体外培养半月板纤维软骨细胞增殖和胶原表达特性的研究

摘要

Objective　To investigate the proliferation of porcine meniscus fibrochondrocytes and the expression of collagen under the condition of monolayer culture to fabricate autologous tissue-engineered menisci.　Methods　Meniscus fibrochondrocytes were obtained from the menisci of 8 Changfeng crossbred pigs with modified Klagsbrun's method and cultured in vitro. Morphological observation, population double time (PDT) and tendency graph of proliferative speed in culture were applied to observe the proliferative activity and phenotype of the cells. And in situ hybridization and immunocytochemistry were selected to determine the phenotype and collagen expression of the cells. 　Results　The average rate of the meniscus cells was (2.31 ± 0.66) ×106/g. The PDT of the original cells to the 6th-generation cells was 2 to 11 days, and the collagen expression was stable. 　 Conclusions　 Meniscus fibrochondrocytes express both types Ⅰ and Ⅱ collagen. The 3rd-generation and 6th-generation autologous fibrochondrocytes can repair meniscus defects and regenerate menisci by tissue engineering approach, respectively.%目的　研究体外单层培养条件下猪半月板纤维软骨细胞增殖和胶原的表达，为构建自体组织工程化半月板奠定基础。　方法　采用改良的Klagsbrun法，从8只45 d龄长枫杂交仔猪的半月板消化获取细胞进行体外单层培养。形态学观察、群体倍增时间(population double time, PDT)、增殖速度趋势图、原位杂交及免疫细胞化学染色法作为评定指标。　结果　半月板组织的平均细胞获得率为(2.31±0.66)×106/g;0～6代细胞的PDT维持在2～11 d之间，且胶原表达稳定。　结论　半月板纤维软骨细胞同时表达Ⅰ、Ⅱ型胶原;第3代和第6代自体纤维软骨细胞的数量和质量可望分别满足组织工程化半月板修复缺损和再造的需求。