羧甲基壳聚糖对白细胞介素-1β刺激的软骨细胞增殖能力和表型的保护作用

Protective effects of carboxymethyl-chitosan on the proliferation and phenotype of interleukin-1β-in-duced chondrocytes

目的评价羧甲基壳聚糖对重组人白细胞介素-1β(rhIL-1β)刺激下的软骨细胞表型、细胞增殖能力的影响,并探讨其作用机制。方法体外培养兔关节软骨细胞,用rhIL-1β刺激,同时加入不同浓度的羧甲基壳聚糖,培养24h后,通过噻唑蓝(MTT)、流式细胞仪检测细胞增殖能力,以Na235SO4蛋白掺入试验检测蛋白多糖的合成,以Greiss反应测定上清液中一氧化氮(NO)浓度,通过反转录聚合酶链反应(RT-PCR)方法分析软骨细胞Ⅰ、Ⅱ型胶原,蛋白多糖聚糖体(Aggrecan)及诱导性一氧化氮合酶(iNOS)mRNA的表达。结果羧甲基壳聚糖能明显拮抗IL-1β对软骨细胞增殖的抑制作用,恢复软骨细胞合成蛋白多糖的能力,减少软骨细胞NO的合成,提高Ⅱ型胶原和蛋白多糖聚糖体mRNA的表达,抑制Ⅰ型胶原、iNOSmRNA的表达,且羧甲基壳聚糖对软骨细胞的保护作用呈剂量依赖性。结论羧甲基壳聚糖能维持IL-1β刺激下的软骨细胞的增殖能力和表型。

Objective To study the influence of carboxymethyl-chitosan （CM-chitosan） on phenotype and proliferation of chondrocytes stimulated by recombinant human interleukin-1β （rhIL-1β）, and explore its mechanism. Methods Chondrocytes were isolated and cultured. 10 ng/ml IL-1β with or without CM-chitosan of varied concentrations were added into the culture medium. After 24 h, changes of proliferative ability of chondrocytes were tested by MTT and flow cytometry. Proteoglycan synthesis was measured by incorporation of Na2^35SO4 into chondrocytes, nitric oxide （NO） production was detected by Greiss reaction, mRNA expression of type Ⅰ , Ⅱ collagens, Aggrecan and inducible NO synthase （iNOS） were examined by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）. Results CM-chitosan could significantly antagonize IL-1β-induced inhibitory effect on proliferation of chondrocytes, restore proteoglycan synthesis of chondrocytes, decrease NO production of IL-1β-induced chondrocytes, increase the mRNA expression of type Ⅱ collagen and Aggrecan, but decrease the expression of type I collagen and iNOS. The effects of CM-chitosan were in a dose-dependent manner. Conclusion CM-chitosan can maintain the proliferation and phenotype of IL-1β- induced chondrocytes.