【作者】 苗宗宁；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2010， 博士

【摘要】 组织工程的原理是利用工程学方法和手段,将具有特定功能或分化潜能的细胞(种子细胞)与可降解生物材料(支架材料)共植入体内,随着种子细胞的不断扩增、细胞外基质的分泌和生物材料的逐渐降解、吸收,最终形成具有正常结构和功能的特定组织或器官。因此种子细胞和支架材料是组织工程研究的主要内容。在组织工程的研究与应用中,种子细胞的来源、分离纯化、规模化扩增等是制约其发展的重要瓶颈问题之一。间充质干细胞(mesenchymal stem cells,MSCs)是来源于发育早期中胚层的一类多能干细胞,在特定诱导条件下可分化为多种类型的结缔组织,形成骨、软骨、骨骼肌、肌腱和脂肪等多种组织,而且具有采集方便、易于体外培养、扩增和诱导等特性,被认为是一种理想的种子细胞。目前,骨髓来源的MSCs(bone marrow-derived MSCs,BMSCs)研究最多,但人BMSCs含量极低。近年来,已有具有干/祖细胞特征的间充质细胞自骨髓、外周血、脂肪及肌肉等组织中分离,这些组织的共同特征是来源于胚胎发育期的中胚层。胎盘由滋养细胞及大量起源于胚外中胚层的间充质和血管共同组成,提示胎盘中存在MSCs成分。对胎盘来源的MSCs(placenta-derived MSCs,PMSCs)的研究也已展开。实验室前期的实验结果表明可以从胎盘组织分离培养间充质干细胞并且这些细胞具有与骨髓间充质干细胞相似的细胞生物学特性。由于可以在体外预先建立胎盘MSCs库,实现即时使用,所以PMSCs在组织工程研究中具有广阔的应用前景。骨组织工程学要求材料具有适宜的降解速率,使支架材料在一定时间内吸收,形成具有功能活性的自身骨组织。骨替代材料降解过快不能提供新骨形成的支架,降解过慢则阻碍新骨的生长及骨塑形,影响功能活性的自身骨组织形成。羟基磷灰石是骨组织主要的矿物质成分,以其良好的生物相容性和生物活性引起研究人员的重视。然而羟基磷灰石材料降解速度非常缓慢,而且脆性大、柔韧性差。丝素具有生物相容性和生物可降解性,丝素特别是家蚕丝素是纯天然,极易获取且价格低廉的一种。以往研究表明丝素材料可以支持人骨髓间充质细胞的贴壁生长和体外增殖,促进间充质细胞分化成为成骨细胞、钙盐沉积以及骨性结构、骨小梁的形成。然而丝素硬度低,在外力作用下极易变形的特性使得单纯的丝素难以适应移植环境。因此,构造一种新型的丝素材料使其同时具有一定的硬度和韧性以及适当的力学性能以适应移植组织的结构并运用于骨组织工程成为必然。羟基磷灰石降解缓慢、脆性大,而丝素蛋白机械强度差。将两者复合,丝素蛋白能改善羟基磷灰石的理化性能,并可调控SF/HA的降解速率,SF/HA复合材料具有良好的组织相容性,有望成为一种新的骨修复替代材料。本研究从胎盘分离培养间充质干细胞(placenta mesenchymal stem cells,PMSCs)并分析其免疫学特性,联合丝素蛋白/羟基磷灰石材料(silk fibroin/hydroxyapatite, SF/HA)对兔桡骨的节段性骨缺损进行修复,以评价PMSCs和SF/HA材料在骨组织工程中应用的可能性和价值。第一部分目的:探索间充质干细胞(mesenchymal stem cells,MSCs)在人胎盘组织中的分布规律以及体外分离培养的方法。方法:取正常剖宫产之足月健康产妇的新鲜胎盘组织,按中央带(A)、中间带(B)、边缘带(C)全层连续切片,以抗CD166、抗CD90、抗CD29分别作免疫荧光和免疫组化染色,显微镜下观察阳性细胞分布区域,采用HPIAS-1000彩色病理图像分析系统进行图象处理,同时应用整体灌注的方法分离细胞,常规的方法进行细胞培养并鉴定培养细胞的表面标志及其分化潜能。结果:3个间充质干细胞标记分子CD166、CD90、CD29阳性表达细胞,主要分布于血管内皮下及血管附近间质内和胎盘中央带,阳性表达的强度在不同部位具有显著性差异(P﹤0.05);整体灌注方法获得的细胞培养后经流式细胞仪检测显示,表达CD73、CD90、CD166和CD105,不表达CD34、CD45、CD14和CD106,呈现PMSCs表型;体外扩增培养的PMSCs成骨诱导后,由长梭形向立方形转变, Von Kossa染色可见细胞呈集落生长并出现钙结节;成脂肪诱导培养的PMSCs油红O染色阳性;成软骨诱导2周后,PMSCs形态逐渐变得扁平,anti-collagen type II免疫组织化学染色可见阳性细胞表达II型胶原;向神经元样细胞诱导1d后大多数PMSCs转变为双极或多极神经元细胞样形态,伸出突触,染色可见NSE、GFAP阳性;向内皮样细胞诱导7天以后,PMSCs逐步回缩,立体感增强,KDR以及v-WF染色结果不同程度阳性。结论:胎盘各部位均存在表达CD166、CD90、CD29阳性的间充质干细胞,细胞的分布与血管有一定的相关性,整体灌注的方法可以分离细胞,脐带附着处下方胎盘组织相对其他部位而言可能为MSCs富集区域,体外扩增培养的PMSCs具有向成骨、软骨、脂肪、神经元样和内皮样细胞分化的潜能。第二部分目的:探讨PMSCs的免疫学特性及与丝素蛋白(silk fibroin,SF)的相容性。方法:采用人以及兔双向混合淋巴细胞反应(Mixed Lymphocyte Reactions,MLR)体系,根据不同比例加入丝裂霉素处理过的PMSCs,3H掺入法测定淋巴细胞增殖率并用ELISA法测定混合培养上清液中IL-2和IFN-γ的含量;运用SF溶液包被的培养瓶培养PMSCs,流式细胞术分析其表型并对其定向分化潜能进行探讨,进一步观察其对MLR体系中淋巴细胞增殖的影响;PMSCs置于SF膜材料培养后通过扫描电镜观察细胞形态变化。结果:PMSCs可以抑制人或兔混合淋巴细胞反应体系中淋巴细胞的增殖,抑制率与加入的PMSCs数量成正比,同时PMSCs可以减少混合淋巴细胞反应中细胞因子IL-2、IFN-γ的分泌;用SF溶液包被的培养瓶培养的PMSCs,其生长特性、表面标志、多向分化潜能无明显变化;PMSCs在SF膜材料上生长良好,培养8d时材料上细胞伸展增殖,分泌大量的的颗粒状、网状基质物质,材料间隙被基质填满。结论:PMSCs具有免疫调节作用;SF材料不影响PMSCs的生长特性、表面标志和多向分化潜能,具有良好的生物相容性。第三部分目的:探讨PMSCs联合丝素蛋白/羟基磷灰石材料(silk fibroin/hydroxyapatite, SF/HA)对兔桡骨的节段性骨缺损修复实验,以评价PMSCs和SF/HA材料在骨组织工程中的应用价值及其机制。方法:应用BrDU标记PMSCs并联合SF/HA材料移植下列兔桡骨缺损部位, 24只新西兰大白兔制成桡骨中段1.5cm长的骨缺损模型,随机分为实验组(植入PMSCs/SF/HA)、对照组(植入SF/HA)、空白组(不植入修复材料)共3组。术后2、4、8、12周分别行大体观察、组织学观察和X线观察,评分比较3组骨缺损修复的情况。结果:兔桡骨缺损植入修复术后2、4、8、12周,影像和组织形态学结果显示,新骨生成在实验组均优于对照组,空白组各时间点均无新骨形成。术后8周时实验组移植部位免疫荧光染色仍可见BrDU阳性细胞存在。术后8周和12周,评分比较实验组和对照组骨折愈合有显著性差异(P<0.05)。结论:SF/HA材料与PMSCs联合移植能够修复兔桡骨缺损,可以作为组织工程骨的支架材料和种子细胞的来源。综上所述,通过本研究发现胎盘各部位均存在表达CD166、CD90、CD29阳性的间充质干细胞,细胞的分布与血管有一定的相关性,整体灌注的方法可以分离细胞,体外扩增培养的PMSCs具有向三个胚层细胞分化的潜能和免疫调节作用;SF具有良好的生物相容性;PMSCs联合SF/HA材料移植能够修复兔桡骨缺损,可以作为组织工程骨的支架材料和种子细胞的新来源。更多还原

