【作者】 王清；

【导师】 徐欣；

【作者基本信息】 山东大学 ， 口腔临床医学（专业学位）， 2017， 博士

【摘要】 组织工程是一门近年来兴起的前沿学科,通过再生出具有正常结构和功能的组织或者器官达到治愈疾病、提高治疗效果或改善生活质量等目标。牙周疾病的治疗是口腔医学的研究和工作中心之一,虽然牙周基础治疗、牙周手术都取得了良好的治疗效果,但是牙周炎导致的骨缺损治疗仍然是一个难题。近年来,关于组织工程和牙周膜干细胞的研究进展给牙周组织生物再生带来了新的希望。除了具有良好的生物学特性和组织再生功能之外,牙周膜干细胞还被发现具有强大的免疫调节能力。以往的研究证实,牙周膜干细胞不会引起同种异体T淋巴细胞的增殖,能剂量依赖性地抑制丝裂原和抗原引起的T淋巴细胞增殖,能够抑制B淋巴细胞的增殖、分化和趋化。异体牙周膜干细胞可以成功地修复牙周炎骨缺损,且没有细胞免疫和体液免疫排斥反应的发生。机体的免疫包括固有免疫和适应性免疫,适应性免疫又可分为细胞免疫(T淋巴细胞主导)和体液免疫(B淋巴细胞主导)。牙周膜干细胞能够抑制细胞免疫与体液免疫,但是牙周膜干细胞对机体固有免疫的影响尚不清楚。中性粒细胞是机体固有免疫的重要组成部分,在维持机体的内环境稳定,抵御病原体入侵方面发挥重要作用。牙周膜干细胞移植到体内后,将不可避免地与中性粒细胞发生接触,而牙周膜干细胞与中性粒细胞的相互作用关系尚不明确。本研究拟探讨牙周膜干细胞和中性粒细胞的相互作用情况及其相关机制,为扩大牙周组织再生种子细胞的来源、促进牙周组织再生生物技术的发展、更安全地应用牙周膜干细胞提供实验依据。本研究包括以下三个部分:第一部分牙周膜干细胞的分离、培养和鉴定目的:通过体外实验研究人牙周膜干细胞的生物学特性。方法:麻醉下无菌拔除人阻生第三磨牙或者正畸减数牙,轻轻剥离其周围的牙周组织,取中段牙周组织,酶消化法分离牙周膜干细胞,通过检测间充质干细胞标志物的表达情况、探讨牙周膜干细胞的骨向、脂肪向、软骨向分化能力来研究牙周膜干细胞的生物学特性。结果:经过流式细胞术检测,牙周膜干细胞不表达造血谱系细胞的标志物CD14,CD34和CD45,表达间充质干细胞的三个标志物STR0-1,CD90和CD146,表达率分别为:26.2%,96.30%,88.6%。在骨向、脂肪向、软骨向诱导培养基的作用下,牙周膜干细胞能够骨向、脂肪向、软骨向分化。PDLSCs在矿化液的诱导下,细胞增殖速度明显减慢,细胞逐渐变成圆形、立方形、多角形,细胞融合逐渐形成单层,培养3周左右出现复层。骨向分化4周后,形成结节样的结构,Von-kossa染色呈现较多的棕褐色钙质阳性反应区域。在脂肪向诱导培养基的作用下,PDLSCs细胞生长速度明显减慢,细胞逐渐失去原有的梭状成纤维细胞样而转变为多角形细胞,细胞肥大、扁平,胞浆内可见小脂滴积聚。脂肪向分化4周后,大多数细胞变成含有较多脂滴的脂肪细胞,油红0染色可见细胞胞浆内橙红色阳性颗粒,脂滴呈串珠状、环状。在软骨向诱导培养基的作用下,PDLSCs的细胞增殖速度显著减弱,细胞呈明显的聚集性生长,形态由梭形逐渐向圆形、多边形、多角形转变,并带有多个突起,10天后,有类似软骨样的结节形成,3周后,番红0染色阳性,表现为细胞外基质红染、细胞内红色颗粒、胞核褐色,Ⅱ型胶原蛋白免疫细胞化学染色呈现较广泛的阳性黄褐色染色,镜下可见胞浆富含棕色颗粒的类软骨细胞。结论:通过酶消化法能较好地分离牙周膜干细胞;牙周膜干细胞不表达造血系细胞标记物CD14、CD34、CD45,表达间充质干细胞的3个标志物STR0-1、CD90、CD146;牙周膜干细胞能够骨向、脂肪向、软骨向分化。第二部分牙周膜干细胞对中性粒细胞生理功能影响的研究目的:研究人牙周膜干细胞对中性粒细胞活力、凋亡、粘附分子表达、吞噬功能、趋化功能等的影响。方法:分离、培养牙周膜干细胞和中性粒细胞。在细胞-细胞直接接触培养/Transwell培养、不同牙周膜干细胞和中性粒细胞数量比的情况下,将牙周膜干细胞和异体中性粒细胞共同培养,然后提取中性粒细胞,检测其细胞活力、凋亡情况、粘附分子表达、吞噬功能、趋化功能;应用酶联免疫吸附测定法检测共培养24小时的牙周膜干细胞+等量中性粒细胞培养上清中IL-6的浓度;将中性粒细胞与不同浓度的IL-6共培养,检测中性粒细胞的凋亡情况;进行中和实验,在牙周膜干细胞+中性粒细胞的培养体系中加入抗IL-6抗体,观察中性粒细胞的凋亡情况。结果:牙周膜干细胞能促使静止期中性粒细胞和IL-8活化中性粒细胞的活力明显增高;随着牙周膜干细胞细胞数量的增多,其促进中性粒细胞活力的能力并没有升高;牙周膜干细胞促进IL-8活化中性粒细胞活力的能力与其对静止期中性粒细胞的作用无显著差异;牙周膜干细胞能够减少静止期中性粒细胞和IL-8活化中性粒细胞的凋亡,且牙周膜干细胞减少两种中性粒细胞的凋亡程度基本相当,无明显差异;无论牙周膜干细胞与中性粒细胞直接接触培养还是Transwell培养,牙周膜干细胞都能够减少中性粒细胞的凋亡,且两种培养方式中牙周膜干细胞减少中性粒细胞凋亡的程度相近,提示PDLSCs的抑制中性粒细胞凋亡作用并不是必须依赖细胞-细胞直接接触的,而可能是通过分泌可溶性因子来实现的;单纯中性粒细胞培养上清液中的IL-6浓度较低,而在牙周膜干细胞与中性粒细胞共同培养24小时后,IL-6浓度明显升高;IL-6能抑制中性粒细胞凋亡;抗IL-6抗体能抵消牙周膜干细胞介导的中性粒细胞凋亡抑制;牙周膜干细胞对中性粒细胞粘附分子的表达情况无影响;牙周膜干细胞不影响中性粒细胞的吞噬功能和趋化功能。结论:牙周膜干细胞能促进静止期中性粒细胞和IL-8活化中性粒细胞的活力、减少其凋亡率;牙周膜干细胞抑制中性粒细胞凋亡的作用并不是依赖细胞-细胞直接接触,而可能是通过分泌可溶性因子IL-6来实现的;牙周膜干细胞不影响中性粒细胞粘附分子的表达、吞噬功能和趋化功能。第三部分中性粒细胞对牙周膜干细胞生物学特性影响的研究目的:研究中性粒细胞对牙周膜干细胞生物学特性的影响。方法:麻醉下无菌拔除人阻生第三磨牙或者正畸减数牙,轻轻剥离其周围的牙周组织,取中段牙周组织,酶消化法分离并培养牙周膜干细胞。将生长密度达80%左右的牙周膜干细胞消化,以5.0×104细胞铺板,在37℃、5%CO2条件下培养2小时,使细胞贴壁。然后与等量异体中性粒细胞共培养24小时。24小时后,提取牙周膜干细胞,然后分别检测以下指标:克隆形成率、细胞增殖率、骨向和脂肪向诱导分化、间充质干细胞标志物的表达情况、超微结构观察、染色体核型分析等。结果:无论是单独培养的PDLSCs,还是与等量异体中性粒细胞共培养24小时后的PDLSCs,细胞呈梭状的成纤维细胞样,形态规则、均一,两者的形态未见明显区别。单独培养牙周膜干细胞的克隆形成率为28/5.0×103细胞,明显高于与等量异体中性粒细胞共培养24小时牙周膜干细胞的克隆形成率(19/5.0×103细胞),两者之间有显著性差异。单独培养牙周膜干细胞的Brdu阳性率为71%±11.20%,明显高于与等量异体中性粒细胞共培养24小时牙周膜干细胞的Brdu阳性率(47%±9.1%),两者之间有显著性差异。两组细胞都可以骨向和脂肪向分化:骨向分化后,RT-PCR发现两组细胞都有骨钙素的表达;脂肪向分化后,RT-PCR发现两组细胞都有PPAR-Y 2、LPL的表达。两组细胞都表达间充质干细胞的标志物STRO-1,CD90和CD146,表达率分别为:29.6%,98.8%,89.5%,与单独培养PDLSCs的表达率无显著性差异。两组细胞的电镜观察结果相近,在扫描电镜下,无论单独培养PDLSCs还是与等量异体中性粒细胞共培养24小时的PDLSCs都有较多长短不一的分支,细胞周围有分泌的细胞外基质。在透射电镜下两种细胞生长旺盛,细胞核形态欠规则,核仁较大,核内染色质丰富。胞浆细胞器丰富,可见丰富的蛋白颗粒、高尔基囊泡、游离核糖小体等。两组细胞的染色体核型检查相同。结论:中性粒细胞能降低牙周膜干细胞的克隆形成率和细胞增殖率,但对其多向分化能力、标志物表达水平、超微结构和染色体核型未见明显影响。更多还原

