成骨细胞复合TCP异体移植成骨情况及免疫学检测

发布时间：2018-01-13 22:20

本文关键词：成骨细胞复合TCP异体移植成骨情况及免疫学检测　出处：《山东大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:研究不同来源成骨细胞体外培养的形态特征和生物学活性,探讨成骨细胞分离培养的最佳方法及作为骨组织工程种子细胞的可行性和应用价值,为骨组织工程的研究提供稳定可靠的种子细胞来源。方法:无菌条件下取新生新西兰兔胫骨骨膜和骨髓,PBS冲洗3次,0.25%胰蛋白酶消化20分钟,以彻底去除残留在组织块上的血污,再将骨膜剪成大小约为1×1mm的组织块,将骨膜组织块放入25ml培养瓶中,加入含10%小牛血清的高糖DMEM培养液2ml,细胞长满瓶底后进行传代培养。注射器肝素化后抽取完全DMEM培养液冲洗骨髓腔,将混合DMEM培养液加入到无菌离心管内,1000转/分钟离心,去除最上面的脂肪层,以1×10~6/cm~2密度接种于100ml培养瓶中进行原代细胞培养。3天后全量换液,细胞汇合成单层后用0.25%胰蛋白酶消化细胞进行传代培养。细胞传代后改用含地塞米松(1×10(-8)mol/l)、β-甘油磷酸钠(10mmol/l)、维生素C(50mg/ml)的条件培养液培养。取第5代生长旺盛的成骨细胞,胰蛋白酶消化后,以10~7/ml的浓度封入冻存管进行超低温冻存,一个月后复苏,以10~6/ml的浓度接种在含有5mlDMEM培养液的培养瓶中培养。每种方法培养的细胞每日在相差显微镜下观察细胞的形态,通过Gomori钙钴法进行碱性磷酸酶染色,von Kossa法进行钙结节染色,观察细胞体外基质分泌和骨化过程。结果:骨膜培养第3天可见少量的梭形细胞从组织块周围游出,随培养时间延长细胞数量逐渐增多,围绕组织块周围呈放射状生长,10天时细胞融合成单层。传代后的细胞呈梭形、多角形,带有粗大的突起,4天时
[Abstract]:Objective: to study the morphological characteristics and biological activity of osteoblasts from different sources in vitro, and to explore the best method for isolation and culture of osteoblasts and the feasibility and application value of osteoblasts as seed cells for bone tissue engineering. To provide a stable and reliable source of seed cells for the study of bone tissue engineering. Methods: the tibia periosteum and bone marrow of newborn New Zealand rabbits were washed with 0.25% trypsin for 20 minutes after washing under aseptic condition. Then the periosteum was cut into a tissue about 1 脳 1mm in size, and the periosteum tissue mass was put into 25ml culture bottle, and the high sugar DMEM culture medium containing 10% calf serum was added to 2ml. After heparinization of the syringe, the complete DMEM culture fluid was extracted to wash the medullary cavity, and the mixed DMEM culture medium was added into the aseptic centrifuge tube to centrifuge for 1000 rpm / min. The top adipose layer was removed and the primary cells were incubated in 100ml culture flask at a density of 1 脳 10 ~ (6) / cm ~ (-2) for 3 days. After synthesis of monolayer, the cells were subcultured with 0.25% trypsin. The cells were transferred to the cells containing dexamethasone (1 脳 10 ~ (-8) mol / L). 尾 -glycerophosphate (10 mmol 路L ~ (-1)), vitamin C ~ (50 mg / ml) was cultured in the conditioned medium. The osteoblasts of the 5th generation were harvested and digested by trypsin. The cryopreservation was carried out with a concentration of 10 / 7 / ml and was resuscitated one month later. The cells were cultured in a culture flask containing 5 ml DMEM at a concentration of 106% ml. The cells cultured in each method were observed under phase contrast microscope every day. Calcium nodules were stained by alkaline phosphatase (ALP) staining von Kossa method with Gomori calcium cobalt method to observe the process of extracorporeal matrix secretion and ossification. Results: on the 3rd day of periosteum culture, a small number of fusiform cells could be seen swimming out from around the tissue mass, and the number of cells gradually increased with the extension of culture time, and the cells grew radially around the tissue mass. The cells were fused into monolayers at 10 days. After passage, the cells were fusiform, polygonal, with a thick protuberance for 4 days.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329;R392

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