【作者】 马钰；

【导师】 李青； 赵大庆；

【作者基本信息】 第四军医大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 战时的复合型、多发性创伤以及平时各种外伤、骨关节病等均可造成全身各处软骨损伤、破坏,直接影响患者的外貌和功能,其稳定及便捷的修复对提高患者的生存质量至关重要。随着生活水平的提高,人们对美容的需求也日渐增长,应用软骨进行的临床整形美容治疗是整形修复外科研究的热点与难点。自体、同种异体软骨以及人工替代材料不断应用于临床,但它们存在来源受限、形态不匹配以及免疫排斥反应等问题。组织工程学是应用细胞生物学和工程学原理,研究和开发用于修复和改善人体各种组织、器官损伤后的功能和形态的一门科学,它可以避免传统治疗方法的缺陷,是解决组织缺损的一项理想途径。组织工程研究的两个关键因素是良好的种子细胞以及合适的支架材料。目前应用的种子细胞中,自体软骨细胞来源有限,会造成机体附加损伤;同种异体软骨细胞来源广泛,获取容易,但免疫排斥反应较强;胚胎干细胞和骨髓基质干细胞是种子细胞较佳的来源,但都受到伦理道德和取材数量的限制,使其在组织工程的应用上难以推广。支架材料方面,目前用于软骨组织工程支架材料主要有胶原海绵、纤维蛋白凝胶、聚丙交酯(polylactide, PLA )、聚乙交酯(polyglycolide, PGA )及其共聚物(Poly lactide-co-glycolide, PLGA )等,它们存在或亲水性差、细胞吸附力弱、降解慢,或免疫反应大,价格昂贵等诸多缺点。脂肪组织基质中存在着具有自我更新和一定的多能分化潜能、处于全能细胞和成熟细胞之间的成体干细胞,称为脂肪基质细胞(adipose tissue-derived stromal cells, ADSCs )。ADSCs具有多向分化的潜能,能向骨细胞、软骨细胞、上皮细胞、心肌细胞、骨骼肌细胞、平滑肌细胞、肝细胞及神经等细胞等定向诱导分化,从而参与组织的损伤修复。其容易获取、采集效率高,对机体损伤小,是一种新型的组织工程种子细胞。羟丙甲基纤维素(Hydroxypropyl methyl cellulose , HPMC)是一种无细胞毒,与机体相容性好,不诱发免疫排斥反应的热敏性水凝胶,其降解速度与组织生长速度相匹配,其可注射性及温度敏感性为创伤修复治疗和重建手术提供了方便、可塑性、微创的生物材料,是一种理想的组织工程生物支架材料。脂肪基质细胞(ADSCs)和羟丙甲基纤维素(HPMC)的联合应用为组织工程修复软骨缺损提供了一个新的途径与方法。材料与方法1.人脂肪基质细胞(ADSCs)的分离和培养取第四军医大学唐都医院整形科超声乳化后的脂肪组织溶液,经消化、过滤、分离、培养得到ADSCs,加入培养基培养,观察原代及传代后细胞形态,并用MTT法绘制生长曲线。2.流式细胞仪检测细胞表面标志物取6代细胞,流式细胞仪检测CD14、CD31、CD34、CD45、CD49d、CD56、CD105以及CD106等表面标志。3.人脂肪基质细胞(ADSCs)向软骨细胞诱导分化ADSCs消化传代,用成软骨培养基重悬培养细胞,接种于新培养瓶中,隔日换液。观察细胞形态变化,细胞爬片行免疫组化和AB/PAS染色以及天狼猩红染色。4.成软骨诱导细胞在裸鼠体内构建工程化软骨配制合适浓度的羟丙甲基纤维素(HPMC)与诱导细胞混和,分别注入3只裸鼠前后肢背部皮下,每只裸鼠有3个注射点为实验组,1个注射点作为对照,注射不含细胞的凝胶。裸鼠分别于饲养3w、5w和7w处死取材,进行组织和形态的观察。结果1.细胞形态与生长曲线原代培养的ADSCs 7d左右即达70%~80%融合,细胞呈漩涡状排列。经体外培养10代以后,细胞的增殖速度减慢。成软骨诱导细胞增殖速度显著快于未诱导细胞。用MTT法分别做ADSCs及成软骨诱导细胞的生长曲线。ADSCs第3d进入指数增长期,群体倍增时间为55 h左右,细胞在第4d进入平台期;成软骨诱导细胞第2d进入指数增长期,群体倍增时间为30 h左右,细胞增殖迅速,在第8d进入平台期。2.流式细胞仪检测结果ADSCs对CD31,CD34以及CD45,CD106表达很低,同时也可观察到CD14,CD56和CD105均呈较低表达,而其对CD49d却有较强的表达,CD49d为ADSCs特异性表达的标志物,而造血系统来源细胞并不表达此标记物。以上均说明实验中所获取的是ADSCs。3.成软骨诱导细胞的免疫组化和AB/PAS染色、天狼猩红染色结果免疫组化显示ADSCs在成软骨诱导后表达II型胶原;AB/PAS染色的细胞分泌有中性及酸性粘多糖成分,其中有较多的软骨细胞所特有的酸性糖蛋白基质;天狼猩红染色细胞爬片在偏振光下可见具有双折光性的红色和黄色纤维,也可见少量呈网状分布的多彩纤维,分别为诱导细胞分泌出的Ⅰ型和Ⅱ型胶原物质。4.裸鼠体内构建工程化软骨的结果裸鼠各个注射点皮肤色泽正常,注射混和凝胶的皮下组织毛细血管增生,在3w、5w和7w所取裸鼠分别在HE染色下观察到软骨细胞以及部分残留凝胶。其中实验组9个点中有4个点出现软骨,对照组3个点均未发现软骨形成。结论1.超声乳化后的脂肪组织溶液经消化、分离后可得到脂肪基质细胞(ADSCs)。2.经成软骨诱导培养后,ADSCs具有软骨细胞特征,可以作为软骨组织工程的种子细胞。3.羟丙甲基纤维素(HPMC)与ADSCs相容性较好,未见其在裸鼠体内引起排斥反应,与诱导细胞混合后移植入裸鼠体内可形成软骨实质,可以作为一种组织工程的支架材料。更多还原

