人髂骨生长板软骨细胞的体外培养及鉴定

Culture and identification of chondrocytes from human iliac growth-plate in vitro

[目的]探讨人髂骨生长板软骨细胞体外培养的可行性并观察其生物学特征。[方法]对手术中获得的10例青少年特发性脊柱侧凸患者髂骨生长板软骨行体外分离、培养,观察细胞形态,MTT法测定细胞增殖,Ⅱ型胶原免疫组化染色,采用逆转录聚合酶链反应检测各代细胞Ⅱ型胶原mRNA的表达水平。[结果](1)体外培养的软骨细胞随着传代次数的增加,细胞形态由原代的多角形逐渐变为长梭形;(2)MTT比色法检测显示,第2代髂软骨细胞的生长曲线近似倒"S"形,在第4～8d细胞呈对数生长,在9～10d达平台期,至第11d开始出现生长抑制。(3)第2代细胞Ⅱ型胶原免疫组化呈强阳性。(4)Ⅱ型胶原mRNA表达水平从第2代后随着传代次数的增加逐渐下降。[结论]采用联合酶序贯消化法将软骨细胞悬液和软骨块共同体外培养髂软骨细胞简单、有效,P2代细胞很好的保持了软骨细胞的特性,可以作为种子细胞来源。

[ Objective] To establish a viable and convenient method for human iliac growth- plate chondrocyte culture in vitro and study the biological characters of the chondrocyte. [ Method] The chondrocytes from 10 human iliac growth -plate were cultured in monolayer and passaged to observe the changes of cell morphology. The growth kinetics was detected by using MTT colorimetric. Immunocytochemistry and RT - PCR were performed to investigate the expression of collagen II at the protein and mRNA level , respectively. [ Result] （ 1 ） The phenotype of chondrocyte was changed from original polygonal shape to fu- siform at 4^th passage. （2） MTT test showed the growth curve of the second passage cells presented inverted “S”, and cells were found logarithmic growth at 4^th -8^th day reached platform stage at 9^th -10^th day, and decreased at the 11^th day. （3） The collagen type II immunohistochemical staining was extensive positive in cells at P2. （4） RT - PCR confirmed that the mRNA level decreased with the cells passaged after P2. [ Conclusion] The modified method of the enzymatic two - step digestion is an effective and simple procedure. The second passage ceils maintain the phenotype of chondrocyte well, could be serving as seeding cells.