反义单核细胞趋化蛋白1对大鼠系膜细胞增生的影响  

Influence of antisense MCP-1 on MCP-1 production and mesangial cell proliferation

作　　者：张新萍[1] 易著文[1] 何小解[1] 何庆南[1] 朱敏[2] 周钢[2] 

机构地区：[1]中南大学湘雅二医院小儿肾脏病研究室, 长沙410011 [2]中南大学分子生物学研究中心

出　　处：《中华肾脏病杂志》2007年第2期95-100,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（39970776）;教育部高等学校博士学科点科研基金（20040533043）;湖南省“十五”重点学科建设项目基金

摘　　要：目的研究反义单核细胞趋化蛋白1（MCP-1）对系膜细胞MCP—1分泌及增生的影响。方法用逆转录病毒感染体外培养的系膜细胞，得到转染反义MCP—1的系膜细胞，经PCR及Southern印迹鉴定外源基因在系膜细胞基因组DNA上的整合。脂多糖（LPS）刺激反义MCP-1系膜细胞，以正常系膜细胞及转染空白载体的系膜细胞为对照，观察其增生情况。用RT-PCR法检测MCP-1、CC趋化因子受体2（CCR2）的mRNA表达。ELISA法检测细胞上清中MCP-1蛋白的分泌。结果经逆转录病毒转染得到的反义MCP-1的系膜细胞，其基因组DNA中有外源基因的整合。在正常的培养条件下，反义组与对照组系膜细胞的增生差异无统计学意义；MCP-1、CCR2的mRNA均有少量表达；MCP-1的蛋白也有微弱表达。在LPS的刺激下，反义组与对照组MCP-1的mRNA的表达均增高；MCP-1蛋白的分泌均增多。与对照组比较，反义组系膜细胞的增生受抑制[（16．83±1．16）×10^4／ml比（19．63±1．85）×10^4／ml]，MCP—1的mRNA的表达较少（0．424比0．866，P〈0．01），MCP—1蛋白的分泌减少。CCR2的mRNA表达与MCP-1的变化一致。结论（1）逆转录病毒载体pLXSN可有效地介导MCP-1 cDNA在系膜细胞的转染。（2）反义MCP-1可降低系膜细胞MCP—1的转录和翻译。（3）反义MCP—1的转染可降低系膜细胞在炎症状态时的增生。（4）大鼠系膜细胞具有CCR2的表达，在炎症状态时形成MCP-1、CCR2的自分泌环路。Objective To examine the influence of antisense MCP-1 on MCP-1 production and mesangial cell proliferation. Methods The rat glomerular mesangial ceils were infected with retroviral supematants of pLXSN-A-MCP-1 containing antisense MCP-1 and verified by Sorthem blot analysis and PCR. Lipopolysaccharide（LPS） stimulated the infected mesangial cells. Cell growth was measured by cell counting method. MCP-1 expression was detected by RT-PCR and enzyme-linked immunosorbent assay （ELISA）. Results Southern blot analysis and PCR demonstrated that antisense MCP-1 cDNA-infected mesangial ceils were established successfully. Treated with LPS, the growth rate of infected cells was significantly lower than that of normal ceils. The level of MCP-1, CCR2 mRNA formation and the level of MCP-1 protein secretion decreased compared with those of normal ceils. Conclusions pLXSN can transfer MCP-1 cDNA into mesangial cells. Antisense MCP-1 decreases MCP-1 transcription and translation. Antisense MCP-1 decreases the proliferation of mesangial cells in inflammation. CCR2 also expresses in mesangial cells. MCP-1 is a autocrine chemokine.

关 键 词：单核细胞化学吸引蛋白质1 细胞增殖 趋化因子 CC 系膜细胞 

分 类 号：R686[医药卫生—骨科学]