【作者】 温秀杰；

【导师】 刘鲁川；

【作者基本信息】 第三军医大学 ， 生物医学工程， 2011， 博士

【摘要】 研究背景牙骨质是牙根表面一薄层矿化组织,在牙齿发育过程中牙骨质基质由成牙骨质细胞分泌而形成的。牙骨质是牙齿赖以稳定在牙槽窝内的重要组织结构之一,在维持牙齿的结构稳定性和生理功能中发挥重要作用。然而,牙骨质的再生往往比较困难的,主要是因为牙骨质中的成牙骨细胞量很少,体外分离培养更加不易。尽管学者们对该领域进行了大量研究,对于成牙骨质细胞的获得,仍局限于牙源性细胞,如牙囊细胞、牙胚来源的上皮根鞘细胞、牙周膜干细胞和颌突间充质细胞等,而这些种子细胞都存在取材受限的缺点。因此,成牙骨质细胞的来源问题在很大程度上制约了组织工程化牙骨质和牙周组织再生的发展。脂肪干细胞(Adipose tissue-derived stem cells: ADSCs)由于储备丰富、取材容易、自我增殖和多向分化潜能强等优点,在再生医学中引起许多学者的关注。研究显示,在不同的体外培养环境中,ADSCs可以向成骨细胞、成软骨细胞、心肌细胞、甚至上皮细胞和神经细胞分化,特别是向成骨细胞分化,体外诱导技术比较成熟。成牙骨质细胞与成骨细胞在形态、表型、矿化能力等方面有着诸多相似之处,包括ALP活性、Runx2、I型胶原(Col-I)、骨涎蛋白(BSP)、骨桥蛋白(OPN)、骨钙素(OCN)等表达。因此,只要能够建立合适的体外诱导微环境,ADSCs向成牙骨质细胞诱导分化为在理论上是可行的。要建立这样一个局部微环境,也叫干细胞壁龛(stem cell niche),就不得不再回顾一下牙骨质的发生、发育过程。尽管成牙骨质细胞的来源问题还一直存在着学术争论,但大家都一致认为:在上皮根鞘发生断裂后,牙囊细胞与根部牙本质发生接触是成牙骨细胞出现的前提。由此可见,牙本质或牙本质基质成份和牙囊细胞对成牙骨细胞分化必不可少。牙本质非胶原蛋白(dentin non-collagenous proteins: dNCPs)是牙本质基质的主要成分,其中含有大量的糖蛋白、涎蛋白、磷蛋白、蛋白多糖和多种生长因子,被认为在成牙骨质细胞增殖和分化以及生成牙骨质过程中发挥重要的调控作用。且已有研究证实,dNCPs能够诱导牙囊细胞向成牙骨质细胞分化。所以,我们推测牙囊细胞条件培养基和dNCPs可能含有大量的成牙骨质细胞分化所需要的活性因子。因此,本实验通过预实验筛选,联合dNCPs和DFCCM建立体外诱导微环境,诱导ADSCs向成牙骨质细胞分化,为牙周组织工程特别是牙骨质组织工程提供新的种子细胞来源与实验方法,并为提示牙骨质发生、发育机制提供实验参考依据。材料与方法1.SD大鼠ADSCs的体外培养与鉴定取出生后6~7天SD仔鼠,脱颈处死,切取腹股沟处脂肪组织,剪碎组织块,消化,离心,去上清,重悬细胞,接种到含10% FBS的DMEM/F12(100μg/ml链霉素和100 units/ml青霉素)培养基、37℃含5%CO2、饱和湿度的恒温培养箱培养。分别从细胞形态、免疫细胞化学、流式以及多向分化潜能等方面对ADSCs进行鉴定。2.SD大鼠ADSCs向成牙骨质细胞分化体外诱导条件的初步筛选SD大鼠牙囊细胞和根尖牙乳头细胞培养、鉴定与条件培养液的制备:取出生后5天的SD仔鼠,体视显微镜下分离第一磨牙牙胚,分别剥离牙囊组织和切取根尖牙乳头组织。细胞培养与鉴定同上。取第3代牙囊细胞和根尖牙乳头细胞,收集并制备条件培养液。SD大鼠ADSCs向成牙骨质细胞诱导分化体外诱导条件的初步筛选:分别用牙囊细胞条件培养基(DFCCM)、根尖牙乳头条件培养基(APCCM)、10μg/ml dNCPs的DMEM/F12(含5%FBS)、10μg/ml dNCPs +DFCCM、10μg/ml dNCPs +APCCM、含5%FBS的DMEM/F12(对照组)培养ADSCs,共6天,每2天换1次液。诱导后对各组进行RT-PCR和Western blotting检测。3.SD大鼠DFCCM联合dNCPs诱导ADSCs向成牙骨质细胞诱分化的体外实验在实验2筛选的基础上,采用10μg/ml dNCPs +DFCCM诱导ADSCs向成牙骨质细胞分化,分别从细胞形态学、细胞增殖活性、ALP活性、体外矿化能力以及相关基因在蛋白和mRNA水平上的表达变化,对诱导后ADSCs进行鉴定。结果1.本实验培养的原代ADSCs原代和传代后生长状态良好,免疫细胞化学法检测,胞浆中Vimentin染色呈强阳性, CK染色呈阴性。流式细胞分析结果为: CD29,CD44,CD90,CD105、STRO-1的阳性检出率分别是93.09%、75.11%、90.20%、95.86%、30.09%。ADSCs体外多向分化鉴定结果,成脂诱导油红O染色可见成红色的脂滴颗粒;成骨诱导茜素红染色可见呈红色钙盐沉积;成神经诱导神经元特异性S100蛋白染色为阳性结果。2.牙囊细胞和根尖牙乳头细胞体外培养生长良好,免疫化学和流式细胞检测鉴定均较高表达Vimentin、CD29、CD44、CD105和STRO-1。在诱导ADSCs分化实验中,牙骨质特异性蛋白——牙骨质粘附蛋白(CAP)和矿化相关蛋白BSP、OPN、OCN、Col-I等在dNCPs +DFCCM实验组表达相对较强。3.在进一步探讨dNCPs+DFCCM诱导作用的实验中,诱导后的ADSCs由原来的成纤维样细胞形态向矮柱状、多角形的成牙骨质样细胞形态改变,且其增殖活性明显降低,而ALP活性和体外矿化能力明显增强,作为牙骨质特异性蛋白,CAP的表达以及其它矿化相关蛋白BSP、OPN、OCN、Col-I等表达也明显增强。结论1.体外分离培养了SD大鼠ADSCs,在细胞生物学鉴定中显示了较强的间充质来源特点和干细胞特性,且能向成脂、成骨和成神经分化。2.体外分离培养囊细胞和根尖牙乳头细胞,通过通过光学显微镜、细胞免疫化学和流式细胞分析等手段对其生物细胞学特性进行了初步鉴定,结合取材部位能够证实本实验所培养的DFCs和APCs具有间充质来源和干细胞特性。3.初步探讨了DFCCM、APCCM和dNCPs在ADSCs向成牙骨质细胞分化中的作用,结果显示DFCCM和dNCPs具有一定的成牙骨质细胞分化诱导作用,而且两者的联合应用更强;而APCCM则显示了更多向成牙本质细胞分化的诱导特性。4.在DFCCM+dNCPs联合诱导下,ADSCs在细胞形态学、增殖活性、ALP表达和体个矿化能力以及基因表达等方面均显示了一些成牙骨质细胞特性。综上所述,在DFCCM+dNCPs联合诱导下,ADSCs在体外初步获得了一些成牙骨质细胞特点,表明DFCCM+dNCPs中可能含有多种成牙骨质细胞分化所需要的生物活性因子。ADSCs储备丰富、获取简单,是一个较为理想的种子细胞来源,在牙周组织工程中显示了较好的应用前景。然而,DFCCM+dNCPs的成分较为复杂,其诱导ADSCs向成牙骨质细胞分化的机制尚不清楚,尚需进一步深入探讨。更多还原

