BMP-7诱导牙髓干细胞向牙源性方向分化的研究

发布时间：2018-07-22 13:57

【摘要】：研究背景：龋病是口腔的一种常见病和多发病。当龋病发展到牙本质层时,致龋菌及其代谢过程中所产生的一些毒性物质可沿着牙本质小管渗入到牙髓组织中。牙髓组织遭遇到外界刺激时,组织中原有的干细胞向受损部位迁移、到达受损部位后,分化为成牙本质细胞并形成修复性牙本质。牙髓组织的这种防御机制可以防止牙髓进一步受损,但牙齿本身的这种自我修复的能力很有限。目前临床上治疗龋病的方法是先去除龋坏的牙体组织,然后使用人工材料对缺损的组织来加以修复。虽然近年来临床上使用的充填和修复材料发展较迅速,材料无论是在生物学性能还是强度上都有了明显的提高,但与天然的牙体组织相比仍然具有较大的差距。而且随着充填治疗时间的延长充填材料会对牙髓组织的活力产生一定的影响。长期的临床观察发现：充填治疗后,在充填体和牙体组织之间会有微渗漏或继发龋的出现,这些最终都会导致充填治疗的失败。干细胞在组织的缺损修复和再生的研究中有着重要作用。牙髓干细胞(dental pulp stem cells,DPSCs)属于成体性干细胞,是目前发现的五种牙源性干细胞中的一种。1990年,Fitzgerald等发现当牙髓组织受到外界入侵时,牙髓组织中的细胞可迁移到受损处并迅速形成修复性牙本质,他当时就提出了牙髓组织中有干细胞的存在。DPSCs作为一种成体性干细胞后来的研究发现其具有成骨、成脂、成牙本质等多向分化的潜能,体外增殖速度快,目前的研究较为广泛。DPSCs在体外较易获得,培养的方法相对较为成熟。且与胚胎干细胞不同的是DPSCs的培养不涉及到任何伦理学方面的问题。目前的研究显示在体外DPSCs可以冷冻保存较长的时间且复苏后细胞的生物学性状无明显的改变。DPSCs的这些生物学特性为牙髓组织损伤修复的研究以及组织工程牙齿的构建提供了重大的依据。在组织工程研究领域,生物性诱导剂必不可少。BMP-7 (bone morphogenic protein 7)作为一种较安全的生物因子,属于转化生长因子-β (TGF-β)家族的一员。首次在19世纪70年代被发现,因其具有成骨作用,也被称为成骨蛋白-1 (osteogenic protein-1,OP-1)。BMP-7作为安全的诱导剂在骨科部分已进入临床研究应用的阶段,发现其在半月板损伤的治疗中成效较为显著。也有研究将BMP-7的基因导入到骨髓间充质干细胞中,可提高细胞的成骨效果。在临床研究中Ashraf Ayoub等将BMP-7负载到Ⅰ型胶原支架上应用到多种类型的牙槽嵴裂缺损的治疗中,结果表明在骨裂的重建修复中该设计无论从影像学的角度还是从临床的角度治疗效果都非常良好。Yang X等将质粒编码的BMP-7基因转染到DPSCs中,然后发现经转染后的细胞可以向成牙本质细胞表型分化并可以在体外形成硬组织。目前国内外有关BMP-7的研究大多数都为体内动物实验,虽然也有研究表明高浓度的BMP-7会抑制细胞的增殖,但BMP-7对DPSCs的增殖和分化是否有影响,以及作用于DPSCs时合适的蛋白作用浓度并不是非常明确。这些问题都有待于进一步的实验研究,为BMP-7应用到牙再生提供理论依据。组织工程的发展基于生物材料的迅猛发展,支架材料是组织再生和重建的基础。首先支架需要在缺损的部位搭建出一个有利于细胞迁移、黏附、生长和分化的空间结构。同时支架的结构也要有利于新生的组织和血管的长入。这些特性要求应用到组织工程的支架材料需要具备良好的三维联通立体构象、机械强度高、生物安全性能优良。在前期我们课题组的研究中采用模板复制法制备的三维联通纳米氧化锆生物陶瓷支架机械强度高,附着骨髓间充质干细胞植入到犬下颌骨缺损部位成骨效果显著,本研究继续使用该支架进行进一步实验。研究希望在体外筛选出作用于DPSCs时合适的BMP-7蛋白浓度,将细胞接种到改善后的纳米氧化锆支架上,评价BMP-7作为诱导剂应用到DPSCs的定向诱导中的作用,从而探讨其对牙体缺损修复及应用到牙组织工程的可行性。目的：探索BMP-7作为一种安全的生物因子在体外是否会影响DPSCs的增殖和分化,并选择出BMP-7作用于DPSCs时合适的浓度。将选择出的合适浓度的BMP-7诱导牙髓细胞后接种到三维联通纳米氧化锆生物陶瓷支架上植入到裸鼠皮下,观察细胞的生长、新生的组织情况以及血管再生的能力,评价BMP-7作为诱导剂应用于牙组织工程的可行性。方法：(1) hDPSCs的体外分离、培养及成骨成脂鉴定选择年轻健康成人的第三磨牙,通过组织块结合酶消化的方法获取hDPSCs,观察细胞的贴壁生长和克隆形成情况。同时通过成骨和成脂诱导实验检测细胞在体外的多向分化能力,鉴定获取的原代细胞中含有干细胞。(2)不同浓度的BMP-7对hDPSCs形态的影响配制含不同浓度BMP-7 (Ong/ml,25ng/ml,50ng/ml, 100ng/ml)的培养基,将细胞在含不同蛋白浓度的培养基中培养。每3-4天换液,连续培养7天,倒置显微镜下观察各组细胞的形态是否有差异。(3)不同浓度的BMP-7对hDPSCs增殖的影响收集处于对数生长期第三代的hDPSCs细胞,消化离心计数后,调整细胞悬液浓度。各组细胞分别加入含不同浓度(Ong/ml,25ng/ml,50ng/ml, 100ng/ml)的BMP-7培养液。连续培养1,3,7d后通过CCK-8方法检测细胞的增殖在各组之间是否有差异。(4)不同浓度的BMP-7对hDPSCs分化的影响通过qPCR、细胞免疫化学染色和Western blot的方法检测牙源性相关基因和蛋白在不同处理组之间的表达差异。(5) 降温速率对纳米氧化锆支架形态影响的研究在原始的支架高温烧结的成形过程中,通过降低支架的降温速率,观察温度对支架的形态完整性是否有影响。(6)BMP-7诱导处理后的细胞接种到纳米氧化锆支架上裸鼠皮下研究将选择出的合适浓度的BMP-7蛋白诱导细胞后接种到生物陶瓷纳米氧化锆支架上植入裸鼠皮下,观察细胞的生长、新生的组织情况以及血管的再生能力。结果：(1) hDPSCs的体外分离、培养及成骨成脂鉴定通过组织块结合酶消化的方法获得的健康年轻人的牙髓细胞在体外贴壁生长迅速且克隆形成率高。细胞表达间充质来源标记物,非上皮性来源。在体外经成骨和成脂诱导后,可以向成骨和成脂方向分化,具备多向分化的能力,说明获得的细胞中有干细胞的存在。(2)不同浓度的BMP-7对hDPSCs形态的影响各组细胞在不同浓度的BMP-7培养基中连续培养7天后,在倒置显微镜下各组细胞在形态上都呈类成纤维细胞样,多角形态,组间未见到明显的差异。(3)不同浓度的BMP-7对hDPSCs增殖的影响细胞增殖在各组随着体外培养时间的延长都呈现上升的趋势。对照组细胞生长情况与不同浓度的BMP-7诱导组之间出现明显的差异,对照组细胞增殖在培养期间都高于诱导组。实验结果表明一定浓度的BMP-7对DPSCs的增殖随着处理时间的延长会抑制细胞的体外增殖能力。(4)不同浓度的BMP-7对hDPSCs分化的影响细胞免疫化学染色显示对照组的DPSCs中DSPP不表达或个别细胞表达,DMP-1低表达,ALP有少量细胞表达。体外BMP-7诱导DPSCs 7d后,DPSCs中大多数细胞DSPP、DMP-1表达呈阳性,ALP染色出现强阳性。qPCR显示细胞经不同浓度的BMP-7在体外处理14d后细胞中成牙本质细胞相关基因(Col I, DSPP, OCN and Runx2)的表达情况为DSPP、OCN和Runx2基因在50ng/ml蛋白处理组的表达量要明显高于对照组,且差异具有统计学意义。DSPP的表达在50ng/ml BMP-7组也高于25ng/ml BMP-7组和100ng/ml BMP-7组。Col I基因在100ng/ml处理组的表达量最高,高于对照组和其它BMP-7诱导组。蛋白结果显示BMP-7处理14天后细胞中DSPP表达明显增加,对照组细胞中DSPP也出现了低表达。