损伤软骨的细胞提取、鉴定及生物学活性研究

Isolation, identification and biological activity of chodrocytes from injured cartilage

摘要:

目的对从膝关节软骨损伤区分离出的细胞进行鉴定，并对其生长活性进行研究，以解决自体软骨细胞移植中软骨细胞来源不足的问题。方法通过膝关节镜，在软骨损伤区取少量损伤软骨组织，不含正常软骨及软骨下骨为实验组；取正常软骨组织为对照组。在胰蛋白酶和Ⅱ型胶原酶作用下从实验组中提取细胞，对照组中提取正常软骨细胞。实验组细胞培养贴壁后在倒置相差显微镜下观察细胞的形态是否与对照组软骨细胞一致。实验组细胞行甲苯胺蓝染色鉴定，与对照组对比；噻唑蓝（MTT）法比较两组细胞生长活性；以具有专利知识产权的Ⅰ/Ⅱ型复合胶原膜为生长载体，培养实验组和对照组第3代软骨细胞，在电镜下观察两组生长活性。结果从少量损伤的软骨组织中提取的细胞为活性正常的软骨细胞，其特征及生长活性与对照组比较，差异无统计学意义（p <0.05）。结论软骨损伤过程中软骨细胞未发生变性，少量损伤的软骨组织仍可获得适量软骨细胞，MTT 法和电镜扫描表明，其生长活性符合自体软骨细胞移植的要求，无需取正常软骨组织作为细胞来源。

Abstract:

Objective To identify the cells isolated from injured cartilage tissue and study their growth activity, in order to provide adequate cells for autologous chondrocyte transplantation. Methods Through the knee arthroscope, a small amount of injured cartilage tissue was taken in the cartilage damage zone as experimental group, which did not include the normal cartilage or the subchondral bone. The normal cartilage was taken as control group. The cells were extracted from the experimental group, and the normal chondrocytes were extracted from the control group by trypsin and type Ⅱcollagenase. The cells of the experimental group were observed under inverted phase contrast microscope to dentify whether the cell morphology was consistent with the chondrocytes of the control group. The cells of the experimental group were identified with toluidine blue staining, and compared with the chondrocytes of the control group. Cell growth activity of the two groups was compared by MTT method. The third generation chondrocytes of the two groups were cultured on type Ⅰ/Ⅱcomposite collagen membrane with patent intellectual property, and their growth activity was observed under electron microscope. Results The cells extracted from injured cartilage tissue were normal chondrocytes, and their characteristics and growth activity were not different from those of the control group (p < 0.05). Conclusions In the process of cartilage injury, the cartilage cells do not degenerate. Appropriate amount of chondrocytes can be obtained from a small amount of injured cartilage tissue, it is proved that the growth activity is in accordance with the requirements of autologous chondrocyte transplantation by MTT method and electron microscopy. As a result, we do not need the normal cartilage tissue as a cell source.