【Abstract】 BackgroundCementum, a thin mineralized tissue produced by cementoblasts, covers the tooth root surface and anchors teeth to surrounding alveolar bone, which plays a crucial role in maintenance of tooth attachment. However, cementum regeneration is generally difficult for the reason that cementoblasts are scarce in root cementum and not easy to isolate. Despite considerable research, sources of cementoblasts for regeneration are largely restricted to dental stem cells, such as dental follicle cells and Hertwig’s epithelial root sheath (HERS) cells from tooth germs, stem cells from periodontal ligament, and ectomesenchymal cells from the first branchial arch, etc, which are not readily available clinically. This shortage of cementoblasts has significantly limited the development of periodontal tissue engineeringAdipose tissue-derived stem cells (ADSCs) have recently been widely studied in regenerative medicine because of their ease of harvesting and high proliferation. ADSCs are capable of differentiating to osteoblasts in an osteogenic microenvironment. Cementoblasts show many similarities in phenotypes to osteoblast and are considered a subpopulation of osteoblasts by some. Therefore, it may be feasible to induce ADSCs to differentiate along the cementoblast lineage if an optimal cementogenic microenvironment is provided.To establish this microenvironment, it is necessary to recapitulate the process of embryogenesis and morphogenesis involved in the developmental formation of cementum. Although the origin of cementoblasts remains a matter of debate, it has been well established that dental follicle cells penetrate disintegrating HERS and contact with unmineralized dentin matrix of the root surface prior to any cementum formation. Thus, dental follicle and dentine matrix may contain some biological mediators which are necessary to differentiation of cementoblasts. Dentin non-collagenous proteins (dNCPs), major component of dentine matrix, include glycoproteins/sialoproteins, phosphoproteins, proteoglycans and growth factors, which are considered to play an important role in differentiation of cementoblasts and cementogenesis.Therefore, we collected dental follicle cell conditioned medium and combined it with dNCPs in this study, to create a cementogenic microenvironment and induce ADSCs to differentiate into cementoblasts. This strategy may provide an alternative stem cell resource for periodontal tissue engineering and a better understanding of regulatory mechanisms for cementogenesis in periodontal development.Materials and Methods1.Culture and identification of ADSCs Adipose tissues were isolated from the inguinal region of 7-day-old Sprague–Dawley(SD) rats and finely minced into small pieces, followed by digestion with type I collagenase solution (0.1 