人骨关节炎软骨细胞的体外培养

Culture in Vitro of Human Osteoarthritis Chondrocytes

目的:对人骨关节炎软骨细胞的分离、消化和培养进行初步研究,并就其生物学活性同人正常软骨细胞进行比较和评价。方法:以含血清培养液配制的0.05%和0.2%Ⅱ型胶原酶顺序消化关节软骨分离细胞,检测细胞存活率;体外传代培养观察细胞形态、生长、增殖和Ⅱ型胶原、蛋白多糖聚集体等的变化,以及用流式细胞仪检测有或无IL-1β诱导的细胞凋亡和周期变化。结果:①消化后骨关节炎组原代细胞活力平均为82%,少于正常组的95%(P<0.01)。②MTT检测骨关节炎组细胞增殖低于正常组(P<0.01),Ⅱ型胶原免疫组化和甲苯胺蓝O异染反应均较弱,传至第4代软骨细胞生物学特性基本消失。③骨关节炎组细胞凋亡率为6.9%,而正常组为0.5%,IL-1β诱导后凋亡率增加了27.4%,正常组只有12.7%。结论:酶二步消化法具有较高细胞存活率、低污染率和操作简便等特点,分离培养的骨关节炎软骨细胞符合人患骨关节炎时软骨细胞退变的表现,能很好的为骨关节炎细胞水平的研究提供最佳实验对象。

Objective： To study the separation, digestion and cultivation of human osteoarthritis chondrocytes, to compare biological characteristics with human normal chondrocytes and to evaluate their biological activity. Methods： The articular cartilage was digested one by one with the 0.05 and 0.2% collagenase Ⅱ to separate chondrocytes in the culture medium including fetal bovine serum. The viability rate of the primary chondrocytes was detected. During the cultivation in vitro, the changes of the chondrocytes shape, growth and proliferation were observed; the changes of the collagen type Ⅱ and aggrecan were investigated and detected. Cell cycle, DNA ploidy and apoptosis of specimens in the presence or absence of IL-1β were evaluated by flow cytometry. Results： ① After the articular cartilage was completely dissolved by the enzymatic two-step digestion, the chondrocytes suspension were harvested. The viability rate of primary osteoarthritis chondrocytes was 82% on average, which was less than 95% of normal（P〈0.01）. ② The primary and passage osteoarthritis chondrocytes proliferated more slowly than the primary normal ones （P〈0.01）. Their immunohistochemical staining of collagen type Ⅱ and heterochromia to toluidine blue O was both weaker than the normal. The fourth generation osteoarthritis chondrocytes had lost their special biological activity. ③ The apoptotic rate of osteoarthritis chondrocytes was 6.9%, but that of normal was only 0.5%. After induced by IL-1β, the apoptotic rate of osteoarthritis chondrocytes increased to 27.4%, but that of normal increased to 12.7% only. Conclusion： The method of the enzymatic two-step digestion has advantages in the high cellular viability rate, low contamination and simple procedures. The osteoarthritis chondrocytes separated and cultured have biological characteristics of human osteoarthritis degenerated cartilage and can be regarded as the best experiment object for the research of osteoarthritis at the cell level.