【Abstract】 Battle wound and various kinds of diseases such as osteoarthrosis caused cartilage defects influence patient’s outward appearance and their function. It is significant to repair it stable and convenient for improve patient’s quality of life. Therefore, autologous, allogeneic cartilade and some artificial materials has been used in clinical. However, the disadvantages such as supply limited, hard to match and immunological rejection had restricted the development. Tissue engineering use the principles and methods of engineering and life sciences to repair and reconstruction of tissue defects. Seed cells and the material of scaffolds are key factors in the tissue engineering. The self-histiocyte has some question about quantity of localization, function aging and so on. The embryonic progenitor cells and marrow mesenchymal stem cells also have some ethical problems. On the other hand, the commonly used scaffolds in the tissue engineering have some disadvantages such as serious immune response, high price and so on.There is a kind of cells in the adipose tissue called adipose tissue-derived stromal cells (ADSCs )have the potential ability to differentiate into bone, cartilage, muscle and so on, ADSCs could be available in large quantities with minimal morbidity and discomfort associate with their harvest. They can be used as a new kind of seed cells in the tissue engineering. The injectable and thermosensitive Hydroxypropyl methyl cellulose (HPMC) has the advantages of convenience, plasticity and mini injury; it might be used as an ideal material of scaffolds in tissue engineering. This study focus on using ADSCs combined with HPMC to fabricate cartilage with tissue engineering technology.Materials and methods1. Cell culture and Differentiation Human adipose tissues were obtained from elective liposuction procedures under local anesthesia, then digested and centrifuge the lipoaspirates to obtain the useful cells. The cellar pellet was resuspended in proper mediums. We observed the appearance of cells and drew the growth curve.2. Flow CytometryWe detected the unique CD maker antigens-CD14, CD31, CD34, CD45, CD49d, CD56, CD105 andCD106 of the 6th generation ADSCs. 3. Cell multiplication and induction.ADSCs cultured separated in different mediums were trypsinized and seeded in two 96-well plates at a density of 2×103/200μl. When the three groups of cells described above were cultured for 1day to 8days, cell proliferation was detected by MTT colorimetric method. 4. Fabrication of engineering cartilage using induced ADSCs in nude mice We injected the mixture of induced ADSCs and HPMC into the 3 mice’s hypodermis. In each mouse, 3 points were experimental group, the other 1 point was control. Mice were killed at the 3w, 5w and 7w after cell injection. Specimens were fixed in buffered formalin.Results1. ADSCs cultured in the two mediums grows and proliferate in different waysADSCs arrange like a swirl, exhibited an average population doubling time of 55h using common medium. The cells could go down to the 9th passage, after that, the cells grew slowly and aging obviously. In contrast, the induction ADSCs grew faster than the former, cells were bigger, and some pseudopodium and nucleoli were observed. These cells went into index stage at the second day, exhibited population doubling time of 30h. The cells turned aging at about the15th passage.2. Phenotypic Characterization of ADSCs population: CD Marker profileCD maker profile was examined for characterize the ADSCs population, no expression of the hematopoietic lineage markers CD31, CD34, CD45 and CD106 was observed in the cells. CD14, CD56 and CD105 were expressed in low level. ADSCs expressed CD49d, whereas this antigen was not expressed in hematopoietic lineage, it can be seen as a significant marker for ADSCs.3. Immunehistochemenical and special staining. After cultured in chondrogenic medium, the cells secreted the specific cartilaginous matrices sulfated proteglycan and collagenⅡ. And we had observed the special staining of chondrocyte induced ADSCs under polarized microscope, which was characteristic cartilage matrix.4. Observation of the nude mice induced ADSCs and HPMCThe skins of all nude mice were normal, the hypodermis became bloodshot. At the 3w, the 5w and 7w, we all observed the cartilage through the microscope.Conclusion1. Adipose derived stromal cells (ADSCs) could be obtained from the adipose tissue in elective liposuction of hypersound. The cells were identificated to be the ADSCs by flow cytometry.2. The ADSCs cultured in induced medium turned to have some characters of cartilage cells, and fabricated the cartilage in nude mice, suggested that the ADSCs were fit for the seed cells in tissue engineering.3. The HPMC and ADSCs were compatible, it wasn’t infected or discharged. It can be considered to be a suitable scaffold in tissue engineering.更多还原