【作者】 刘欧胜；

【导师】 唐瞻贵； 王松灵；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2012， 博士

【摘要】 牙齿缺失修复及牙周组织再生是口腔科学的工作和研究中心之一,目前临床上对牙周炎致骨缺损的治疗仍然是一个难题,而利用义齿包括固定义齿、活动义齿和种植义齿等修复是一类赝复体修复,缺乏真正意义上的生物修复。近年来,关于牙齿干细胞及组织工程技术的研究进展给牙齿和牙周组织生物再生带来了新的希望。诸多研究提示牙齿相关干细胞是牙齿再生和牙周组织再生的最佳种子细胞来源,具有较广阔的临床应用前景。然而,自体牙齿干细胞来源有限,如果牙齿干细胞能异体应用,就能明显扩大牙齿再生种子细胞来源,极大推进牙齿和牙周组织再生的发展。机体的免疫分为细胞免疫和体液免疫两部分,本课题组的前期研究发现牙齿相关干细胞不会引起细胞免疫应答,但是牙齿相关干细胞对机体体液免疫应答(B淋巴细胞)的影响尚不清楚。为了同种异体牙周膜干细胞能够将来安全地用于临床,本研究拟探讨牙周膜干细胞对同种异体B淋巴细胞功能的影响,并利用小型猪模型,观察牙周膜干细胞异体移植修复牙周炎骨缺损的能力及有无体液免疫排斥。本研究包括以下三个部分：第一部人牙周膜千细胞的培养与鉴定目的：分离培养及鉴定人牙周膜干细胞,为下一步对牙周膜干细胞的其他性能进行研究提供必要的基础和前提。方法：选取首都医科大学附属北京口腔医院口腔颌面外科门诊拔除阻生第三磨牙的患者,无菌条件下剥离牙根外牙周组织,参照以往关于人牙齿相关干细胞的培养方法及条件,分离培养人牙周膜干细胞,并通过倒置显微镜、流式细胞仪等对其生长特性、诱导分化能力,表面标志物进行观察。结果：原代培养的人牙周膜干细胞生长良好,呈梭性。牙周膜干细胞的克隆形成率为(15-28个/105)。流式细胞仪结果显示,分离培养的牙周膜干细胞中的STRO-1,CD146, CD90, SSEA-4, OCT-4,其表达阳性率分别为13.34±1.83%,80.61%士5.41%,98.67%±1.03%,88.67%±4.03%,95.33%士4.53%。经诱导分化后,牙周膜干细胞在体外有诱导分化成骨和成脂肪的能力。结论：人牙周膜干细胞可在体外成功培养,表现出良好的生长状态,并有横向分化的潜能。第二部分人牙周膜干细胞对同种异体B淋巴细胞的影响目的：在体外研究人牙周膜干细胞对同种异体B淋巴细胞功能的影响。方法：正常人外周血的B淋巴细胞与人牙周膜干细胞共培养,通过对B淋巴细胞增殖、凋亡、分化、趋化、分泌功能和共刺激分子表达的检测,从而研究牙周膜干细胞对B淋巴细胞功能的影响,同时用hBMSCs作同型对照,并通过Transwell实验,CBA、抗体中和实验等来研究牙周膜干细胞影响B淋巴细胞增殖和凋亡的可能机制。结果：经流式细胞术检测,牙周膜干细胞抑制由CpG ODN、 rCD40L、Anti-immunoglobulin (IgA+IgG+IgM)、IL-2和IL-4引起的B淋巴细胞增殖,且这种抑制具有浓度依赖性；延迟加入牙周膜干细胞也能抑制由上述刺激所引起的B淋巴细胞增殖。本研究还发现,牙周膜干细胞对B淋巴细胞表面的共刺激分子(CD40、CD80、CD86和HLA DR)的表达和分泌功能不产生影响,但对B淋巴细胞的分化和趋化功能有抑制作用。通过Transwell实验发现：牙周膜干细胞抑制B淋巴细胞增殖的机制可能是通过细胞接触和可溶性分子共同作用。通过流式细胞术检测发现：共培养后,牙周膜干细胞表面PD-1、 PD-L1和PD-L2表达发生改变；进一步利用抗体中和实验证明,牙周膜干细胞可能是通过PD-1和PD-L1来部分抑制B淋巴细胞增殖。凋亡实验表明,牙周膜干细胞并非通过促进B淋巴细胞凋亡来发挥免疫抑制作用,相反,牙周膜干细胞通过分泌IL-6抑制B淋巴细胞的凋亡。结论：hPDLSCs和hBMSCs类似,能够抑制B淋巴细胞的增殖,而且这种抑制作用呈浓度依赖性；hPDLSCs对B细胞的分化、趋化功能也有抑制作用,但对B细胞的抗原提呈功能和分泌功能无明显影响；hPDLSCs通过分泌IL-6抑制B淋巴细胞的凋亡；hPDLSCs通过PD-1及PD-L1部分抑制B淋巴细胞的增殖,与B淋巴细胞的凋亡无关。第三部分小型猪牙周膜干细胞同种异体移植后对机体体液免疫应答的影响目的：在体内研究牙周膜干细胞的异体移植后对机体体液免疫应答的影响。方法：选用雌性五指山小型猪12只,建立牙周炎骨缺损模型,同时分离、培养、扩增五指山小型猪和雄性贵州小型香猪牙周膜干细胞。建模后1个月开始治疗,分为4组：(1)空白对照组：建立实验性牙周炎模型后不做任何处理;(2)HA/TCP组：在建模后做翻瓣刮治+HA/TCP修复,覆盖以明胶海绵；(3)自体牙周膜干细胞+HA/TCP组：建模后做翻瓣刮治+自体牙周膜干细胞+HA/TCP修复,覆盖以明胶海绵；(4)异体牙周膜干细胞+HA/TCP组：建模后做翻瓣刮治+雄性贵州小型香猪牙周膜干细胞+HA/TCP修复,覆盖以明胶海绵。观察指标包括临床观察(牙周探诊深度、附着丧失等)、影像学检查、血液学检查、组织学观察和体液免疫指标等的检测。结果：成功建立牙周炎模型,各组模型之间无统计学差异；治疗后12周,发现自体牙周膜干细胞组和异体牙周膜干细胞组的牙周指数(牙龈退缩、牙槽骨缺失和牙周袋深度)与空白对照组、HA/TCP组相比有统计学差异,但治疗后自体牙周膜干细胞组和异体牙周膜干细胞组牙周指数之间无统计学意义。CT扫描发现,治疗后12周,自体牙周膜干细胞组和异体牙周膜干细胞组都有明显的新生骨质影像,而空白对照组和HA/TCP组却不明显。血液学研究表明,无论是治疗前还是治疗后各个时间点,各治疗组的血常规、血生化、免疫球蛋白和体液免疫学指标(CD20, CD25, B220)都没有统计学差异,同时检测龈沟液和牙周组织的免疫球蛋白也没有统计学差异,说明异体干细胞移植组在治疗后无炎性病变产生,无肝肾功能的损伤,无近期或者远期体液免疫反应产生。组织学观察表明,在自体牙周膜干细胞组和异体牙周膜干细胞组,都有明显的牙槽骨、牙骨质和牙周膜再生,骨缺损基本修复,而在空白对照组和HA/TCP组,仍可见明显的典型牙周炎表现,包括深牙周袋、缺乏新骨和新牙周膜形成。最后通过细胞移植术后1周和2周,对自体移植组和异体移植组的局部牙周组织IL-6、PD-1、PD-L1、PD-L2的mRNA检测,发现IL-6的mRNA在两组间无统计学意义；而PD-1、PD-L1、PD-L2的mRNA,异体组均明显高于自体组。结论：异体牙周膜干细胞能够使牙周骨质再生；异体牙周膜干细胞在小型猪体内不会引起机体体液免疫反应,可能是通过PD-1及其配体来起作用。更多还原