定量结果显示在50ng/ml和100ng/ml BMP-7处理组中DSPP表达量与对照组相比具有统计学差异。(5) 降温速率对纳米氧化锆支架形态影响的研究支架在首次高温烧结时,通过减缓降温速率后,肉眼观支架高温烧结后首次成形效果明显改善,支架塌坯的现象没有出现,完整性破坏较少。电镜下观察延长降温时间后,支架呈现出三维网状联通结构,内部孔隙大小分布较均匀,但支架表面的微裂纹及断裂现象仍然存在。高倍镜下氧化锆颗粒形态与原烧结程序下相比未见到明显的差异。(6)BMP-7诱导后的细胞接种到纳米氧化锆支架上裸鼠皮下研究细胞接种到支架上后在支架表面附着伸展。随着体外培养时间的延长,细胞增殖迅速,铺满支架的内部和表面。术后2个月取材所有标本被血管及纤维结缔组织所包绕,支架周围未见明显的炎性组织。肉眼观四组之间未见明显的差异。髓染色显示无细胞组中有纤维结缔样组织的长入,细胞组中都在靠近支架边缘有类似成牙本质细胞样细胞及小血管的出现,但均未观察到成熟的牙本质样结构的形成。结论：(1)在体外BMP-7对DPSCs的增殖有时间和剂量的联合效应。50ng/ml和100ng/ml的BMP-7在体外具有诱导DPSCs向牙源性方向分化的潜能且不会明显的抑制细胞的增殖速率。(2)BMP-7作用于DPSCs时需要掌控好蛋白的使用剂量和作用时间,以便有利于细胞的生长和分化。(3)三维联通纳米氧化锆多孔支架可以为DPSCs提高良好的细胞外微环境,显示出了良好的生物学性能。(4) DPSCs附着到生物陶瓷纳米氧化锆支架上植入裸鼠皮下后,有望再生牙本质样组织,有待进一步的研究。
[Abstract]:Background: caries are a common and frequently occurring disease in the oral cavity. When caries develop to the dentine layer, the cariogenic bacteria and some toxic substances produced in the process of metabolism can infiltrate into the dental pulp tissue along the dentinal tubules. When the dental pulp tissues encounter external stimuli, the original stem cells in the tissue migrate to the damaged part and reach the recipient. When the site is damaged, it divides into odontoblast cells and forms a restorative dentin. This defense mechanism of the dental pulp tissue prevents the dental pulp from further damage, but the tooth itself has limited ability to repair it. At present, the clinical treatment of caries is to remove caries first, and then use artificial materials for the defect group. Although the materials used in the bed have developed rapidly in recent years, materials have been significantly improved in both biological performance and strength, but there is still a big gap compared with natural dental tissues. Stress has a certain effect. Long term clinical observation shows that after filling, there will be microleakage or secondary caries between the filling body and the tooth tissue, which will eventually lead to the failure of the filling treatment. The stem cells have an important role in the study of tissue defect repair and regeneration. Dental pulp stem cells, DPSCs) belongs to adult stem cells. It is a.1990 year of five odontogenic stem cells found. Fitzgerald, etc., found that when the dental pulp tissues were invaded by the outside, the cells in the pulp tissue could migrate to the damaged area and quickly form a restorative dentin. At that time, he proposed the presence of.DPSCs as a stem cell in the dental pulp tissue. A later study of adult stem cells found that it has the potential of multiple differentiation of osteogenesis, fat formation, dentin formation, and so on. The proliferation rate is fast in vitro. The current research on.DPSCs is relatively easy to obtain in vitro, and the methods of culture are relatively mature. And different from embryonic stem cells, DPSCs culture does not involve any ethical aspects. Problems. Current studies show that in vitro DPSCs can be frozen for a long time and after resuscitation, the biological properties of cells have no obvious changes in.DPSCs. These biological characteristics provide a significant basis for the repair of dental pulp tissue damage and the construction of tissue engineering teeth. In the field of tissue engineering, biological inducers Essential.BMP-7 (bone morphogenic protein 7), as a safer biological factor, is