松质骨源性人成骨细胞的分离培养和细胞密度效应

Isolation and culture of human osteoblast from trabecular bone and the cell density effect

目的:通过对人松质骨成骨细胞的培养,寻找人成骨细胞培养最佳途径中细胞密度的相关影响因素,以期掌握人成骨细胞扩增的规律。方法:实验于2005-03/12在江苏省血液病研究所(苏州大学附属第一医院血研所)血栓室完成。①以人工髋关节置换术中切下的股骨头和膝关节置换术中切下的股骨髁的松质骨作为成骨细胞来源,分离培养和鉴定。②以第1代成骨细胞分别按1∶2;1∶3;1∶4的比例传代培养,观察成骨细胞的生长周期。③计数培养后的细胞数,观察细胞密度不同对细胞生长的影响。结果:①细胞的生长及形态特征:原代细胞种植40h后,可见有少部分成骨细胞贴壁并伸出伪足,80h后,可见较多的细胞贴壁,以梭形或多角形多见。7～12d左右细胞形成单层,此时成骨细胞多为梭形,胞浆丰富,透明度高,有核及明显的核仁,细胞整体形态与成纤维细胞相似。传代培养后,细胞多为梭形或多角形,细胞核大,有多个突起,突起相互交叉。随着时间推移细胞逐渐连接成片。②成骨细胞的生长周期:人成骨细胞的生长周期约4d。③人成骨细胞不同比例传代培养生长情况:1×106个细胞分别按1∶2,1∶3和1∶4传代培养4d后,收集细胞总数分别为:(2.025±0.063)×106,(1.688±0.052)×106,(1.352±0.059)×106,统计学比较细胞数差异有显著性(P<0.001)。结论:同等数量细胞在密度不同时用相同的时间培养获得细胞总数并不相同,细胞的生长速度与细胞的密度相关,人成骨细胞培养以1∶2传代较好。以松质骨取材来源人成骨细胞培养方便和快捷,细胞扩增要达到最佳速度必须掌握好细胞传代的密度。

AIM： To search the factors influencing the concentration of cells cultured by the best way and find the characteristic of cell amplification of human osteoblast by culturing the osteoblast from human trabecubar bone. METHODS： The experiment was performed in the thrombus room of Hematonosis Research Institute of Jiangsu province （the Hematonosis Research Institute of the First affiliated Hospital of Suzhou University） from March to December 2005. （1）Human osteoblasts were isolated from trabecular bone of femoral heads taken during total hip arthroplasty and condyles of femur taken during total joint replacement for isolating culture and identification. （2） Osteoblasts were cultured according to the ratio of 1：2, 1：3, 1：4 from the first generation, and osteoblast growth cycle was observed. （3）The cell population was counted after culture and the effect of cell density on cell growth was observed. RESULTS： （1）Growth and morphologic character of cells： After the primary osteoblasts were planted for 40 hours, a few osteoblasts were observed to adhere the bottle wall and stretch out the spurius. After 80 hours, there were more adherence cells of fusiform or polygon. After 7-12 days, the cells formed monolayer and the major displayed fusiform with abundant kytoplasm and high clarity; there were nuclear and visible plasmosome in the cells. The appearance of osteoblast was similar to fibroblast. After serial subcultivation, the major cells displayed fusiform or polygon, nucleus was big and there were more ecphymas, which were overlapping. The osteoblasts gradually linked to lamellar as time. （2） Growth cycle of human osteoblast： It was about 4 days. （3）Growth of human osteoblast cultured with different ratios： 1 ×10^6 cells serial subcultivation were taken according to the radio of 1：2, 1：3 and 1：4 respectively for 4 days, the number of total cells collected was （2.025±0.063）×10^6; （1.688±0.052）×10^6 and （1.352±0.059）×10^. There were significant differences （P 〈 0.001 ）. CONCLUSION： Cell number obtained is different if cells are cultured in different density under the same condition. Cell growth velocity is related to cell density. It is better to culture the human osteoblast in the ratio of 1：2. It is convenient and shortcut to isolate and culture human osteoblast from trabecular bone. But cell density in serial subcultivation is very important to reach the best cell amplication velocity.