【Abstract】 Restoration of lost teeth and regeneration of periodontal tissue are one of the key issues in dental clinic and research. At present, the treatment of bone defect of periodontitis is still difficult. Artificial teeth including fixed teeth, removal teeth and dental implant are prosthetic appliances, which are not truly biological restoration. Recently, the new prospect on bio-regeneration of teeth and periodontal tissue is brought about by the progress of dental related stem cells and tissue engineering technology. Currently, many studies present that dental stem cells are regarded as the best seed cells resource for the regeneration of teeth and periodontal tissue with a booming clinical application. However, autologous dental stem cells are limited in the patients. If dental stem cells can be used in allogeneic individuals, the source of seed cells can be expanded greatly which will greatly promote the development on restoration of lost teeth and regeneration of periodontal tissue. Adaptive immune divides into two parts including cellar immune and humoral immune. Our previous studies have proved that PDLSCs possessed low immunogenecity and immunosuppressive function through secretion of PGE2. But there is no study on humoral immune response to transplantation of allogeneic periodontal ligament stem cells. The aim of this study is to address the immunological characteristics of PDLSCs which effect on B lymphocyte functions, the mechanisms underlying the PDLSCs-mediated immunological suppression, observe the ability of regenerating bone defect of periodontitis by using allogeneic PDLSCs in the miniature pig model and the present of humoral immune rejection in vivo. Part1Culture and identification of the periodontal ligament stem cells of human in vitroObjective:To culture and identify the periodontal ligament stem cells of human in vitro for providing the necessary foundation and prerequisite for the next step to study other characteristics of the periodontal ligament stem cells.Methods:Normal impacted third molars of healthy individuals were extracted, and periodontal ligament stem cells were separated and cultured referring to the methods of human’s related stem cells. The growth characteristics of these stem cells were observed, the surface markers were detected and the abilities of trans-differentiation were investigated.Results:The cultured human periodontal ligament stem cells shaped in typical fibroblast cells under light microscope. The colony forming units-fibroblasts were from15to28units/105in periodontal ligament cells. These cells were positive for STRO-1, CD146, CD90, SSEA-4and OCT-4,5MSCs markers, identifying them as PDLSCs. Moreover, the cultured human periodontal ligament stem cells had the abilities to be induced to form calcium and adipose materials in vitro.Conclusion:The human periodontal ligament stem cells, which can be cultured successfully in vitro showed good growth and could undergo multilineage differentiation. Part2Modulation of B lymphocyte function in vitro by hPDLSCsObjective:To study hPDLSCs modulate B lymphocyte function in vitro.Methods:B lymphocyte were extracted from healthy human and cultured with hPDLSCs in the absence or presence