【Abstract】 The principle of tissue engineering is to use the engineering methods and tools to build and regenerate various tissues and organs to replace the lesions organization, which means to implant the seed cells with the specific function or differentiation potential and the biodegradable materials (scaffolds) to form a specific tissue or organ with normal structure and function at last, as the seed cells proliferating, secreting extracellular matrix, and the biological material gradual degradated and absorbed. Therefore, the seed cells and the scaffold were main research contents in the Tissue Engineering.The important problems in bone tissue engineering are the source, isolation, and expansion of seed cells. Mesenchymal stem cells (MSCs) are derived from the mesoderm at the early development stage. It can be adopted as an ideal source of seed cells which have the potential to be induced into osteogenic, chondrogenic, and adipogenic cells or even tendon and adipose tissues. MSCs are easy to be obtained, cultured, expanded in vitro as well as to be easily induced into designed tissues. Currently, the bone marrow-derived MSCs (BMSCs) are widely used. However, the amount of MSCs in bone marrow is extremely low and accounts for about 0.01~0.001% of the bone marrow derived cells. Increasing evidence suggests MSCs with osteogenic potential have been isolated from a diverse range of tissues including adipose tissue and perinatal tissues such as umbilical cord, placenta, and umbilical cord blood. Previous findings from our group indicate that MSCs from the placenta (PMSCs) could be obtained and expanded in vitro. In vitro cultured PMSCs still well maintain the same biological characteristics as the BMSCs. In addtion, the cell bank of PMSCs could be set up in advance for the clinical trials, suggesting that PMSCs have a wide application prospect.The biomaterial scaffolds used in bone tissue engineering should be biocompatible and osteoconductive. In addition, they should have proper porosity and pore size for cell attachment and flow transport of nutrients and metabolic waste, as well as proper mechanical properties to match bone tissues. Hydroxyapatite (HA) is the main mineral component of bones. HA has been attracted much attention for its excellent biocompatibility and bioactivity. However, the biodegradation rate of sintered HA ceramics is very slow and the porous HA ceramics is fragile.Silk fibroin (SF), especially, produced by the Bombyx mori silkworms is one of the most abundant natural proteins and can be obtained easily and inexpensively. Previous studies indicated that silk fibroin matrices could support human bone marrow-derived MSCs (BMSCs) attachment and proliferation, the calcium deposition on them and the development of bone-like trabeculae with cuboid cells. However, because of the low hardness and easy deformation under stress, pure silk fibroin materials are difficult to adapt to environmental tissue. Thus, it is necessary to fabricate new silk materials which have highly hardness and toughness and suitable mechanical properties to fit hard tissue and be applied as bone tissue engineering or bone tissue inducing matrices. The low degradation rate, high brittleness of HA, and poor mechanical strength of SF can be well solved through combining HA and SF to be SF/HA scaffold with an appropriate degradation rate and favorable biocompatibility.In Our study we isolated and cultured PMSCs in vitro, explored its immunological characteristics, then produced the bio-composite materials with cultured PMSCs and SF/HA porous materials to repair the rabbit bone defect. Through this study, we want to evaluate the value and