a member of the transforming growth factor - beta (TGF- beta) family. It was first discovered in 1870s for its osteogenic effect and also known as osteogenic protein -1 (osteogenic protein-1, OP-1).BMP-7 as a safe inducer in the Department of orthopedics. In the stage of clinical application, it has been found that it is more effective in the treatment of meniscus injury. There is also a study to introduce BMP-7 gene into bone marrow mesenchymal stem cells, which can improve the osteogenic effect of cells. In clinical study, Ashraf Ayoub, etc., applied BMP-7 to type I collagen scaffold to apply to a variety of types of alveolar cleft. In the treatment of damage, the results showed that the design, both from the angle of image and from the clinical point of view, was very good in the reconstruction and repair of the bone fissure. The plasmid encoded BMP-7 gene was transfected into DPSCs, such as.Yang X, and then the transfected cells could differentiate into the odontoid cells and form hard in vitro. Most of the research on BMP-7 at home and abroad is in vivo animal experiments, although there are also studies showing that high concentration of BMP-7 inhibits the proliferation of cells, but the effect of BMP-7 on the proliferation and differentiation of DPSCs, as well as the appropriate concentration of protein acting on DPSCs, is not very clear. These problems need to be further studied. The experimental study provides a theoretical basis for the application of BMP-7 to tooth regeneration. The development of tissue engineering is based on the rapid development of biomaterials, and the scaffolding material is the basis for tissue regeneration and reconstruction. These characteristics require that the scaffolds applied to the tissue engineering need to have good three-dimensional conformation, high mechanical strength and good biological safety performance. In the previous research group, the three-dimensional zirconia bioceramic scaffold prepared by template replication method was used in the research group. High mechanical strength, adherent bone marrow mesenchymal stem cells implanted into the mandible defect site of the dog bone formation effect is significant. This study continues to use the scaffold for further experiments. We hope to screen out the appropriate BMP-7 protein concentration in vitro, and inoculate the cells to the improved nano zirconia scaffold, and evaluate BMP-7 as a lure. The application of the guide to the directional induction of DPSCs to explore the feasibility of dental defect repair and application to dental tissue engineering. Objective: To explore whether BMP-7 as a safe biological factor will affect the proliferation and differentiation of DPSCs in vitro, and select the appropriate concentration of BMP-7 to act on DPSCs. The BMP-7 induced dental pulp cells were implanted into the subcutaneous tissue of nude mice on the three dimensional nano zirconia bioceramic scaffold, observed the growth of the cells, the new tissue and the ability of vascular regeneration, and evaluated the feasibility of BMP-7 as an inducer for dental tissue engineering. (1) the separation, culture and osteogenesis in vitro of (1) in vitro The third molar of young healthy adults was identified by lipid identification. HDPSCs was obtained by tissue block binding enzyme