【Abstract】 Tissue engineering is an emerging discipline in recent years,which curing the diseases,promoting the treatment effects,and improving the quality of life by regenerating new tissues or organs with normal structures and functions.The treatment of periodontal tissues diseases is one of the key issues in dental clinic and research.Although the periodontal initial therapy and operations obtained good effects,the treatment of periodontitis related bone loss is still a difficult problem.Recently,the new prospect on bio-regeneration of periodontal tissue is brought about by the progress of tissue engineering technology and periodontal ligament stem cells.Except their well studied biological properties and regenerative functions,periodontal ligament stem cells have been found to possess profound immunomodulatory capabilities.PDLSCs could not initiate allogeneic T cells proliferative responses,inhibited the mitogen-induced T cells proliferation in a dose dependent manner,and successfully restore the periodontitis related bone loss without immunological rejections of cellular immunity.In addition,periodontal ligament stem cells are capable of inhibiting the proliferation,differentiation,and chemotaxis of B lymphocytes.Immunity consists of innate immunity and adaptive immunity,and adaptive immunity is composed of cellular immunity(T lymphocytes)and humoral immunity(B lymphocytes).Although inhibiting the cellular immunity and humoral immunity,the effects of periodontal ligament stem cells on innate immunity are largely unknown.Neutrophils are important parts of innate immunity,and it is inevitable to contact neutrophils for periodontal ligament stem cells after transplantation in vivo.However,the mutual effects between periodontal ligament stem cells and neutrophils are not clear.The aim of this study is to address the interplay of periodontal ligament stem cells and neutrophils and the underlying mechanisms,thus providing experimental data for enlarging the seed cell of periodontal tissue regeneration,improving the development of periodontal tissue biological regeneration,and safe application of periodontal ligament stem cells.Part 1 Isolation,culture and identification of periodontal ligament stem cellsAim:To acquire the in vitro biological characteristics of human periodontal ligament stem cells.Methods:Normal premolars and impacted third molars of healthy individuals were extracted,and periodontal ligament stem cells were separated by enzyme digestion method and cultured as previously reported.The expression profiles of mesenchymal stem cells related markers,and the exploration of osteogenic,adipogenic,chondrogenic differentiation of periodontal ligament stem cells were performed to identify the