potentiality in application of both PMSCs and SF/HA scaffold in bone tissue engineering.PartⅠObject:To investigate the distribution and isolation of MSCs in human placenta and to establish the stable expanding culture system for PMSCs in vitro.Method: To take three tissue samples from the term (38–40 weeks’gestation) placentas of healthy donor mothers, sample one (A) is from the central part of the placenta where the umbilical cord attached, sample two (C) is the fringe part of placenta, and the sample three (B) is the part between part A and part C, to do the full-thickness serial sections, separately, then, immunofluorescence and immunohistochemical staining were adopted to observe the distribution of CD166,CD90 and CD29 positive cells, finally, the HPIAS-1000 color pathological image analysis system was used for the image analysis. Besides, the overall perfusion method was adopted to isolate PMSCs and the cell surface markers and multi-differentiation potential of the cultured PMSCs was identified.Result: Comparing with other area (part B, and part C), CD166, CD90 and CD29 positive cells were found mainly distributed in part A , the OD value in this group was statistically significant (P<0.05), and all these CD166, CD90 and CD29 positive cells were found gathered around the blood vessels. The cultured PMSCs obtained by overall perfusion method were CD73、CD90、CD166 and CD105 positive, while CD34、CD45、CD14 and CD106 were negative. Exposure to osteogenic inductive medium resulted in secretion of extracellular calcium crystals, identified by von Kossa staining, indicating osteogenic differentiation. When cells were cultured in adipogenic inductive medium, intracytoplasmic lipid vacuoles were observed, and confirmed by oil red O staining. After been cultured in chondrogenic inductive medium, chondrogenic differentiation was identified demonstrated by positive type II collagen staining. After been exposed to the neural inducer, most PMSCs showed the typical morphology of neurons with many projections, and the induced cells were NSE、GFAP, neural cell specific markers, positive by immunofluorescence staining, while if change the inductive medium to the endothelial one, 7 days later, cell body was found gradually retracted, three-dimensional sense enhanced, and the endothelial cell-specific markers KDR and v-WF were expressed on the induced cells by immunofluorescence staining.Conclusion: CD166, CD90 and CD29 positive cells were found in placenta, moreover there was a certain correlation between the distribution of these cells and blood vessels in the placenta. The region where the umbilical cord attached was proved to be the MSCs-rich region. Besides, overall perfusion method could be adopted to culture the PMSCs in vitro, and the cultured cells were proved having the ability to differentiate into bone, cartilage, fat, nerves and endothelial cell.PartⅡObject:To study the immunological characteristics of PMSCs and its compatibility with silk fibroin (SF).Method: The two-way mixed lymphocyte reaction (MLR) system both for human and the rabbits were established. After adding PMSCs which was treated by the mitomycin in the MLR system, the lymphocyte proliferation rate were measured by 3H incorporation, and the content of IL-2 and IFN-γin the supernatant was detected by ELISA to observe the effects of human PMSCs on lymphocyte proliferation both from human and the rabbits. Meanwhile, PMSCs were cultured in the SF coating flasks, then its phenotype was analysised by