【作者】 张磊涛； 殷学民； 黄洪章；

【Author】 (1.Department of Stomotology,Nanfang Hospital Southern Medical University,1838 Guang-Zhou Road Eest, Guangzhou,Guangdong,510515,PR China.2.Department of Oral and Maxillofacial Surgery, Guanghua College of Stomatology,Sun Yat-senUniversity,Guangzhou,Guangdong,510055,PR China)

【机构】 南方医科大学南方医院口腔科； 中山大学光华口腔医学院口腔颌面外科；

【摘要】 目的:探讨基质金属蛋白酶组织抑制剂-2(TIMP-2)基因转染体外培养的人成釉细胞瘤(AM)细胞侵袭性生物学行为的改变,进一步研究MMP-2、TIMP-2与AM局部侵袭性的关系。方法:pcDNA3.1(+)/GFP-TIMP-2质粒转染人AM细胞。倒置相差荧光显微镜观察转染情况,流式细胞仪测定转染效率。明胶酶谱法检测基质蛋白酶-2(MMP-2)的活性。逆向明胶酶谱法检测TIMP-2的活性。RT-PCR分析MMP-2以及TIMP-2 mRNA的表达。Western-blot分析MMP-2及TIMP-2蛋白的表达情况。三维培养观察分析细胞生长状况。Transwell微侵袭分析。结果:与对照组比较,不同浓度质粒转染组的MMP-2均有不同程度的抑制,差异有显著性,P<0.05;72时MMP-2抑制率为54.38%。TIMP-2的活性均有不同程度的增加,差异有显著性,P<0.05;72时的TIMP-2增加率为43.75%。TIMP-2蛋白表达水平明显低于P1、P2组,经统计学分析表明,差异有显著性,P<0.05;与对照组比较,转染72h后,不同质粒组的MMP-2蛋白表达水平的抑制率分别为16.1%,45.3%,39.6%。不同浓度组的TIMP-2蛋白表达水平的增加率分别为26.1%,61.3%,32.6%。在Transwell中培养72h后,实验组与各对照组之间细胞的细胞计数无统计学差异,P>0.05;与对照组比较,质粒转染组Transwell下室细胞的细胞数明显减少,P<0.05。两组的相对侵袭抑制率分别为53.7%、46.9%。结论:TIMP-2基因转染AM细胞后,TIMP-2的mRNA及蛋白表达增加,蛋白活性亦增加;MMP-2mRNA表达量无明显改变,其蛋白表达量及活性降低。细胞的侵袭性被部分抑制,可能机制是TIMP-2基因过表达使MMP-2活性降低所致。TIMP-2、MMP-2以及二者的关系可能是影响AM局部侵袭性生长的原因之一。更多还原

【Abstract】 Objectives:To explore the invasive behaviors of ameloblastoma after TIMP-2 transfection Methods:Ameloblastoma cells were cultured in RMPI 1640 medium.pcDNA3.1(+)/GFP-TIMP-2 expression plasmid constructed in vitro was transfected into ameloblastoma cells.Fluorescence microscope was used to detect the result of transfection.Flow cytometry was used to check the transfection rate.Gelatinolytic zymography analysis was used to investigate the activity of MMP-2 in supernatant of culture medium. Reverse zymography analysis was used to investigate the activity of TIMP-2.Reverse transcription polymerase chain reaction(RT-PCR) analysis was performed to check the expression of MMP-2 and TIMP-2 mRNA.Western-blot analysis was performed to investigate the expression of MMP-2 and TIMP-2 proteins.The invasiveness of ameloblastoma cells was detected by three dimension culture system. Transwell analysis was done to measure the invasiveness of ameloblastoma cells.Cell adhesion rate on fibronectin was detected by cell adhesive analysis.The analysis of students t test was used to analyze the data.Results:MMP-2 and TIMP-2 are all expressed in cytolymph of ameloblastoma cells.pcDNA3.1(+)/GFP-TIMP-2 expression plasmid was successfully transfected into ameloblastoma cells. At 72h,the rate of transfection was 53.9%in group P3.MMP-2 mRNA expression was no significant different in every groups.P>0.05. TIMP-2 mRNA was respectively increased 20.7%,64.6%,53.8%in group P1,P2 and P3.MMP-2 protein expression was reduced to45.3%in group P2 at 72h after transfection.TIMP-2 protein expression was added to 61.3%in group P2 at the same time.The activity of TIMP-2 was increased 43.75%in group P2 at 72h after transfection,at the same time the activity of MMP-2 was decreased 54.38%.The inhibition rate of the invasiveness of ameloblastoma cells was 53.7%.The inhibition rate of adhesion of ameloblastoma cell was 47.4%.P<0.05.Conclusion:TIMP-2 gene was successfully transfected into ameloblastoma cells.After transfection TIMP-2 was over-expressed and the activity of MMP-2 was inhibited.The invasiveness of ameloblastoma cells in vitro was deduced after TIMP-2 gene transfection.TIMP-2 and MMP-2 may has close relation with the invasiveness of ameloblastoma cells in vitro.更多还原