biological characteristics of human periodontal ligament stem cells.Results:The flow cytometry assays showed that human PDLSCs expressed STRO-1 CD90 and CD 146,three mesenchymal stem cells related markers,and the positive rates are 26.2%,96.3%,and 88.6%,respectively.However,PDLSCs were negative for CD 14,CD34 and CD45,three hematopoietic lineage molecules.PDLSCs could differentiate along bone,adipose and cartilage pathways under the corresponding inductive medium.Von Kossa staining of PDLSCs in osteogenic medium for 4 weeks showed black nodular structures of calcium deposition.PDLSCs formed Oil Red O-positive lipid clusters following 4 weeks of adipogenic induction,and bead-like and ring-like lipid droplets were formed.Three weeks post chondrogenic induction,safranin O experiments showed positive staining in extracellular matrix with brown nucleus and red paticles in the cytoplasm.Immunocytochemical staining of type Ⅱ collagen displayed extensive brown dyeing.Conclusion:PDLSCs can be obtained by enzyme digestion method.PDLSCs expressed STRO-1,CD90 and CD 146,three mesenchymal stem cells related markers.However,PDLSCs were negative for CD 14,CD34 and CD45.PDLSCs could differentiate along bone,adipose and cartilage pathways.Part 2 The effect of periodontal ligament stem cells on the physiological functions of neutrophilsAim:To investigate the effect of periodontal ligament stem cells on the viability,apoptosis,expression of adhension molecules,phagocytosis function,and chemotaxis function of neutrophils.Methods:Normal premolars and impacted third molars of healthy individuals were extracted,and periodontal ligament stem cells were separated by enzyme digestion method and cultured as previously reported.Neutrophils were isolated and cultured as document statement.Under cell-cell contact culture or Transwell culture conditions,different ratios of periodontal ligament stem cells and neutrophils were co-cultured together.Then neutrophils were got and examined their viability,apoptosis,expression of adhension molecules,phagocytosis functions,and chemotaxis functions.Enzyme-linked immunosorbent assays were used to detect the concentration of IL-6 in the supernatant of co-culture of periodontal ligament stem cells and equal amount neutrophils post 24 hours.The apoptosis of neutrophils was investigated after culture with different concentrations of IL-6.Anti-IL-6 antibody was added into the co-culture of periodontal ligament stem cells and neutrophils,then the apoptosis of neutrophils was examined.Results:Periodontal ligament stem cells could promote the viability of resting neutrophils and IL-8-activated neutrophils.However,periodontal ligament stem cells-mediated viability promotion of neutrophils did not rise with the amount increase of periodontal ligament stem cells.There is no significant difference between periodontal ligament stem cells-mediated viability promotion of resting neutrophils and IL-8-activated neutrophils.Periodontal ligament stem cells could decrease the apoptosis of resting neutrophils and IL-8-activated