of the following stimuli:the CpG synthetic oligonucleotide, recombinant CD40L, anti-human immunoglobulin goat antibodies, interleukin2and IL-4, co-cultured with hBMSCs as a positive control. Proliferation, apoptosis, differentiation, chemotatic function, expression of costimulatory molecules and secretion of B lymphocyte were detected by flow cytometry to study hPDLSCs modulate B lymphocyte function. Furthermore, transwell culture experiments, quantification of soluble factors, neutralization experiments, and detection of apoptotic B cells were performed to identify the possible mechanisms of the immunosuppressive and apoptosis function of hPDLSCs on B lymphocyte.Results:HPDLSCs could not initiate B lymphocyte proliferative responses. B lymphocyte proliferation was significantly inhibited by hPDLSCs in a dose dependent manner in the presence of CpG ODN, rCD40L, anti-immunoglobulin, IL-2, and IL-4. Moreover, proliferative of B lymphocyte was inhibited by delaying to co-cultured with hPDLSCs. The experiment also proved that the expression of costimulatory molecules(CD40, CD80, CD86, HLA DR) and secretion of B lymphocyte was not impacted by hPDLSCs, but the differentiation and chemotactic function of B cell was inhibited. By using transwell chambers, we found that the inhibition of B cells proliferation was mediated by cell-to-cell contact and soluble factors. By using flow cytometry, we found that the expression of PD-1, PD-L1and PD-L2on the surface of hPDLSCs was changed. Furthermore, neutralization experiments showed that anti-PD-1antibody, anti-PD-L1antibody and anti-PD-L2antibody partially restored B cells proliferation inhibited by hPDLSCs. Apoptosis showed that inhibition B cells proliferation by hPDLSCs was not mediated by inducing apoptosis of B cells. On the contrary, hPDLSCs inhibited apoptosis of B cells by IL-6secretion. HBMSCs as a positive control, the results on the effect on B cells were the same to hPDLSCs.Conclusion:HPDLSCs, similar to hBMSCs, can inhibit the proliferation of B lymphocyte in a dose dependent manner partially by PD-land PD-L1and have no relationship with B cells apoptosis. They also can inhibit apoptosis, differentiation and chemotatic function of B cells. But they cannot modulate the expression of costimulatory molecules(CD40, CD80, CD86, HLA DR) and secretion of B lymphocyte.Part3Humoral immune response to transplantation of allogeneic periodontal ligament stem cells of minipig in vivoObjective:To study humoral immune response to transplantation of allogeneic periodontal ligament stem cells of minipig in vivo.Methods:We generated periodontitis-lesions in12female Wuzhishan inbred minipigs, totally24defects. Then the miniature pig’s periodontal ligament stem cells were separated and cultured in vitro. The24defects were randomly assigned to four different groups:(1) Control group (6defects in3minipigs):no treatment;(2) HA/TCP group (6defects in3minipigs):flap surgery, transplantation of HA/TCP scaffolds, and covering of the defects with