PEMF联合BMP9对牙周膜干细胞增殖和成骨分化的影响

发布时间：2018-03-09 16:00

本文选题：牙周膜干细胞　切入点：BMP9　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景与目的:慢性牙周炎、肿瘤、外伤、不恰当的正畸治疗均可导致牙槽骨缺损。近年来,组织工程技术用于牙槽骨缺损的治疗被证实具有一定效果,并且拥有巨大潜力。牙周膜干细胞(periodontal ligament stem cells,PDLSCs)因具有低免疫原性、自我更新能力和多向分化潜能等特点被证实为牙周组织工程中的首选种子细胞。如何更好的促进种子细胞的增殖和成骨分化,并且发挥修复组织缺损的生物学功能,是目前亟需解决的问题。脉冲电磁场(Pulsed Electromagnetic Fields,PEMF)物理刺激和骨形态发生蛋白(bone morphogenetic proteins,BMPs)细胞因子均被证实具有促进干细胞增殖和诱导成骨分化的能力。体外实验表明,适宜磁场参数的PEMF与其他生化因子(如分化因子和细胞生长因子)联合作用时可明显提高细胞增殖活性及成骨标志分子的表达及矿化结节的生成。然而,目前尚没有PEMF单独刺激或联合生长因子在牙槽骨修复方面的体内外研究。因此,本课题探索性的研究不同磁场强度的PEMF单独以及联合骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)对PDLSCs的增殖能力和成骨分化的影响,期望为牙周骨组织再生提供一种无创的辅助性疗法。实验方法:1.在患者知情同意下,收集因正畸治疗需要拔除的健康前磨牙,共20颗。采用组织块贴壁法体外培养原代牙周膜细胞,采用CD146免疫磁珠分选获得CD146阳性PDLSCs,然后通过流式细胞鉴定细胞表面抗原CD146和STRO-1的表达。通过免疫荧光染色鉴定细胞的组织来源,通过茜素红S染色和油红O染色鉴定细胞的成骨和成脂分化能力。2.构建重组过表达BMP9腺病毒(adenoviruses overexpressing BMP9,Ad-BMP9),确定适宜的病毒感染浓度(multiplicities of infection,MOI)检测感染PDLSCs的BMP9基因和蛋白的表达变化。然后通过细胞计数试剂盒(Cell counting kit-8,CCK8)检测连续10天内的细胞增殖活性,并绘制细胞增殖曲线。检测Ad-BMP9对PDLSCs中早期和中期成骨标志分子如碱性磷酸酶(alkaline phosphatase,ALP)、Runt相关转录因子2(Runt-related transcription factor 2,Runx2)、骨桥蛋白(osteopontin,OPN)的基因和蛋白表达影响,以及对成骨后期矿化结节形成的影响。3.设置15 HZ,5组不同场强的PEMF(0.6、1.2、1.8、2.4、3.0 m T)辐照体外培养的PDLSCs或者感染Ad-BMP9的PDLSCs,每天辐照1h,使用CCK8试剂盒连续10天检测细胞的增殖活性,并绘制细胞增殖曲线。4.设置15 HZ,5组不同场强(0.6、1.2、1.8、2.4、3.0 m T,1h/d)的PEMF辐照PDLSCs,检测ALP、Runx2、OPN基因表达,筛选适宜的磁场强度。筛选出的PEMF(1.8 m T和2.4 m T)辐照转染了Ad-BMP9的PDLSCs,检测ALP、Runx2、OPN基因和蛋白表达,采用茜素红S染色检测分化后期的钙盐沉淀情况。实验结果:1.流式细胞检测显示,CD146免疫磁珠分选所得的同时表达CD146阳性和STRO-1阳性的细胞比例约占32.2%。免疫荧光染色显示角蛋白阴性、波形蛋白阳性。茜素红S染色和油红O检测表明分选后的PDLSCs具有成骨和成脂的多向分化潜能。2.腺病毒感染PDLSCs的MOI确定为30-50时具有较高的转染效率70%-80%;RT-PCR和Western blot结果显示感染Ad-BMP9的PDLSCs明显高表达BMP9基因和蛋白;CCK8检测发现Ad-BMP9明显促进对数生长期的PDLSCs增殖活性;ALP定量检测显示Ad-BMP9明显促进了成骨分化早期细胞内ALP蛋白的表达,通过实时定量PCR(quantitative real-time polymerase chain reaction,q RT-PCR)检测表明Ad-BMP9具有诱导PDLSCs表达ALP、Runx2、OPN等成骨标记基因的能力。3.CCK8检测发现PEMF对PDLSCs的细胞数量没有明显影响,而且PEMF没有增强Ad-BMP9对PDLSCs增殖活性的影响。4.q RT-PCR结果显示15Hz,1.8m T和2.4m T场强的PEMF明显促进了PDLSCs的早期和中期成骨基因的表达。q RT-PCR、Western blot、茜素红S染色结果显示PEMF联合BMP9诱导PDLSCs表达ALP、Runx2、OPN的成骨分化能力以及成骨后期矿化结节的形成均高于单独因素作用组。结论:本课题证实了BMP9具有促进PDLSCs的增殖活性和诱导成骨分化的能力;单独作用或在BMP9存在的前提下,PEMF刺激均没有对细胞增殖活性产生显著影响;适宜磁场参数下的PEMF具有诱导PDLSCs成骨分化的能力,而且联合BMP9诱导PDLSCs成骨分化时存在交互作用。因此,适宜参数的PEMF可以增强BMP9诱导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.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782.1

【参考文献】