不同类型人软骨细胞体外生物学特性比较

Comparison of the celluar properties of different human chondrocytes in vitro

目的 通过研究人耳软骨和肋软骨两种不同类型软骨细胞体外分离、增殖、老化规律 ,为选择合适的组织工程种子细胞提供依据。方法 取小耳畸形残耳软骨和肋软骨 ,体外分别用0 .0 5 %和 0 .15 %Ⅱ型胶原酶消化 16h分离 ,台盼蓝染色计活细胞数 ,得原代细胞获得率。体外单层培养 6代 ,观察形态学改变 ,群体倍增时间 (PDT ) ,免疫细胞化学染色及逆转录 聚合酶链反应(RT PCR)检测Ⅱ型胶原和Aggrecan评定软骨细胞老化规律。 结果 人残耳软骨组织平均细胞获得率为 ( 1.5 4± 0 .14 )× 10 6/ g ,肋软骨平均获得率为 ( 0 .46± 0 .0 9)× 10 6/ g ,两类软骨细胞P1的PDT最短 ,前 3代增殖力较强 ,P3 以后PDT明显延长 ,P6代细胞不再增殖。免疫细胞化学及RT PCR均证实耳软骨细胞 3代内、肋软骨细胞 4代内软骨细胞表型稳定。结论 第 2代的耳软骨细胞与第 3代肋软骨细胞可作为人体内组织工程化软骨构建的种子细胞。

Objective[WT5'BZ] To observe the proliferation and aging of chondrocytes isolated from human auricular remnant and rib cartilage,then to evaluate these two types of chondrocytes as a potential source of seeding chondrocytes in cartilage tissue engineering.Methods Human auricular remnant and rib cartilage specimens were obtained from patients of microtia for auricular reconstruction.Auricular and costal chondrocytes were isolated by digestion with 0.05% and 0.15% collagenase type Ⅱ for 16 h respectively.The methods of Trypan Blue staining and cell counting were applied to determine the original cell viability.The proliferation of chondrocytes cultured in vitro from P 0 to P 6 was calculated by the population double time (PDT) determination.The phenotype of chondrocyte was analyzed by the expression of collagen type Ⅱ and Aggrecan using the method of immunohistochemistry and RT-PCR.Results The average yield of chondrocytes from auricular and rib cartilage was (1.54±0.14)×10 6/g and (0.46±0.09)×10 6/g respectively.The PDT of P1 was shortest,and that of P 5 was longest.No chondrocyte proliferation could be detected in Passage 6.The expression of collage Ⅱ and Aggrecan could not be detected in auricular chondrocytes until passage 3,either in costal chondrocytes until passage 4 by the method of immunohistochemistry and RT-PCR.Conclusion The human auricular chondrocytes of P 2 and costal chondrocytes of P 3 can be used respectively as seeding cells in the engineered cartilage construction.