digestion. The cell adhesion and formation of cells were observed. At the same time, the differentiation ability of cells in vitro was detected by osteogenesis and lipid induction. (2) different concentrations of BMP were found. The influence of -7 on the morphology of hDPSCs was prepared with different concentrations of BMP-7 (Ong/ml, 25ng/ml, 50ng/ml, 100ng/ml). The cells were cultured in the medium containing different protein concentrations. The cells were cultured every 3-4 days and cultured for 7 days. Under the inverted microscope, the morphology of the cells in each group was observed. (3) the effect of BMP-7 on the proliferation of hDPSCs in different concentrations was collected. HDPSCs cells in the third generation of logarithmic growth period were collected. After digestion centrifuge counting, the concentration of cell suspension was adjusted. Each cell was added to BMP-7 culture solution containing different concentrations (Ong/ml, 25ng/ml, 50ng/ml, 100ng/ml). After continuous culture 1,3,7d, the proliferation of cells was detected by CCK-8 method. (4) BMP- at different concentrations. 7 Effect on hDPSCs differentiation through qPCR, cellular immunochemistry staining and Western blot method to detect the difference in expression of odontogenic genes and proteins between different treatment groups. (5) the study on the effect of cooling rate on the morphology of nano zirconia scaffold in the process of high temperature sintering of the original scaffold by reducing the cooling of the scaffold Rate, whether the temperature has an effect on the morphological integrity of the scaffold. (6) the cells after BMP-7 induction were inoculated into nude mice subcutaneously on the nano zirconia scaffold, and the suitable concentration of BMP-7 protein was selected to induce the cells to be implanted subcutaneously on the bioceramic nano zirconia scaffold to observe the growth of the cells and the new group. Results: (1) in vitro isolation, culture and identification of hDPSCs in vitro, the dental pulp cells of healthy young people obtained by tissue block binding enzyme digestion are fast growing and cloned in vitro. The cell expression of mesenchymal origin markers, non epithelial sources. After bone and lipid induction, it can differentiate into osteogenesis and fat formation, with the ability to multiform differentiation, indicating the existence of stem cells in the obtained cells. (2) the effects of different concentrations of BMP-7 on the morphology of hDPSCs in each group of cells in different concentrations of BMP-7 medium after 7 days of continuous culture, the cells in each group are in shape under the inverted microscope. (3) the effect of BMP-7 on the proliferation of hDPSCs in each group increased with the prolongation of the culture time in each group. The cell growth of the control group was significantly different from the BMP-7 inducement group with different concentrations, and the cells in the control group increased. The results showed that the proliferation of DPSCs in a certain concentration of BMP-7 inhibited the proliferation of cells in vitro. (4) the effect of BMP-7 on the differentiation of hDPSCs in different concentrations showed DSPP non expression or individual cell expression in DPSCs of the control group and DMP-1 low table. ALP has a