【作者】 李萍；

【导师】 吴补领； 王常勇； 余擎；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2008， 博士

【摘要】 牙成釉细胞不仅可以分泌釉基质形成牙釉质,而且参与调控成牙本质细胞的分化和分泌基质,在牙齿发育中发挥着重要的作用。环境因素、营养障碍、外伤、炎症以及遗传缺陷等可造成牙成釉细胞的分化异常,其表现形式多种多样,如氟牙症、四环素牙、釉质发育不全、牙本质形成不全等。进行成釉细胞的细胞生物学相关研究对于揭示成釉细胞的分化、牙釉质的形成和对成牙本质细胞调控的分子机制具有重要意义,对牙齿组织工程研究具有重要的理论指导意义。成釉细胞为终末分化细胞,在牙冠发育形成后逐渐退化为缩余釉上皮,牙齿萌出后自然脱落,因而成釉细胞的获得和体外培养难度较大。迄今为止,牙成釉细胞的细胞生物学及分子调控机制的研究成果皆建立在大家所熟悉的方便、廉价的二维细胞培养基础上。虽然体外分离培养成釉细胞的方法较多,但均存在一定的缺陷。另外,二维培养条件下细胞的基因表达和生物学特性发生了变化,导致细胞去分化、部分蛋白的表达丧失、蛋白合成能力下降。如何获得更接近于体内细胞生长微环境、保持细胞生物学特性的体外细胞培养方法是急需解决的问题。三维立体培养系统不仅可提供接近于体内的培养微环境,还可以高通量地研究特定基因改变与细胞表型的关系及其生物学行为的变化。三维培养的细胞在基因表达图谱和其他生物学特性如细胞形态及排列方式等方面比二维培养更接近于活体组织。本课题研究的目的是明确RCCS是否可以用于培养牙成釉器细胞;观察牙成釉器细胞在RCCS三维立体培养条件与传统静态培养条件下其增殖、分化能力的差异;研究RCCS三维立体培养条件下,间充质细胞对牙成釉器细胞增殖、分化和细胞生物学行为的改变;探讨完全解离的牙成釉器细胞和牙乳头细胞在人工细胞外基质内的相互识别、自组装。本研究包含两个大部分内容:第一部分大鼠牙成釉器胞在RCCS中的的的细胞生物学特性研究本部分实验将原代分离大鼠成釉器细胞接种至旋转式生物反应器内进行三维立体培养,通过RT-PCR、免疫组化、免疫荧光、血气学分析、生化分析,研究旋转式生物反应器三维立体培养条件下大鼠牙成釉器细胞的生物学特性。首先利用Dispase酶和细胞差速贴壁的方法分别纯化1-6天日龄大鼠牙成釉器上皮细胞,常规DMEM/F12培养基培养。出生后4天大鼠牙成釉器细胞细胞生长特性典型、细胞增殖能力强,呈铺路石样生长,细胞连接紧密,上皮岛之间散在少量星形细胞,免疫细胞化学检测显示该细胞为CK14阳性,vimentin阴性。因此,将出生后4天大鼠作为实验动物进行以下实验。随后进行了成釉器上皮细胞在RCCS内的三维立体培养。RCCS特别适合用于培养终末分化细胞,尤其上皮细胞样细胞。由于RCCS生物反应器为悬浮培养系统,因此需要使用微载体提供贴壁细胞生长所需表面积。通过光镜观察细胞的生长情况调整微载体浓度和成釉细胞接种密度,掌握在RCCS内三维立体培养成釉器上皮细胞的关键技术。倒置显微镜观察和扫描电镜检测发现:成釉细胞可在微载体表面黏附生长并且细胞/微载体相互聚集形成三维立体结构。成釉细胞的最低接种密度为1×10~5个/mL;cytodex-3微载体的浓度为1mg/mL即可为成釉细胞提供充足的生长表面积。通过比较RCCS和静态培养条件下细胞生长曲线,发现RCCS培养系统可显著提高成釉器细胞体外扩增倍数,终浓度可达5×10~6个/mL而静态二维培养系统仅为1×10~6个/mL。旋转式生物反应器对细胞的接种密度要求比静态培养条件高,当以最小接种密度(0.5×10~5个/mL)接种细胞时,无法进行细胞的大量扩增。通过台盼兰染色活细胞和AO/PI荧光染色发现RCCS生物反应器内细胞活性高,通过免疫荧光技术检测发现随着培养时间的延长,成釉细胞特异性功能蛋白-成釉蛋白表达阳性细胞量高于静态培养组。通过RT-PCR检测釉原蛋白的mRNA表达情况也显示RCCS生物反应器培养系统可显著促进细胞分化、增加细胞外基质的分泌。生化和血气学分析结果显示该培养系统的生化指标均在生理范围之内,不构成该系统应用的限制。最后,在RCCS内将大鼠牙乳头细胞与牙成釉器细胞共培养,比较该培养系统与静态培养条件下两种细胞共培养的差别和三维培养条件下上皮-间充质相互作用对成釉细胞活性和分化能力的影响,为研究三维立体培养条件下间充质细胞对牙成釉细胞增殖、分化等细胞生物学特性的影响。实验结果显示RCCS生物反应器系统可显著促进共培养细胞的增殖:RCCS内共培养细胞终浓度可达8.6×10~6个/mL,而静态培养条件仅为3.14×10~6个/mL;牙乳头细胞可显著提高RCCS内成釉细胞的扩增速度和扩增倍数,单独培养成釉细胞和与牙乳头细胞共培养的终浓度分别为5×10~6个/mL和8.6×10~6个/mL。RCCS共培养成釉器细胞与牙乳头细胞的微载体接种密度需提高至3mg/mL。扫描电镜结果显示:在该培养体系内可形成大量细胞/微球聚集体,细胞可在该聚集体表面三维立体生长,形成类似体内的细胞生长微环境。成釉蛋白荧光染色阳性细胞所占上皮细胞比例较静态培养组显著提高。RT-PCR结果显示:RCCS和静态培养组p75NGFR、成釉蛋白和釉原蛋白的表达趋势相同。于体外培养第5天p75NGFR的表达量达到最大值;而成釉蛋白和釉原蛋白的表达量则逐渐上升,于培养的第7天达到最大值,但RCCS内表达量显著高于静态培养组。通过血气学分析和生化分析可实时监控该三维培养体系内的变化,研究发现除滞留期较高以外,乳酸脱氢酶的释放量始终处于较低水平,说明死亡细胞数量较少。虽然葡萄糖含量、pH值和氧分压较静态培养条件低,但均处于生理范围内,且与RCCS单独培养成釉器上皮细胞的生化分析和血气学分析结果没有显著学差异。本部分实验通过联合使用Dispase酶消化和差速贴壁的方法成功分离纯化大鼠成釉器上皮细胞,大大简化实验步骤、显著降低培养成本;成功将RCCS体外三维立体培养系统应用于大鼠成釉器细胞的体外培养,建立了大鼠成釉器体外三维立体培养研究模型,该特殊培养系统可避免传统静态培养系统的自身缺陷,促进成釉细胞的增殖、分化,可增加细胞外基质的分泌;该培养系统内可形成大量细胞聚集体,该聚集体提供了模拟体内的细胞生长微环境。因此,是进行上皮-间充质细胞相互作用形成器官和进行分子调控机制研究的良好模型。第二部分离散牙胚细胞在人工ECM内生物学行为特点的研究本部分实验利用人工细胞外基质进行细胞三维立体培养,模拟牙胚细胞体内生长微环境,研究离散牙成釉细胞和牙乳头细胞之间的相互识别与自组装及其生物学行为的变化。首先制备人工细胞外基质蛋白凝胶。0.1%醋酸溶解成年大鼠尾腱获得5mg/mL的Ⅰ型胶原溶液,并掌握联合使用Ⅰ型胶原和Matrigel胶原制备人工细胞外基质蛋白凝胶的关键技术,为研究离散大鼠上皮-间充质细胞在三维蛋白凝胶内的细胞生物学行为特点的研究提供实验基础。其次为优化实验条件,通过大体观察和组织学检测,获得最佳Ⅰ型胶原/Matrigel配比和最佳大鼠牙胚细胞接种密度。实验数据表明:Matrigel可显著促进上皮细胞的生长,9:1为Ⅰ型胶/Matrigel最佳混合比例,在该凝胶内上皮细胞和间充质细胞生长旺盛、增殖均衡;接种细胞浓度过高可因细胞-凝胶复合结构收缩程度过大,导致部分断裂,丧失静态拉伸力,造成中心部分坏死;而细胞浓度过低,细胞分布过于松散不利于细胞增殖培养,实验结果提示细胞最佳接种密度为5×10~6个/mL。应用免疫组织化学的方法研究发现在该人工细胞外基质凝胶内上皮细胞存在两种方式生长:一种为上皮细胞移行至胶原表面局部增生形成上皮条索,间充质细胞围绕其周围增殖,该结构类似于牙胚发育早期的牙蕾结构;另一种为上皮细胞停留在基质内部局部增殖形成上皮细胞团,而Vimentin阳性牙乳头细胞则在上皮细胞团周围紧密包绕生长,该结构类似于成釉器与牙乳头的组织构成。由此可见,完全离散的牙成釉器细胞和牙乳头细胞在体外三维立体培养条件下保持了其原有的生物学行为特性,在三维立体培养条件形成了牙胚不同时期的类似结构,具有形成牙胚结构的趋势。但由于牙齿发育过程中大量细胞因子参与调控作用,因此尚需要不断优化培养条件,提供更接近于牙齿发育过程中特殊的生长环境。总之,本实验的研究结果表明RCCS和人工细胞外基质是研究牙成釉器细胞在三维立体培养条件下细胞生物学特性的有力工具。对研究牙源性上皮细胞三维立体条件下的生物学特性和与间充质细胞相互识别、自组装等细胞行为特点进行了探索性研究,该三维细胞培养系统是进行上皮-间充质细胞相互作用形成器官和进行分子调控机制研究的良好模型。更多还原

