摘要目的软骨细胞培养观察白介素-10(IL-10)对软细胞的直接保护作用.方法取Wistar乳鼠(7-10天)股骨髁关节软骨分离软骨细胞.子一代细胞用含10%胎牛血清(FBS)DMEM培养液在24孔培养板中培养.2-4天后换含0.1%FBS DMEM培养液,分别加白介素-1,100?μ/ml,IL-1 100?μ/ml+重组鼠白介素-10,20?ng/ml,rmIL-10 20?ng/ml,继续培养48小时,行扫描电镜观察以及免疫组化和原位杂交,分别观察软骨细胞的生长和形态、一氧化氮合酶2(NOS2)和基质金属蛋白酶3(MMP3)的表达.从软骨细胞培养开始到3周,倒置显微镜下观察细胞生长和形态.结果 IL-1刺激软骨细胞浆内颗粒形成,细胞培养2周后细胞死亡,存活细胞减少.IL-10直接对抗IL-1,细胞结构清晰,细胞培养2周后细胞死亡少见,存活细胞数多,并维持细胞持续生长.扫描电镜观察,IL-1刺激软骨细胞形成密集细胞突起,加IL-10的软骨细胞突起无变化.免疫组化和原位杂交显示IL-10抑制NOS2和MMP3的表达.结论 IL-10不仅抑制致炎细胞因子合成,而且直接对抗IL-1,对软骨细胞有直接保护作用.IL-10可望成为治疗0A的新的有效途径.

abstractsAbstract:Objective To assess whether interleukin-10 (IL-10) is chondroprotective in vitro. Methods Chondrocytes were isolated from femoral cartilage of rats (7-10 days) by digestion with collagenase Ⅱ. The first passage cells were grown in 24- well plates with DMEM, supplemented with 10% fetal bovine serum, for 2-4 days. The cells were then cultured in 0.1% fetal bovine serum DMEM medium, and given respectively interleukin-1 (IL-1) 100?μ/ml, IL-1 100?μ/ml+recombinant murine interleukin-10 (rmIL-10) 20?ng/ml, rmIL-10 20?ng/ml, and cultured for 48 hours. Scanning electron morphology and immunohistochemical study of nitric oxide synthase 2 and matric metalloproteinase 3 mRNA in situ hybridization were performed. Cell proliferation and morphology were observed under inverted microscope from the beginning of cell culture for three weeks. Results IL-1 stimulated granule production in the cytoplasma of chondrocytes, and the cells died in the second and third weeks of culture. IL-10 antagonized IL-1, protected the cells from death and maintained chondrocyte proliferation. Scanning electron morphology showed that IL-1 stimulated the formation of numerous microvilli on the cell surface, while thin and less numerous microvilli were found in cultures with IL-10. Immunohistochemical study and in situ hybridization showed that IL-10 inhibited NOS2 and MMP3 expression.Conclusion IL-10 not only inhibits the synthesis of inflammatory cytokines, but also directly protects chondrocytes by antagonizing IL-1.