中国实验用小型猪成骨细胞的培养与鉴定  

Culture and identification of osteoblasts from Chinese experimental mini-pigs

作　　者：姚晖[1] 张林朴[1] 濮礼臣[2] 张洪杰[3] 王冠华[1] 

机构地区：[1]天津市口腔医院组织工程实验室,天津市300041 [2]天津市口腔医院颌面外科,天津市300041 [3]天津市整形外科医院,天津市300041

出　　处：《中国临床康复》2006年第45期43-46,I0005,共5页Chinese Journal of Clinical Rehabilitation

基　　金：天津市自然科学基金资助(003609011)~~

摘　　要：目的:分离、诱导培养中国实验用小型猪成骨细胞作为骨组织工程种子细胞,并进行形态学与生物学性能鉴定,为骨组织工程研究提供新型种子细胞。方法:实验于2002-08/2004-08在天津市口腔医院暨天津市整形外科医院中心实验室组织工程实验室完成。采用胶原酶消化法和诱导条件培养法自新生中国实验用小型猪四肢骨松质分离成骨细胞,经细胞生活形态观察、细胞倍增时间测定、透射电镜超微结构分析、裂解上清碱性磷酸酶检测、矿化结节形成测定等方法进行成骨细胞的生物学性能鉴定,以获取中国实验用小型猪成骨细胞作为骨组织工程种子细胞的必备的增殖活性与成骨活性数据。结果:①原代培养中国实验用小型猪成骨样细胞具有很强的贴壁率,细胞分裂旺盛,2～3d即可达到完全融合。细胞形态呈多角形和星形、胞体大、胞核圆,核仁丰富,通常为两三个核仁;胞浆中核周见散在黑色颗粒。②条件培养液诱导后的猪成骨样细胞培养6d左右可见细胞呈旋涡状复层生长,胞突伸展互相连接并集结成细胞簇团,中心区形成褐色结节;细胞核周围胞浆中出现大量黑色颗粒。③细胞增殖曲线显示猪成骨样细胞在播种3d后进入对数生长期,15d左右达到生长峰值U经细胞倍增时间公式计算猪成骨样细胞倍增时间为67.2h。④透射电镜观察显示:中国实验用小型猪成骨样细胞表面突起极丰富;核大,有较多和较深的内陷U核仁大而多窗孔,胞质内细胞器常常分布于核的一侧,扩张的粗面内质网池内有中等电子密度的蛋白质沉淀,其膜表面的核糖体密集分布。Golgi氏复合体发达,并见胞质内微丝及多量的游离核糖体。核仁特征为高转录活动的核仁。细胞外形、核谱型、核位置以及胞质内细胞器的特征性分布符合成骨细胞的超微结构特征。⑤猪成骨样细胞的碱性磷酸酶活性在β-磷酸甘�AIM： To separate, induce and culture osteoblasts from Chinese experimental mini-pigs for bone tissue engineering as seed cells, and to carry out performance evaluation of morphology and biology so as to provide new-type seed cell for bone tissue engineering research. METHODS： The experiment was performed in the Laboratory of Tissue Engineering, Central Laboratory, Tianjin Stomatological Hospital, which was Tianjin Orthopedics Hospital from August 2002 to August 2004. Osteoblasts were isolated from spongy bone of four limbs of newborn Chinese experimental mini-pigs with collagenase digestion method and induction and culture method. Biological performance of osteoblasts was evaluated by the observation of cell living formation, assessment of cell doubling time, uhrastructural analysis under transmission electron microscope, measurement of alkaline phosphatase in split supernatant, evaluation of mineralized tuberculation and so on. The data of necessary proliferative activity and osteoplastic activity of osteoblasts, as seed cells, from Chinese mimi-pigs were obtained for bone tissue engineering. RESULTS：① Primary cultured osteoblast-like cells from Chinese experimental mini-pigs had powerful adherence rate. The osteoblast-like cells with productive fission could coalesce completely from 2-3 days. These cells were polygon or starlike, big cell body, round nucleus, abundant nucleolus, usually two or three nucleoli; Black granules were scattered around nucleus in cytoplasm. ② After being induced with conditioned medium, osteoblast-like cells from pigs at about day 6 grew to multiple lays in spiring shape. Cytoplasmic process extended, connected each other, built up cell cluster; Brown node appeared in the center; A mass of black granules appeared around nucleus in cytoplasm.③Cell growth curve showed that osteoblast-like cells from pigs entered logarithmic phase at day 3 after seeding, reached peak growth value at about day 15. The doubling time of osteoblast-like cells from pigs was 67.2 hours calculated w

关 键 词：组织工程 猪 雏型 成骨细胞 细胞 培养的 生物学鉴定法 

分 类 号：R394.2[医药卫生—医学遗传学]