【Abstract】 The ameloblast plays a very important role during the development of tooth. The ameloblast not only secrets enamel matrix to form the enamel, but also controls the differenciation and secretion of matrix of the odontoblast. There are a lot of reason that can cause the ameloblast differentiation abnormal,such as envinronment factor、dystrophia、trauma, or a variety of genetic disorders. The ameloblast differentiation abnormal can cause a lot of disease, such as mottledenamel, tetracycline discoloration, enamel hypoplasia, dentinogenesis imperfect, et al. Selection and culture of ameloblast lineage cell within the enamel organ epithelium are necessary for studies of ameloblasts function, pathology and enamel formation.The ameloblast is a kind of differenciated cells. It degenerated into induced enamel epithelium when the crown has formed, and losed when the tooth eruption.So it is very difficult to get and culture in vitro. Until now, two-dimensional monolayer cell culture method is the most common method for the research of cellular biology and molecular biology. There are a lot of methods of ameloblast lineage cell culture in vitro, but all are based on the two-dimensional cell culture system. There are a lot of significant differences on the gene expression, cellular secretion and cell functioned activities of cells cultured in the two-dimensional culture system and in the three-dimensional culture system. It is the most important that how to obtain the cell culture method which can mimic the in-vivo environments and facilitate observing the interactions between epithelial cells and mesenchyme cells.The objective of this study were to separate and purify the dental EOEs; to identify whether RCCS can be used for culturing the dental EOEs; to observe the differences of cell proliferation and differentiation characteristics of dental EOEs cultured in RCCS and in the traditional cell culture system; to observe the differences of cell proliferation and differentiation characteristics of dental EOEs in RCCS from in the traditional cell culture system;to study the effect of dental papilla cells on the cell proliferation, cell viability and the differentiate of dental EOE in the special three-dimensional cell culture system-RCCS; to investigate the identify and self-assemble of dental EOEs and papilla cells in ECM. The present study consists of two parts:Part one: the cellular biology charactorastic of the rat enamel organ epithelium under the three dimensional cell culture systemIn order to study the effect of three-dimensional cell culture system on the characteristic of ameloblast cellular biology, we culture the rat enamel organ epithelium in the RCCS, and analysis the effect of three-imensional culture and the interaction of mesenchyme on the EOE, respectively.Firstly, we used dispasnase to separate the papilla from the enamel organ, and used the different speed of epithelium and mesenchyme to stick to the plate to purify enamel organ epithelium, and culture in DMEM/F12 medium. The post-natal 4 day rats enamel organ epithelium purificated rapidly and present cobblestone morphology, cellular connection is conjected tightly, epithelium grow like lamellar layer Several stellate cells grow between epithelium islands. Immuocytochemistry analysis reveals cobblestone morphology cells are positive in CK14 staining, but negative in vimentin staining. The pnd-4 rat pups