摘要
目的观察羧甲基壳聚糖（CMCS）对硝普钠（SNP）诱导的大鼠椎间盘髓核细胞增殖、诱导型一氧化氮合酶（iNOS）及基质金属蛋白酶-3（MMP-3）表达的影响。方法使用酶消化法提取大鼠椎间盘髓核细胞并进行体外培养，Ⅱ型胶原免疫组织化学鉴定髓核细胞。通过不同浓度（1、2、3mmol／L）SNP诱导髓核细胞，并在经SNP诱导的髓核细胞内加入不同浓度（100、200、500mg／L）CMCS进行处理，作用24h后通过细胞计数试剂盒（CCK一8）检测各组细胞增殖。通过荧光定量聚合酶链反应（Real．timePCR）及Westernblot法检测不同浓度（100、200、500mg／L）CMCS对SNP诱导髓核细胞内iNOS及MMP-3表达的影响。结果培养的细胞经Ⅱ型胶原免疫组织化学染色鉴定显示其表达阳性，表明为髓核细胞；通过CCK-8检测发现不同浓度SNP可诱导髓核细胞使其增殖活性降低，其中3mmol／LSNP使髓核细胞增殖降低62％（P〈0．05），通过100、200、500mg／LCMCS作用后髓核细胞增殖分别增加17％、35％和58％；Real—timePCR检测结果表明3mmol／LSNP可诱导髓核细胞iNOS及MMP-3mRNA表达增加，分别增加1．84倍和1．24倍，经100、200、500mg／LCMCS作用后其表达分别降低17％、61％、69％和12％、47％、51％（P〈0．05）；Westernblot检测结果表明3mmol／LSNP可诱导髓核细胞iNOS及MMP-3蛋白表达增加，经100、200、500mg／LCMCS作用后其表达有不同程度的降低。结论CMCS对SNP诱导的髓核细胞增殖抑制具有保护作用，并对SNP诱导髓核细胞内iNOS及MMP-3表达增高具有抑制作用，从而抑制髓核细胞发生退变。
Objective To observe the effect of carboxymethylated chitosan （CMCS） on prolifera- tion, expression of inducible nitric oxide synthase （ iNOS）, and matrix metalloproteinase-3 （MMP-3） in degenerative nucleus pulposus ceils of rats induced by sodium nitroprusside （SNP）. Methods Nucleus pulposus cells of rats were isolated by enzyme digestion and cultured in vitro. Different concentrations of SNP were used to induce the nucleus pulposus cells. 100, 200 and 500 mg/L CMCS were added into the SNP-treated nucleus pulposus cells. After treatment for 24 h, the cell counting kit-8 （CCK-8） assay was used to determine the proliferation of nucleus pulposus cells. The real-time polymerase chain reaction （PCR） and Western blotting were used to detect the expression of iNOS and MMO-3 mRNA and protein, respectively. Results The cells in this study were collagen-2 positive by the immunohistochemistry stai- ning. The CCK-8 assay results showed that different concentrations of SNP could inhibit the proliferation of nucleus pulposus cells, and the maximum response occurred at the 3 mmol/L SNP-treated group, 62% （P 〈0. 05）. After treatment with 100,200, and 500 mg/L CMCS, the proliferation was increased to some extent （ 17% , 35% and 58% respectively）. Real-time PCR revealed that 3 mmol/L SNP could induce the expression of iNOS and MMP-3 mRNA, 1.84 and 1.24 fold, respectively. After CMCS treatment the ex- pression was decreased to some extent. The Western blotting results indicated that 3 mmol/L SNP induced the expression of iNOS and MMP-3 protein. Conclusion CMCS at a certain concentration can protect the proliferation, and inhibit the increased expression of iNOS and MMP-3 effectively and reduce the degrada- tion of proteoglycans and collagen in nucleus pulposus cells induced by SNP in vitro.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第12期2545-2548,共4页
Chinese Journal of Experimental Surgery
关键词
羧甲基壳聚糖
髓核细胞
一氧化氮合酶
基质金属蛋白酶-3
Carboxymethylated chitosan
Nucleus pulposus cells
Nitric oxide synthase
Matrix metalloproteinase-3
