【作者】 尹航；

【导师】 张锡庆；

【作者基本信息】 苏州大学 ， 外科学， 2007， 博士

【摘要】 一、骺板组织工程种子细胞——兔骺板软骨细胞体外培养及其细胞表型的研究目的探索体外培养兔骺板软骨细胞的方法,并对其细胞表型进行初步研究,为骺板组织工程寻找良好的种子细胞。方法取两周龄,体重250g左右幼兔胫骨上端骺板,经过机械剪切、胰酶、胶原酶的消化获得细胞后,1640培养基体外培养。应用倒置显微镜、透射电镜、Alcian blue染色、蕃红花“O”--亮绿染色,免疫组织化学染色、PCR、MTT等方法,对细胞进行定性、定量检测。结果在体外观察到了骺板软骨细胞贴壁生长及传代的生长过程,多次传代后增殖能力降低。PCR检测有II型胶原的表达、蕃红花“O”--亮绿染色阳性、Alcian blue染色显示第三代细胞蛋白聚糖含量高,MTT显示第三代细胞生长曲线呈倒“S”型,于第12天出现生长抑制。结论成功地建立了骺板软骨细胞的体外培养方法,并证实所培养出的细胞是骺板软骨细胞。二、组织工程骺板的构建——兔骺板软骨细胞分别与多孔聚乳酸、多孔丝素构建两种组织工程骺板并比较其体外相容性目的探索兔骺板软骨细胞与生物支架材料复合构建组织工程骺板的可行性。方法将体外培养的第三代兔骺板软骨细胞分别与多孔聚乳酸材料、多孔丝素材料复合,构建两种组织工程骺板。应用扫描电镜、激光共聚焦显微镜、MTT等方法,对细胞与材料的相容性进行观察,并对两种生物支架材料进行比较。结果兔骺板软骨细胞在支架材料上生长良好,随着培养时间的延长,分裂增殖的细胞逐渐增多。细胞在两种材料上生长情况类似,统计学显示无显著性差异。结论多孔聚乳酸材料和多孔丝素材料均具有良好的生物相容性,可以作为骺板组织工程理想的载体材料。三、组织工程骺板治疗骺板损伤的动物实验研究——两种组织工程骺板分别治疗骺板损伤并对其进行比较目的探索组织工程骺板在治疗骺板损伤中的作用。方法将两种组织工程骺板分别植入骺板损伤的模型中,同时设单纯植入材料组、空白对照组。应用组织学、BrdU细胞示踪、X线等方法,观察成角畸形和短缩畸形情况。同时对两种组织工程骺板、以及两种支架材料进行比较。结果空白组,骨桥明显,短缩畸形和成角畸形显著;单纯植入支架材料组,短缩畸形和成角畸形明显;组织工程骺板组,存在较小的短缩畸形和成角畸形。统计学显示三种方法存在显著性差异,两种组织工程骺板之间,两种支架材料之间无显著性差异。结论组织工程骺板治疗骺板损伤是一种有效的方法,其疗效优于单纯植入生物支架材料。四、人骨骺软骨细胞体外培养及鉴定的初步研究目的建立体外培养及鉴定人骨骺软骨细胞的方法。方法取人多指切除手术之多指骨骺,先行机械剪切,后用胰蛋白酶及II型胶原酶消化,再接种于1640培养基中体外培养。应用蕃红花“O”--亮绿染色,Alcian blue染色,免疫组化II型胶原染色和MTT检测,对其生物学特性进行了观察和鉴定。结果在体外观察到了人骨骺软骨细胞贴壁生长及传代的生长过程,多次传代后增殖能力降低。蕃红花“O”--亮绿染色阳性。Alcian blue染色阳性。免疫组化II型胶原染色阳性。MTT显示生长曲线呈倒“S”型,至第15天细胞出现生长抑制。结论成功地建立了人骨骺软骨细胞的体外培养方法,并证实所培养出的细胞是人骨骺软骨细胞。更多还原

【Abstract】 Part I: The seed cells of epiphyseal plate tissue engineering -- the methods of culture the epiphyseal plate chondrocyte in vitro and its cellular phenotype in rabbits.Objective To establish a kind of method about culture the epiphyseal plate chondrocyte in vitro, so that we can obtain the seed cells for the useage of epiphyseal plate tissue engineering.Methods The cells we used come from proximal epiphyseal plate of tibia of rabbits. Before we culture the cells, we used Trypsin and Collagenese II . Checking up biological characteristics of the cells by applying the ways of microscope and MTT test. Identifying the cells by applying the ways of safrarine“o”staining and PCR test.Results We have observed the conditions of the growth of the epiphyseal plate chondrocyte. The test of safrarine“o”staining shows positive. The test of PCR shows the expreesion of II collagen. The test of MTT shows the biological properties of the cells decrease from the 12th day.Conclusions We have established a method of culture the epiphyseal plate chondrocyte, at the same time we have identified that the cells were the epiphyseal plate chondrocyte.Part II: The construction of engineering epiphyseal plate ----the study on the biocompatibility of epiphyseal plate chondrocyte with scaffold PLA and epiphyseal plate chondrocyte with scaffold SF.Objective To explore the way of construction about engineering epiphyseal plate using epiphyseal plate chondrocyte and scaffold.Methods To construct two engineering epiphyseal plates by culturing the epiphyseal plate chondrocyte on scaffold PLA and scaffold SF. Checking up biological characteristics of the cells by applying the ways of scanning electron- microscope, laser scan confocal microscope and MTT test.Results The epiphyseal plate chondrocyte grew well on scaffold PLA and scaffold SF. The function of the tow scaffold is similar.Conclusions Both scaffold PLA and scaffold SF have good biocompatibility and can be used as ideal scaffold in tissue engineering.Part III: The animal experimental study on the application in epiphyseal plate injury of the two engineering epiphyseal plates.Objective To observe the role of engineering epiphyseal plate in the treatment of epiphyseal plate injury.Methods The two engineering epiphyseal plates were implanted into the model of epiphyseal plate injury. Evaluating the role by histology, BrdU and X-ray. At the same time, comparing engineering epiphyseal plate to scaffold .Results The degree of deformity in engineering epiphyseal plate group was less that scaffold group ,whereas the degree of deformity in scaffold group less than control group .The two engineering epiphyseal plate groups are similar , and the two scaffold groups are also similar.Conclusions It is feasible to treat epiphyseal plate injury by engineering epiphyseal plate, and it is better than by scaffold.PartⅣ: The methods of culture and identification of the human epiphyseal chondrocyte.Objective To establish a kind of method about culture and identification of the human epiphyseal chondrocyte.Methods The cells we used come from epiphysis of polydactyly. Before we culture the cells, we used Trypsin and Collagenase II . Checking up biological characteristics of the cells by applying the ways of MTT test. Identifying the cells by applying the ways of safrarine“o”staining, Alcian blue test and immuno- histochemistry staining for type II collagen.Results We have observed the conditions of the growth of the human epiphyseal chondrocyte. The test of safrarine“o”staining and Alcian blue shows positive. The test of immunohistochemistry staining for type II collagen shows the expression of II collagen. The test of MTT shows the biological properties of the cells decrease from the 15th day.Conclusions We have established a method of culture the human epiphyseal chondrocyte , at the same time we have identified that the cells were the human epiphyseal chondrocyte.更多还原