gelatin membranes;(3)HA/TCP scaffolds+autologous PDLSCs group (6defects in3minipigs): transplantation of autologous PDLSCs combined with HA/TCP scaffolds, and covering of the defects with gelatin membranes;(4) HA/TCP scaffolds+allogeneic Guizhou minipig PDLSCs group (6defects in3minipigs):transplantation of allogeneic Guizhou minipig PDLSCs combined with HA/TCP scaffolds, and covering of the defects with gelatin membranes. The regeneration of bone defect was observed by clinical assessments including probing depth (PD), gingival recession (GR) and attachment loss (AL), CT scanning, and histopathological study. Blood routine test, biochemical routine test, immunoglobulin in serum, gingival crevicular fluid and periodontal tissues test and B cells related markers, i.e. percentage of CD20+cells, CD25+cells, B220+cells, and the expression of activated B cells marker.Results:We successfully established periodontitis model and there was comparable between them. At12weeks post-transplantation, either autologous or allogeneic PDLSCs treatment significantly improved periodontal tissue regeneration in comparison with HA/TCP and the control groups, and there was no difference between autologous and allogeneic PDLSCs groups. CT scanning showed that the height of alveolar bone in autologous and allogeneic PDLSCs groups recovered to approximately the normal levels. In contrast, HA/TCP groups and the control groups showed very limited or no bone regeneration. In addition, histopathological photomicrographs showed increased new bone and periodontal tissues, including cementum and periodontal ligament, were regenerated in the periodontal defect area in autologous and allogeneic PDLSCs groups. However, typical periodontitis status, including deep periodontal pocket and shortage of new bone and periodontal ligament fibers, was still found in the HA/TCP and control groups. Furthermore, the data of blood routine tests, biochemical routine test, immunoglobulin test and B cells related immunological markers(CD20,CD25and B220) were almost the same as those of4weeks pre-transplantation, suggesting there were no marked immunological rejections in the animals received allogeneic PDLSCs transplantation. By using ELISA, we found that there was no different on immunoglobulin in serum, gingival crevicular fluid and periodontal tissues between autologous and allogeneic PDLSCs groups. Moreover, by using real time-PCR, we detected the mRNA of IL-6, PD-1, PD-L1and PD-L2in autologous and allogeneic PDLSCs groups at1week and2week after transplantation. We found there was no difference between them on the expression of IL-6at different time. However, there was significantly difference of the mRNA expression of PD-1, PD-L1and PD-L2between autologous and allogeneic PDLSCs groups at1week and2week after transplantation.Conclusion:Allogeneic PDLSCs can effectively repair bone defect of periodontitis in minipig and do not induce humoral immunological rejections in which PD-1and PD-L1maybe play an important role.更多还原