mg/ml, Sigma, USA) for 1 h at 37°C. The cell suspension was centrifuged at 1000 r/min for 5 minutes. The supernatant was discarded and the cell pellet was suspended in Dulbecco Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 IU/ml penicillin and 100μg/ml streptomycin), then cultured at 37°C in 5% CO2. Cells were used at the third passage for the following experiments.Before used in the induction experiments, ADSCs isolated and cultured in this study were identified by morphological analysis, immunohistochemical staining, flow cytometry and multi-lineage differentiation capacity assays.2.Primary selection of conditions for cementogenic differentiation of ADSCsCell culture and conditioned media preparation: 5-day-old SD rats were selected and killed. Dental follicle and apical papilla tissue was dissected respectively from the first molars under a dissecting microscope. Culture and identification of these two cells were the same as above. Cells at the third passage were cultured. Once the cells reached confluence, culture medium was changed every day and collected for 3 days. The collected media were filtered through a 0.2μm Millipore strainer and mixed with an equal volume of fresh DMEM supplemented with 10% FBS, then stored at ?80°C before used as dental follicle cell conditioned media (DFCCM) and apical papilla cell conditioned media (APCCM).Primary selection of induction conditions: ADSCs at the third passage were cultured in DFCCM, APCCM, 10μg/ml dNCPs+ DMEM/F12 with5%FBS, 10μg/ml dNCPs +DFCCM, 10μg/ml dNCPs +APCCM, DMEM/F12 with 5%FBS(control) respectively for six days. Culture medium was changed every other day. Treated ADSCs were assayed by RT-PCR and Western blotting.3.In vitro experiment of cementogenic differentiation of ADSCs treated with dNCPs/DFCCMBased on primary selection of induction conditions above, the effects of dNCPs/DFCCM on cementogenic differentiation of ADSCs were further studied in this experiment. The changes on morphology, proliferative capacity, alkaline phosphatase activity, in vitro mineralization behaveiors, protein and gene expression for cementum attachment protein (CAP) and mineralization-related markers were assayed in ADSCs treated with dNCPs/DFCCM.Results1.ADSCs isolated from 7-day-old SD rats for in vitro primary culture and the 4–6th passages showed high proliferative capacity. Positive expression of vimentin was detected in ADSCs by immunohistochemical staining but no expression of CK was detected. Flow cytometry assay showed that ratios of CD29, CD44, CD90, CD105, STRO-1 positive cells in ADSCs were 93.09%, 75.11%, 90.20%, 95.86%, 30.09% respectively. In adipogenic, osteogenic, neuron-like differentiation experiments, ADSCs isolated in this study showed the ability of multipotential differentiation.2.Dental follicle cells and apical papilla cells for in vitro primary culture