应用基因芯片技术研究硫酸氨基葡萄糖对骨性关节炎相关炎性基因表达谱的影响

Study on the different genes expression of chondrocyte in patients with OA treated by the glucosamine sulfate under inflammatory situation by the microarray

目的利用基因表达谱芯片技术对硫酸氨基葡萄糖作用于骨性关节炎关节软骨胶原酶和磷脂酶A2基因表达谱的改变进行研究,为硫酸氨基葡萄糖的作用机制提供基因学证据,并验证基因芯片技术检测胶原酶和磷脂酶A2基因表达差异的可靠性。方法收集9例软骨样本,其中正常膝关节软骨样本3例,6例来源于髋关节骨性关节炎关节置换软骨样本,体外分离培养组织样本。将每株细胞分为正常对照组、炎症对照组(炎症介质刺激)、药物干预组(硫酸氨基葡萄糖+炎症介质)。行基因芯片杂交,并进行芯片扫描和数据分析,分别对比正常对照组与炎症对照组、炎症对照组与药物干预组,筛查出细胞样本中与前列腺素E2代谢相关表达有差异的基因,并对胶原酶和磷脂酶A2基因筛查结果进行验证。结果细胞株样本的炎症对照组与药物干预对照组筛选出共同的差异表达基因143条,其中与前列腺素E2及磷脂酶A2、胶原酶代谢有关的差异表达基因PTGS1基因上调,CXCL13、TNFSF17、PTGS2、PTGES表达下调。通过酶联免疫吸附剂测定及反转录聚合酶链反应验证炎症介质能激活前列腺素E2的表达,进而促进软骨分解代谢中胶原酶和磷脂酶A2的异常表达趋势;而硫酸氨基葡萄糖能够抑制胶原酶和磷脂酶A2的表达,抑制前列腺素E2表达。结论炎症介质能激活前列腺素E2的表达,促进关节软骨炎症的发生及关节软骨细胞的分解代谢;硫酸氨基葡萄糖抑制磷脂酶A2和胶原酶活性,减少前列腺素E2生成的同时,能够调节前列腺素E2基因的表达,从而起到对抗炎症保护关节软骨作用。

Objective To compare and look for the different gene expression of chondrocyte in patients with OA treated by glucosamine sulfate and non-treated by glucosamine sulfate by Affymetrix microarray,supllying gene evidences for the action of glucosamine sulfate,verifying the difference of the genes expression conncened with the MMP13 and PLA2.Methods There were nine articular cartilages,three specimens were from normal cartilage of knee and six specimens came from THA surgery for OA.The condrocytes of cartilage were taken to culture and divided into three groups,including the normal group,inflammatory group(inflammatory medium)and drug intervention group(glucosamine sulfate+inflammatory medium).Hybridization was executed by Affymetrix microarray.The scanning of image and analysis of data were executed,comparing the data between normal group and inflammatory group,the inflammatory and the drug intervention group,verifying differential expression conncened with the PGE2 genes.At last the discrepant genes of hybridization conncened with the MMP13 and PLA2 genes were proven.Results The affymetrix microarray system found 143 differential expression genes between the inflammatory group and drug intervention group.Differential expression genes associated with MMP13 and PLA2:PTGS1 was upregulated,CXCL13、TNFSF17、PTGS2、PTGES were downregulated.It was verified that inflammatory medium increased the PGE2 expression,hence promoted the expression conncened with the degeneration of the cartilage by ELISA and RT-PCR.The glucosamine sulfate decreased the expression of MMP13、PLA2 and PGE2.Conclusion Inflammatory medium increased PGE2 expression,promoted the degeneration of the cartilage.glucosamine sulfate restrained the biological activity of MMP13 and PLA2 meantime decreased the expression of PGE2,protected the articular cartilage,prevented the degeneration of cartilage from inflammation.