负向调控基质金属蛋白酶-2的表达抑制成釉细胞瘤的侵袭性  

Negative regulation of matrix metalloproteinases-2 gene expression to inhibit the invasiveness of ameloblastoma cells

作　　者：曾东林[1] 黄洪章[1] 张磊涛[2] 

机构地区：[1]中山大学光华口腔医学院.中山大学口腔医学研究所,广州510055 [2]南方医科大学南方医院口腔颌面外科

出　　处：《中华口腔医学研究杂志（电子版）》2008年第1期19-22,共4页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：国家自然科学基金(30471896);广东省自然科学基金(04300340;06021272)

摘　　要：目的探讨基质金属蛋白酶-2(MMP-2)靶向小干扰RNA(siRNA)对成釉细胞瘤(AM)细胞MMP-2基因的负向调控作用及对AM侵袭性的抑制作用。方法培养成釉细胞瘤细胞,应用免疫荧光法检测成釉细胞瘤中MMP-2的表达;体外构建MMP-2靶向siRNA质粒表达载体,并转染成釉细胞瘤细胞,激光共聚焦显微镜检测siRNA质粒表达载体的转染效果,流式细胞仪检测转染效率,逆转录聚合酶链反应检测MMP-2mRNA的改变,免疫印迹法检测细胞MMP-2蛋白表达,侵袭小室微侵袭分析检测细胞的侵袭性,对统计结果进行方差分析。结果成釉细胞瘤中MMP-2呈阳性表达,siRNA质粒表达载体对成釉细胞瘤细胞的转染率为63.6%;转染后,成釉细胞瘤细胞的MMP-2mRNA表达减少69.3%,MMP-2蛋白表达减少64.2%,P<0.05,细胞的侵袭抑制率为61.2%。结论MMP-2靶向siRNA表达质粒成功转染成釉细胞瘤细胞并沉默MMP-2基因,MMP-2与细胞的侵袭性密切相关。Objective To study the inhibition of the invasiveness of ameloblastoma cells by negative regulation of MMP-2 gene using RNA interference, Methods Ameloblastoma cells were cultured in Defined Keratinocyte-SFM medium in vitro and MMP-2 was detected by immunofluorescence. Plasmid-vector for MMP-2 targeted siRNA was constructed and transfected into ameloblastoma cells. Laser confocal microscope was used to detect the positive transfected cells. Flowocytometer was used to analyze the transfection rate. Reverse transcription polymerase chain reaction（RT-PCR） analysis was performed to detect the expression of MMP-2 mRNA. Western-blot analysis was performed to investigate MMP-2 protein. Transwell analysis was done to explore the invasion of ameloblastoma cells. The analysis of variance （ANOVA） was used to analyze the data. Results Positive expression of MMP-2 was observed in ameloblastoma. The transfection rate of plasmid-vector for siRNA into ameloblastoma cells was 63.6%, After transfection with MMP-2 targeted siRNA, the expression of MMP-2 mRNA and MMP-2 protein decreased 69.3% and 64.2%, respectively, P 〈 0.05. The inhibition rate against the invasion of ameloblastoma cells was 61.2%. Conclusion MMP-2 targeted plasmidvector for siRNA is successfully transfected into ameloblastoma cells and MMP-2 gene is silenced by siRNA. MMP-2 has close relation with the invasiveness of ameloblastoma cells.

关 键 词：MMP-2 RNA干扰 小干扰RNA 基因沉默 侵袭 

分 类 号：R739.8[医药卫生—肿瘤]