neutrophils,and there is no significant difference between periodontal ligament stem cells-mediated apoptosis decreasement of resting neutrophils and IL-8-activated neutrophils.Periodontal ligament stem cells could decrease the apoptosis of neutrophils under cell-cell contact culture or Transwell culture conditions.The concentration of IL-6 in supernatant of periodontal ligament stem cells alone is low,whereas is elevated significantly post 24 hours co-culture of periodontal ligament stem cells and neutrophils.In addition,IL-6 could inhibit the apoptosis of neutrophils.Anti-IL-6 antibody is able to counteract the periodontal ligament stem cells-mediated apoptosis decreasement of neutrophils.Periodontal ligament stem cells did not influence the expression of adhension molecules,phagocytosis function,and chemotaxis function of neutrophils.Conclusion:Periodontal ligament stem cells could promote the viability and decrease the apoptosis of resting neutrophils and IL-8-activated neutrophils,which was achieved by the secretion of IL-6.The expression profiles of adhension molecules,phagocytosis function,and chemotaxis function of neutrophils were not affected by periodontal ligament stem cells.Part 3 Effect of neutrophils on biological characteristics of periodontal ligament stem cellsAim:To study the effect of neutrophils on biological characteristics of periodontal ligament stem cells.Methods:Normal premolars and impacted third molars of healthy individuals were extracted,and periodontal ligament stem cells were separated by enzyme digestion method and cultured as previously reported.Periodontal ligament stem cells of 80%confluence were digested and plated with the density of 5.0 ×104 at 37 ℃ and 5%CO2 for 2 hours to make the cells adhenrence.Periodontal ligament stem cells were co-cultured with equal amount of neutrophils for 24 hours.Then periodontal ligament stem cells were obtained and examined the following items:colony-forming efficiency,cell proliferation rate,multi-potential differentiation,expression of mesenchymal stem cell markers,ultra-structure,and karyotype analysis.Results:The colony-forming efficiency of periodontal ligament stem cells alone and co-cultured periodontal ligament stem cells were 28/5.0 ×103 and 19/5.0×103 respectively,and the Brdu positive rate were 71%±11.2%and 47%±9.1%respectively,there was significant difference between the two groups.Both periodontal ligament stem cells alone and co-cultured periodontal ligament stem cells could differentiate along bone and adipose pathways,and were positive for STRO-1,CD 190,and CD 146,and the expression rates were 29.6%,98.8%,and 89.5%,respectively.Moreover,two groups had the same manifestation of ultra-structure and G-band karyotype.Conclusion:Neutrophils could decrease the colony-forming efficiency and cell proliferation rates of periodontal ligament stem cells markedly,but did not affect the multi-potential differentiation,expression of mesenchymal stem cell markers,ultra-structure,and karyotype analysis of periodontal ligament stem cells.更多还原