胎儿软骨细胞体外培养过程中生长及代谢的变化

目的探讨体外培养的软骨细胞形态及Ⅰ、Ⅱ型胶原和聚集蛋白聚糖的表达变化,为组织工程软骨种子细胞选择合适的回植时机。方法取体外培养的第1-5代胎儿软骨细胞,观察细胞形态和细胞增殖率;用爱茜蓝(alcianblue)法检测软骨细胞聚集蛋白聚糖中糖胺聚糖含量;分别用免疫细胞化学和逆转录聚合酶链反应检测Ⅰ、Ⅱ型胶原及聚集蛋白聚糖在蛋白和mRNA水平的表达。结果体外培养的胎儿软骨细胞从第3代起逐渐向成纤维样细胞转换;各代软骨细胞糖胺聚糖的含量随传代次数的增加而逐渐降低,第3代后处于较低水平;第2代以前Ⅱ型胶原表达较强,Ⅰ型胶原表达较弱,之后随传代Ⅱ型胶原表达减弱,Ⅰ型胶原表达逐渐增强;而聚集蛋白聚糖在第3代以前表达较高,从第4代后明显下降。结论人胎儿软骨细胞体外培养过程中,第2代软骨细胞从形态和功能上最接近体内状况,可作为软骨组织工程的种子细胞。

Changes in growth and metabolism of human fetal chondrocytes cultured in vitro

Objective To investigate changes in the morphology and expression of collagen typeⅠand Ⅱ and core protein of aggrecan of chondrocytes during the in vitro culture, and to select the best time of chondrocytes transplant in the tissue engineering. Methods Human fetal chondrocytes cultured from different passages were harvested to observe the morphology and proliferation rate of cells. Alcian blue precipitation was utilized for quantification of glycosaminoglycans (GAG) in aggrecan. Immunocytochemistry and RT-PCR were performed to detect the expression of collagen type Ⅰand Ⅱ and core protein of aggrecan at the protein and mRNA level, respectively. Results Human fetal chondrocytes changed into the fibroblast-like cells at passage 3. The GAG content dropped with passages after passage 3. Before passage 3, the expression of collagen type Ⅱ was high, but the expression of collagen type Ⅰ was low. After passage 2, expression of collagen type I became higher, while that of and collagen type Ⅱ became lower. The expression of core protein of aggrecan decreased after passage 4. Conclusion Cultured human fetal chondrocytes of passage 2 are the most similar to those in vivo in respect of morphology and metabolism, and can be used as seed cells for cartilage tissue engineering.