were used to do the next study.Secondly, the rat enamel organ cells were cultured in RCCS three-dimensional culture system. To scale-up anchorage-depended cell in RCCS bioreactor must use the microcarrier as the scaffold. The microcarrier can provide the nessessary growth areas for anchorage-depended cells. The RCCS is suitable for differentiated cell culture, particularly for cells with an epithelial-like morphology. The concentration of microcarriers and EOE density were adjusted by observing the cell growth by phase-contrast microscopy, obtain the key techniques of EOE culture in RCCS. We find that the cell can attached on the surface of the microcarriers and form cell/microcarriers three-dimensional aggregation ; cytodex-3 can provide efficient cell growth surface for EOE at the concentration of 1mg/mL; the lowest inoculation indensity of EOE is 1x105cells/mL。By comparing cellular growth curve in RCCS and dynamic culture condition, we find that RCCS culture system can significantly increase the number of enamel organ epitheliums, 5x106cells/mL and 1x106cells/mL, for RCCS and dynamic traditional culture, respectively. Much higher inoculation density was required in RCCS than in dynamic traditional culture condition did. cell can’t amplify in the condition of lowest inoculation indensity （0.5x105cells/mL）. We also find that in RCCS the cellular activity is higher and EOE characteristic functionalized protein （ameloblastin protein） expression is higher than the dynamic culture group. We also confirm this result by analyzing the mRNA of ameloblastin and amelogenin. Throughout the experiments, glucose was staying within the accepted physiological range. This suggests that glucose is not a limiting factor in these cultures.Lastly, the dental development is the result of epithelium-mesenchyme interaction. The in vitro co-culture system is the helpful tool to study the mechanisms of epithelium-mesenchyme interaction. RCCS is the only equipment which can provide co-culture study until now. In this part, we co-culture rat enamel organ epitheliums with dental papilla cells in RCCS, contract with the dynamic traditional culture systems. The results reveal that RCCS system can improve the proliferation of EOE at the finial density of 8.6×10⁶cells/mL and 3.14×10⁶cells/mL, for RCCS and dynamic traditional culture, respectively.It can significantly improve the EOE cellular growth speed and increase folds in the condition of co-culture with dental papilla, the concentration of microcarriers needed in RCCS co-culture system was 3mg/mL. The scanning electron microscropy analysis results show that there are a lot of cells/beads aggregations, and the cell can growth three dimensionaly. The positive cellular number of ameloblastin protein fluorescent staining is higher than the dynamic culture condition. The RT-PCR result showed the similar trend of marker protein mRNA expression :the expression of p75NGFR reach the maximum on day 5, but the mRNA expression of ameloblastin and amelogenin increased constantly, reaching the maximum on days 7. We can monitor the glucose level、LDH release rate、the lactic acid level、pO₂、pCO₂、pH changes in this three-dimensional culture system by biochemistry and Blood Gas Analyzer, the results show that the level of LDH is the low condition all the time except for lag phase. Although the level of glucose, PH value and oxygen pressure is lower than the dynamic situation, but still in the physical range. There was no significant difference of biochemistry