and the 4-6th passages showed good proliferative capacity and characteristics of mesenchymal stem cells. In immunohistochemical stainingand flow cytometry assay, vimentin, CD29, CD44, CD90, CD105 and STRO-1 were detected to positively express in both cell populations. In the experiment to optimize culture conditions for cementogenic differentiation of ADSCs, relative high expressions of CAP, bone sialoprotein(BSP), osteocalcinOCN, type I collagen (Col I),osteopontin(OPN) and osteonectin(ON) were detected at mRNA or protein levels in dNCPs/DFCCM treatment group.3.To further confirm the effects of dNCPs/DFCCM on cementogenic differentiation of ADSCs, more detailed experiments were performed. After treratment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and an in vitro mineralization assay indicated that dNCPs/DFCCM greatly enhanced the mineralization behaviour of differentiated ADSCs, in which mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype.Conclusions1.ADSCs isolated and cultured in this study show high proliferative capacity and characteristics of mesenchymal stem cells. They could differentiate into multi-lineage cells in corresponding media, such as adipocyte, osteoblast lineage, neuron-like cell lineage.2.Dental follicle cells and apical papilla cells were successly isolated and cultured in vitro. They were identified by immunohistochemical stainingand flow cytometry assay. The results and tissue extraction sites confirmed that dental follicle cells and apical papilla cells were originated from mesenchymal progenitors and showed some characteristics of mesenchymal stem cells.3.The effects of DFCCM, APCCM and dNCPs on cementogenic differentiation of ADSCs were investingated in a primary study. DFCCM and dNCPs showed promotion effect on cementogenic differentiation of ADSCs, especially combination of them, while APCCM showed promotion effect more of odontogenic differentiation.4.After treatment with dNCPs/DFCCM, ADSCs underwent morphological change from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and an in vitro mineralization assay indicated that dNCPs/DFCCM greatly enhanced the mineralization behaviour of differentiated ADSCs, in which mineralization-related markers including CAP, BSP, OCN, OPN and ON were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype.In summary, the lines of evidence presented here suggest that ADSCs are capable of differentiating to the cementoblast lineage following treatment with dNCPs/DFCCM, which includes multiple growth factors and other molecules necessary for signaling cementogenic differentiation. ADSCs, easily harvested clinically, could be an optimal source for cementogenic cells and very feasible for periodontal regeneration. However, further studies are necessary to address the underlying mechanisms involved in dNCPs/DFCCM–mediated cementogenesis.更多还原