2 factor, Runx2), osteopontin (osteopontin, OPN) The expression of gene and protein, and set the 15 effects of HZ on osteogenic late mineralized nodule formation of.3., 5 groups of different intensity of PEMF (0.6,1.2,1.8,2.4,3.0 m T) irradiation in vitro PDLSCs infection or Ad-BMP9 PDLSCs, a day of irradiated 1H, use CCK8 kit for 10 consecutive days to detect cell proliferation and cell activity. The proliferation curve of.4. is set to 15 HZ, 5 groups of different intensity (0.6,1.2,1.8,2.4,3.0 m T, 1h/d) PEMF irradiation PDLSCs, detection of ALP, Runx2, OPN gene expression, screening magnetic field strength suitable. The screened PEMF (1.8 m T and 2.4 m T) irradiation Ad-BMP9 transfected with PDLSCs, detection of ALP, Runx2, expression OPN gene and protein, calcium salt by alizarin red S staining was used to detect the differentiation of late precipitation. The experimental results show: the detection of 1. flow cytometry sorting, CD146 immunomagnetic the simultaneous expression of CD146 positive and STRO-1 positive cells proportion of about 32.2%. free Immunofluorescence staining showed negative keratin, vimentin positive. Alizarin red S staining and oil red O showed that after the separation of PDLSCs with osteoblasts and 70%-80% transfection efficiency of lipid differentiation.2. adenovirus PDLSCs MOI was determined as 30-50 has higher; RT-PCR and Western blot results showed that Ad-BMP9 infection was PDLSCs the high expression of BMP9 gene and protein; CCK8 assay showed that Ad-BMP9 significantly promoted the proliferation of PDLSCs in logarithmic growth phase; quantitative detection of ALP showed that Ad-BMP9 could promote the expression of osteogenic differentiation of early intracellular ALP protein, using real-time quantitative PCR (quantitative real-time polymerase chain reaction, Q RT-PCR) assay showed that Ad-BMP9 has induced the expression of PDLSCs ALP, Runx2 OPN, such as.3.CCK8 detection of bone marker gene PEMF of PDLSCs found that the number of cells had no obvious effect, but PEMF did not enhance Ad-BMP9 .4.q effect of RT-PCR on proliferation of PDLSCs showed that 15Hz, 1.8m T and 2.4m of T PEMF can promote the early and mid PDLSCs osteogenic gene expression of.Q RT-PCR, Western blot, alizarin red S staining showed that the expression of ALP, PDLSCs induced by PEMF and BMP9 Runx2 OPN, osteogenic differentiation and formation later bone mineralization nodules were higher than that of group action factors. Conclusions: This study confirmed that BMP9 can promote PDLSCs proliferation and induce osteogenic differentiation capacity; alone or in the premise of the existence of BMP9, PEMF showed no significant impact on cell proliferation activity; the suitable parameters of the PEMF with a magnetic field the ability of osteogenic differentiation induced by PDLSCs, and combined with BMP9 induced osteogenic differentiation of PDLSCs during the interaction. Therefore, the optimum parameters of PEMF can enhance BMP9 PDLSCs induced osteogenic differentiation capacity.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R782.1

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