flow cytometry, its adipogenic, chondrogenic, and osteogenic differentiation potential were determined by specific staining, and its immune regulation ability was detected in the MLR system, finally its growth condition was observered under the electric microscope.Result: The proliferation of T lymphocytes could be inhibited to different extents by adding different ratios of PMSCs to human or rabbit PBMCs, and the inhibition rate increased as the ratio of PMSCs to PBMCs increased. Meanwhile, PMSCs could reduce cytokines IL-2, IFN-γsecretion in the mixed lymphocyte reaction system. After being cultured on the SF film, the growth characteristics, surface markers, multi-differentiation capacity of PMSCs were not changed; the electric microscope observation results showed that PMSCs proliferated in the SF film materials after been co-cultured for 8 days, secreted large amounts of granular, mesh matrix substances well filled in the material clearance.Conclusion: The cultured PMSCs had the immune regulation ability and inhibited xenogeneic immune cells as well. PMSCs also held the cross-mesoderm differentiation ability, and could be induced to differentiate to neural cells and endothelial cells. SF had good biocompatibility and had no effects on the PMSCs growth characteristics, surface markers and multi-differentiation capacity.PartⅢObject:To explore the silk fibroin/hydroxyapatite (SF/HA) porous materials serving as a delivery vehicle for human PMSCs in a rabbit bone defect model so as to evaluate its value and mechanism of application in bone tissue engineering.Method: The BrDU labeling PMSCs were cultured in SF/HA porous materials, then transplanted them to the radial defect animal model. A total of 24 healthy adult New Zealand rabbits were randomly divided into experimental group, control group and the blank control group, and a 15-mm length of radius defect model was established. The gross observation result, histological result and the radiographic examination result were analyzed on 2nd, 4th, 8th, and 12th week after transplantation to evaluate fracture healing.Result: Radiological and histological examination results of 2, 4, 8, 12 weeks after implantation showed that new bone formation in the experimental group were better than the control group, while in the blank control group, no new bone formation was detected at each time point. Immunofluorescence staining showed BrDU-positive cells were still visible exists 8 weeks after transplantation in the experimental group. Moreover, results from radiographic examination showed significant differences in the radiographic score between experimental groups and control groups at the 8th week and the12th week(P<0.05).Conclusion: PMSCs/SF/HA composites materials could be adopted to construct tissue-engineered bone which can repair the bone defect. And it proved that SF/HA porous materials could be applied as a scaffold for bone tissue engineering, and PMSCs were a new source of seed cells.Summary: CD166, CD90 and CD29 positive cells were found in placenta, moreover there was a certain correlation between the distribution of these cells and blood vessels in the placenta. Besides, overall perfusion method could be adopted to isolate the PMSCs in placenta, and the cultured cells held the cross-mesoderm differentiation ability and the immune regulation ability. SF had good biocompatibility and SF/HA porous materials could be applied as a scaffold for bone tissue engineering, and PMSCs were a source of seed cells.更多还原