骺板软骨细胞的体外培养及鉴定  

In vitro culture and identification of chondrocytes from the epiphyseal plate

作　　者：尹航[1] 张锡庆[1] 王晓东[1] 张亚[1] 王科文[1] 

机构地区：[1]苏州大学附属儿童医院骨科,江苏省苏州市215003

出　　处：《中国组织工程研究与临床康复》2007年第6期1025-1027,J0002,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江苏省卫生厅135工程课题资助项目(38RC2002038)~~

摘　　要：目的:建立体外培养及鉴定骺板软骨细胞的方法。方法:实验于2005-03/2006-05在苏州大学附属儿童医院骨科实验室完成。选用3只生后14～28d的新西兰幼兔,空气栓塞处死,暴露股骨下端和胫骨上端,分别取2处的骺板组织,将其剪切成1～3mm3的小块,经胰蛋白酶消化,接种于含150g/L牛血清的1640培养基中,饱和湿度培养,传代。①细胞接种后3h在倒置显微镜下观察细胞生长情况,见细胞贴壁后每天观察2次。②第2代细胞达到80%～90%左右汇合时采用常规苏木精-伊红染色,光镜观察细胞爬片情况。③第3代细胞爬片至细胞达到80～90%左右汇合时,采用苏木素染色3min,3%亮绿染色5min,蕃红花“O”染色5min,光镜观察细胞产生蛋白聚糖情况。④采用PCR检测细胞Ⅱ型胶原的表达。⑤采用四唑盐MTT比色法检测细胞活性。结果:①骺软骨细胞刚接种后呈大小不等之圆形悬浮于培养液中,3h后见大部分细胞贴壁,24h后贴壁细胞呈短梭形、圆形、三角形和不规则形,见细胞分裂相。48h后见细胞伸展明显,细胞分裂相每高倍镜视野可见多个。经隔日换液细胞生长至第5天,细胞呈聚集生长,达到汇合状态,将细胞传代。接种后第1代细胞2d后呈梭形,培养4d传至第2代,第2代细胞长满瓶底后,90%呈胞膜较厚的圆形,10%为梭形,第3代、第4代亦如此。传至第5代见肥大细胞增多,细胞松散,折光性减弱,呈凋亡状态。②细胞爬片后观察骺软骨细胞形态以梭形居多,同时也有圆形、三角形和不规则形。可见细胞分裂及细胞中的分泌小泡。③见细胞呈红色,无绿色,证明蕃红花“O”-亮绿染色阳性,显示所培养的细胞可以分泌蛋白聚糖。④PCR检测术所养细胞含有Ⅱ型胶原,电泳带在440bp上。⑤四唑盐MTT比色法检测显示,第3代骺软骨细胞的生长曲线近似倒“S”形,在第4,5,6天细胞呈对数生长,约在7,8,9,10d达平台期,至第12天细胞出现�AIM： To establish a method for the culture and identification of the epiphyseal plate chondrocytes. METHODS： The experiment was carried out in the laboratory of Department of Orthopedics, Children Hospital affiliated to Suzhou University between March 2005 and May 2006. Three New Zealand rabbits born 14-28 days were selected and killed by aeroembolism to expose the distal part of femur and proximal part of tibia. The epiphyseal plate tissues ware harvested and cut into pieces of 1-3 mm^3, which were seeded into 1640 culture medium containing 150 g/L fetal bovine serum after digested with trypsin, and cultured at saturated humidity and passaged. （1）Three hours later, the growth condition of cells was observed under inverted microscope, and twice daily after the cells attached. （2）When the second passage cells fused to 80%-90%, the cells ware given HE staining to observe the growth condition on slides. （3） When the third passage cells grew to fusion of 80%-90%, the cells ware subjected to HE staining for 3 minutes, 3% bright green staining for 5 minutes, safrarine ＂O＂ staining for 5 minutes to observe the proteoglycans under light microscope. （4）The expression of collagen Ⅱ was detected by PCR. （5）Cell activity was detected by MTT colorimetric method. Checking up biological characteristics of the cells by applying the ways of microscope and MTT test. Identifying the cells by applying the ways of histology, and PCR test. RESULTS： （1）Round chondrocytes at different sizes were found in the culture solution after seeded; 3 hours later, most of the cells adhered and 24 hours later, the cells presented short fusiform, round, triangle and irregularity, and cell division phase was observed. Forty-eight hours later, cell stretch was found, and many cell division phases were observed in every high-fold field. On the 5^th day, the cells showed aggregation growth after changing the solution, and gradually fused and passaged. The first passage cells presented fusiform after 2 days culture

关 键 词：骨骺 软骨细胞 组织工程 体外发生 细胞培养 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学]