small amount of cell expression. After BMP-7 induction of DPSCs 7d in vitro, most of the cells in DPSCs are DSPP, DMP-1 expression is positive, ALP staining appears strong positive.QPCR display cells in different concentrations of BMP-7 in vitro. The expression of the 50ng/ml protein treatment group was significantly higher than the control group, and the difference was statistically significant.DSPP expression in the 50ng/ml BMP-7 group was also higher than the 25ng/ml BMP-7 group and 100ng/ml BMP-7 group.Col I gene expression in 100ng/ml treatment group, higher than the control group and other BMP-7 induction groups. Protein results showed 14 days after treatment. The expression of DSPP in the cells was significantly increased, and the expression of DSPP in the control group also showed low expression. The quantitative results showed that the DSPP expression in the 50ng/ml and 100ng/ml BMP-7 treatment groups was significantly different from that of the control group. (5) the effect of cooling rate on the morphology of nano zirconia scaffold in the first high temperature sintering was reduced by slowing down the cooling rate. After the high temperature sintering of the naked eye view, the first forming effect was obviously improved, the phenomenon of the stents collapsed and the integrity failure was less. Under the electron microscope, the stent presented a three-dimensional network connecting structure and the internal pore size distribution was more uniform, but the micro crack and fracture still existed on the surface of the support frame. The morphology of the zirconia particles was not significantly different from that under the original sintering procedure. (6) the cells after BMP-7 induction were inoculated to the nude mice on the nano zirconia scaffold and were inoculated on the scaffold to extend on the scaffold surface. With the extension of the time of culture in vitro, the cells proliferated rapidly and covered the internal and surface of the scaffold. 2 All specimens were wrapped around the vascular and fibrous connective tissue, and no obvious inflammatory tissue was found around the scaffold. No significant difference was found between the four groups. Pulp staining showed that there were fibrous connective tissue in the cell group, and the cell groups were similar to odontoblast like cells and small vessels near the edge of the scaffold. No mature dentin like structure was observed. Conclusion: (1) the combined effect of time and dose of BMP-7 on the proliferation of DPSCs in vitro,.50ng/ml and 100ng/ml BMP-7 have the potential to induce DPSCs to differentiate into odontogenic direction in vitro and do not significantly inhibit the proliferation rate of the cells. (2) BMP-7 is required to act on DPSCs. The dosage and time of the protein should be controlled in order to help the growth and differentiation of the cells. (3) the three-dimensional porous nano zirconia porous scaffold can improve the good extracellular microenvironment for DPSCs and show good biological properties. (4) it is hopeful that DPSCs attach to the subcutaneous of nude mice on the ceramic nano zirconia scaffold. The dentin like tissue of regenerated teeth needs further study.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781

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