and Blood Gas Analysis between co-culture and EOE cultured in RCCS system.In short, we have grasped the key culture techniques of rat enamel organ epithelium in vitro RCCS successfully, and establish the study model of rat enamel organ three-dimensional culture system, and have studied the growth characteristic of rat EOE in this culture system, It is the experimental basis for further study.Part two: The identification and self-assemble of dissociated dental germ cells in the Collagen/Matrigel ScaffoldTo imitate the in vivo microenvironment of dental germ cells, the self-made artificial ECM was used to culture the dissociated dental germ cells three dimensionally and to study the identification and self-assemble of dissociated dental germ cells and dental papilla cells, and also the biological behaviors.Firstly, we prepared artificial ECM protein gel by mixing Matrigel and type I collagen solution, which was the solution of rat tail tendon with 0.1% acetic acid. and have gotten the key techniques of preparing artificial ECM protein gel with Matrigel and type I collagen by adjusting pH. Secondly, rat tooth germ cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During the days of in vitro culture, the dental constructs were observed under microscope and analyzed using histological and immunohistochemistry examination. We find that Matrigel can improve the proliferation of EOE. The EOEs and mesenchyme cells grown healthy in artificial ECM protein gel with the best mixed ratio of 9 type I collagen to 1 Matrigel.Secondly,cell density is vital to determine the intensity of contraction. At high cell density, the constructs break down. Because of intensively stretching the constructs break and center cellular necrosis. The optimal concentration was about 5×10⁶cells/ml. With this concentration, most structures containing *and* structures formed.There are two growth type of EOE in artificial ECM gel: one type is the EOE creped to the surface of gel to form epithelium layer and proliferated at a point to form a deepen epithelium strip into gel and mesenchyme cells surrounding them, this structure is similar to tooth bud; the other type is CK 14 positive EOE cells proliferated inside the ECM and formed epithelial pearl-like structures, which vimentin positive dental papilla cell surrounding these epithelial pearl-like structures. These structures are similar to enamel organ and dental papilla tissue.Dissociated enamel organ cells and dental papilla cells keep the primary biological behaviors to form similar architecture in different dental germ periods, have tendency to form tooth in vitro three-dimensional culture condition. We need to optimize culture conditions continually as the limitation of growth environment provided by in vitro three-dimensional culture system.In short, the results revealed that RCCS and artificial ECM is the helpful tool for studying the interaction between dental germ cells and mesenchyme cells in three-dimensional culture condition. This research obtains the key techniques of enamel organ cells- mesenchyme cells, enamel organ endothelial cells in RCCS. This system can amplify high quality enamel organ cells and the research tool for studying interaction between dental germ cells and mesenchyme cells in three-dimensional culture condition. We have exploratory study biological characteristic and interaction recognition, self-assemble of epithelium cellula intersitialis. It can provide biological theory basis for dental regeneration.更多还原