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目的:比较研究不同代次间兔半月板纤维软骨细胞生物学特性的改变,为构建组织工程半月板和细胞治疗中的种子细胞优选提供进一步的理论和实践基础。方法:采用机械分离与酶连续消化相结合的方法体外分离兔半月板纤维软骨细胞,单层培养传代至第5代。采用相差倒置显微镜和SEM观察各代细胞的形态变化和超微结构,MTT法检测细胞增殖情况并描绘生长曲线,采用细胞化学染色和免疫组化鉴定细胞分泌的蛋白聚糖和Ⅰ、Ⅱ型胶原蛋白。结果:半月板纤维软骨细胞原代接种后8~12 h开始贴壁,48~72h大部分细胞贴壁,胞质逐渐展开,细胞变大伸长,形成突起,呈多角形,细胞形态规则,轮廓清晰,镜下观察有立体感,7~10 d后,细胞长满传代。传代后细胞贴壁生长能力和增殖速度明显加快,原代至第2代细胞形态较规则,多呈多角形,大小均一。第3代后随着传代次数的增加,细胞中梭形细胞增多,分泌和增殖能力下降,传代周期延长。第5代分裂相少见,密度稀疏,细胞形态不佳,约80%呈长梭形,分泌和增殖能力下降。SEM示第1代半月板细胞形态规则,呈多极性,表面有突起;第5代细胞形态不规则,多呈梭形。生长曲线可见第4代前半月板细胞生长速度及生长周期均相似,第5代后细胞生长速度减慢,增殖减缓。细胞化学和免疫组化染色分析胶原和蛋白聚糖的表达:随传代的进行,Ⅱ型胶原蛋白和蛋白聚糖的表达逐渐减弱而Ⅰ型胶原表达逐渐增强。结论:体外单层培养条件下,随着传代的进行,细胞活力逐渐下降,生长速度减慢,传代周期延长,逐渐变为梭形,形态不规则。体外单层培养系统中半月板纤维软骨细胞表型随传代的进行,Ⅱ型胶原蛋白和蛋白聚糖的表达逐渐减少而Ⅰ型胶原表达逐渐增多,体外单层培养条件下培养的半月板纤维软骨细胞从第3代开始逐渐失去其特异性表型,发生去分化,逐渐变为成纤维细胞的表型。体外单层分离培养的第5代前半月板细胞基本维持纤维软骨细胞的表型和生物学特性,可作为组织工程半月板的种子细胞。
OBJECTIVE: To comparatively study the changes of biological characteristics of rabbit meniscal fibrochondrocytes between different generations, and to provide further theoretical and practical basis for constructing tissue engineering meniscus and seed cell selection in cell therapy. Methods: Rabbit meniscal fibrochondrocytes were isolated in vitro by means of mechanical separation and enzymatic digestion. The monolayer culture was passaged to passage 5. Morphological changes and ultrastructure were observed by inverted phase contrast microscopy and SEM. Cell proliferation was measured by MTT assay and the growth curve was drawn. Cytochemical staining and immunohistochemistry were used to identify the expression of proteoglycans and type I and type II collagen protein. Results: The primary fibroblasts of the menisci began to adhere 8 to 12 hours after inoculation, and most of the cells adhered to the wall at 48 to 72 hours. The cytoplasm gradually expanded, the cells grew larger and formed protuberances with polygons, Clear outline, three-dimensional observation under the microscope, 7 ~ 10 d, cells covered with passage. After passage, the ability of adherent cells to grow and proliferate significantly faster. The morphology of primary passage to passage 2 cells was more regular and polygonal in shape with uniform size. After passage 3, the number of spindle cells increased, the ability of secretion and proliferation decreased, and the passage period prolonged with the increase of passage times. The fifth generation of split phase rare, sparse density, cell morphology is poor, about 80% spindle fusiform, secretion and proliferation decreased. SEM showed the first generation of meniscus cell morphology rules, was multipolar, the surface has protrusions; 5th generation cells irregular shape, mostly fusiform. The growth curve shows that the growth and growth cycles of plate cells in the first half of the fourth generation were similar. After the fifth passage, the cell growth rate slowed down and the proliferation slowed down. Cytochemistry and immunohistochemistry analysis of collagen and proteoglycan expression: With the passage of time, the expression of type Ⅱ collagen protein and proteoglycan gradually weakened and the type Ⅰ collagen expression gradually increased. CONCLUSION: Under monolayer culture conditions, the cell viability gradually decreases, the growth rate slows down, the passage cycle prolongs, and gradually becomes fusiform with irregular shape. In vitro monolayer culture system, the phenotypes of meniscus fibrochondrocytes with passaged, the expression of type Ⅱ collagen protein and proteoglycan decreased and the expression of type Ⅰ collagen gradually increased, the culture of meniscus fibrocartilage Cells from the third generation gradually lost its specific phenotype, the occurrence of dedifferentiation, and gradually become the phenotype of fibroblasts. In vitro monolayer culture of the fifth generation of pre-meniscus cells to maintain the basic phenotype and biological characteristics of fibrous chondrocytes, tissue engineering can be used as seed cells of the meniscus.