软骨组织工程软骨细胞分离方式的优化

摘要

目的：探讨不同软骨分离方法获得软骨细胞的细胞存活率及细胞数量，优化软骨细胞获取方法。方法采用 SD 大鼠20只，获取关节软骨，机械剪碎后称重，每组使用相同重量的软骨组织（1 g），4组采用不同方法获取软骨细胞：实验1组：0．25％胰蛋白酶消化3 h，0．2％Ⅱ型胶原酶消化10 h。实验2组：4℃下0．25％胰蛋白酶消化12 h，0．25％胰蛋白酶消化72 h。实验3组：0．2％Ⅱ型胶原酶消化16 h。实验4组：0．08％Ⅱ型胶原酶消化12 h。加入原代细胞培养液进行体外培养增殖。使用化学特殊染色法鉴定获取细胞是否为软骨细胞。计算每组软骨细胞存活率及细胞数。结果经过4种方法消化细胞，所获得细胞在外形上无明显差异，细胞均呈四方形，甲苯胺蓝染色鉴定所获取细胞为软骨细胞。细胞存活率：实验1组：原代（P0）为（79．6±2．1）％，一代（P1）为（78．6±8．4）％，三代（P3）为（82．7±2．1）％。实验2组：P0为（81．0±6．9）％，P1为（80．7±3．5）％，P3为（85．0±3．0）％。实验3组：P0为（84．7±4．0）％，P1为（85．3±1．2）％，P3为（89．7±2．5）％。实验4组：P0为（82．0±2．6）％，P1为（79．0±7．8）％，P3为（86．3±2．5）％，实验3组的细胞存活率明显高于其他3组，差异具有统计学意义（P ＜0．05）。活细胞数：实验1组（364776±81743）个，实验2组（306111±49768）个，实验3组（472580±42644）个，实验4组（361906±78405）个，实验3组的活细胞数明显高于其他3组，差异具有统计学意义（P 均＜0．05）。结论4种实验方法均能获取一定数量的软骨细胞，0．2％Ⅱ型胶原酶消化16 h 的方法在细胞存活率及细胞数量均高于其他方法，适合用于软骨组织工程中软骨细胞的获取。%Oojective To investigate the optimized chondrocyte isolation method.Methods The chondro-cytes were obtained from the same quality of rat knee jointscartilage and cut into pieces,then adopted the differet enzyme digestion method from reference.Toluidine bluestaining were used to identify the articular cartilage and thenassess cell viability and the number of cells in each group.Results Articular cartilage were successfully isolated from different digestion methods.No significant differences in cell morphology which represented a square-like appearance.Four groups were expressed in cartilage-specific staining.Cell viability were P0 (79.6±2.1)%,P1(78.6±8.4)%,P3(82.7±2.1)% in group 1;P0(81.0±6.9)%,P1 (80.7±3.5)%,P3(85.0±3.0)% in group 2;P0(84.7 ±4.0)%,P1 (85.3 ± 1.2)%,P3(89.7 ±2.5)% in group 3;P0(82.0±2.6)%,P1(79.0±7.8)%,P3(86.3±2.5)% (P <0.05)in group 4.Number of viable cells were:(364 776±81 743)in group 1,(306 111±49 768)in group 2,(472 580±42 644)in group 3, (361 906±78 405)(P <0.05)in group 4,respectively.The third group were the highest of cell viability and cell number and which had statistically significant in all groups.Conclusion Though all groups can harvest chondrocytes,group 3 were higher than other groups in cell survival rate and cell number,so it is suitable for use in cartilage tissue engineering.