三步酶消化法高效分离兔原代关节软骨细胞及体外培养观察

Efficient isolation of chondrocytes from rabbit articular cartilage with three-step enzymatic digestion and observation of their biological characteristics during cultivation in vitro

目的 观察设计的三步酶消化法分离原代关节软骨细胞的效果,并对体外培养不同代次细胞的生物学活性进行评价。方法 三步酶消化法设计为以培养液配制的1g/L胰蛋白酶及1g/LEDTA混合液、1g/L透明质酸酶和2g/LⅠ型胶原酶顺序消化关节软骨分离细胞,检测细胞收获效率和存活率;体外传代培养观察细胞形态、生长及胞外基质中Ⅰ型和Ⅱ型胶原、蛋白多糖聚集体等的变化。结果 (1)关节软骨经三步酶消化基质逐步解离和降解,细胞得以完全释放和分离,每克软骨的细胞收获量平均为50 3×106 个细胞,细胞存活率平均为98. 8%。(2)原代和第一代细胞附壁生长呈三角形或多角形,生长融合时呈卵圆形,Ⅱ型胶原免疫组化和甲苯胺蓝异染反应均呈阳性,原代细胞外基质有高的硫酸糖胺多糖含量为(92±10)μg/cm2;第三代后细胞逐渐变为梭形,Ⅱ型胶原免疫组化为阴性,甲苯胺蓝异染反应明显减弱,第四代细胞外基质的硫酸糖胺多糖含量为(48±12)μg/cm2。结论 (1)三步酶消化法可使关节软骨基质完全消化降解,具有高细胞收获率、高细胞存活率和操作简便等特点。(2)原代和第一代软骨细胞具有良好的生物学活性,而第三代以后的细胞生物学活性低下。

Objective To observe the effect of isolating the chondrocytes from articular cartilage with the method of three-step enzymatic digestion, and the biological characteristics of the isolated chondrocytes during cultivation in vitro in order to evaluate their biological activity. Methods The method of three-step enzymatic digestion was designed that the articular cartilage was digested one by one with the 1g/L trypsin and 1g/L EDTA, 1g/L hyaluronidase and 2g/L collagenaseⅠ in the culture medium to isolate chondrocytes. The harvesting and viability rate of the primary chondrocytes were detected. During the passage cultivation in vitro, the changes of the chondrocytes shape and growth were observed, the changes of the collagen typeⅠ and Ⅱ and aggrecan in the extracellular matrix were investigated and detected. Results (1) The extracellular matix of articular cartilage was completely dissolved by the three-step enzymatic digestion, and the chondrocytes were completely isolated from the solid matrix. The number of the harvested chondrocytes from every gram of wet cartilage was 50.3×10~6 on average,and their viability rate was 98.8% on average. (2) The primary and first passage chondrocytes had triangle or multi-angle shape, and became elliptic shape at the growing confluence with the positive immunohistochemical stain of collagen type Ⅱ and the strong heterochromia to toluidine blue. The content of sulfate glycosaminoglycans(GAG) in the extracellular matrix of the primary passage cells was (92±10) μg/cm^2. The chondrocytes after the third passaging gradually became spindle shape with the negative stain of collagen typeⅡand the weak heterochromia to toluidine blue. The content of sulfate GAG of the fourth passage cells was (48±12) μg/cm^2. Conclusion (1) The method of three-step enzymatic digestion can make the extracellular matix of articular cartilage completely degraded , and has advantages in the high efficiency of harvesting primary chondrocytes, high cellular viability rate and simple manipulation. (2) The primary and first passage chondrocytes have fine biological activity, and the chondrocytes after the third passaging have lost their special biological activity.