摘要目的 检测整合酶相互作用分子1(INI1)基因在糖尿病大鼠下肢缺血性疾病血管中的表达,探讨其对血管内皮细胞影响的分子机制.方法 以8周龄的雄性SD大鼠为研究对象,通过尾静脉注射链脲佐菌素(STZ)的方法获得16只糖尿病大鼠模型,以完全随机分组方法将大鼠分为2组(每组8只):实验组部分结扎双侧股外动脉制作下肢缺血模型,对照组仅进行糖尿病造模,不作其他处理.以实时荧光定量逆转录聚合酶链反应(RT-PCR)和Western blotting技术检测2组下肢动脉壁INI1的mRNA和蛋白表达情况.合成针对INI1的小干扰RNA(siRNA-INI1),并转染入人脐静脉血管内皮细胞株HUVEC-12,对照组分别为无关序列转染及脂质体对照；应用噻唑蓝(MTT)比色法、Transwell侵袭小室实验、RT-PCR、Western blotting等技术检测转染INI1-siRNA前后HUVEC-12细胞迁移及血管生成能力的变化,进一步检测迁移相关基因细胞间黏附分子1(ICAM-1)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶抑制物1(TIMP-1)和血管生成相关基因血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、血管紧张素2(Ang-2)的变化情况.以t检验与方差分析做组间数据对比.结果 糖尿病下肢缺血大鼠的动脉壁内INI1 mRNA和蛋白表达均较对照组明显增加(分别为0.83±0.07、0.21±0.03和0.92±0.08、0.31 ±0.03,t=23.03、20.19,均P＜0.01)；转染后与脂质体及无关序列对照组相比,INI1-siRNA组HUVEC-12细胞INI1 mRNA及蛋白表达均显著降低(INI1mRNA分别为0.26±0.01、0.75 ±0.08、0.80 ±0.07,INI1蛋白分别为0.11 ±0.02、0.79 ±0.12、0.82±0.14,F =36.49、24.17,均P＜0.01)；INI1-siRNA组迁移能力明显增强(分别为66 ±5、35±3、37 ±5,F=19.39,P ＜0.01).与两对照组相比INI1-siRNA组HUVEC-12中ICAM-1、MMP-2、VEGF、bFGF和Ang-2的mRNA和蛋白表达明显增强(均P＜0.05),TIMP-1的mRNA和蛋白表达显著降低(F=36.48、237.91,P＜0.05).结论 INI1可抑制血管内皮细胞的迁移和血管生成能力,该效应可能是通过调控细胞中ICAM-1、MMP-2、TIMP-I、VEGF、bFGF和Ang-2的表达实现的.

abstractsObjective To study the expression of integrase interactor 1 (INI1) gene in the arterial wall of diabetic foot rats and its molecular mechanism in the blood vessel endothelial cells.Methods The 8-week old male Sprague-Dawley rats were used.The rats were injected with streptozotocin (STZ) through caudal vein to establish diabetic rat models.Sixteen diabetic rats were randomly assigned to 2 groups(8 rats in each group):operation group received bilateral ligation while control group received sham operation.The expression of INI1 mRNA and protein in the arterial wall cells were evaluated by using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting methods in the two groups.INI1 specific siRNA (INI1-siRNA) was synthesized and transfected into human umbilical blood vessel endothelial cell (HUVEC-12) while other 2 group of HUVEC-12 cells received siRNA transfection by non-related sequence and blank sequence.The HUVEC-12 cell migration,angiogenesis and the gene expression of intercellular adhesion molecule 1 (ICAM-1),metalloproteinase-2 (MMP-2),tissue inhibitor of metalloproteinase 1 (TIMP-1),vascular endothelial growth factor (VEGF),basic fibroblast growth factor (bFGF) and angiopoietin-2 (Ang-2) before and after transfection were evaluated and compared among the three HUVEC cell groups by t test and analysis of variance.Results The expression of INI1 mRNA and protein in the arterial wall of diabetic rats is much higher than those in the control group(mRNA:0.83 ± 0.07 vs 0.21 ± 0.03,t =23.03,P＜0.01 ;protein:0.92 ±0.08 vs 0.31 ±0.03,t =20.19,P ＜0.01).Compared with lipoplast control group and non-related sequence group,the expression of INI1 mRNA and protein in INI1-siRNA transfected group of HUVEC-12 cells was suppressed (mRNA:0.26 ± 0.01,0.75 ± 0.08,0.80 ± 0.07,respectively;F =36.49,P ＜ 0.01 ; protein:0.11 ± 0.02,0.79 ± 0.12,0.82 ± 0.14,respectively; F =24.17,P ＜0.01),while the cell migration capacity was improved after INI1-siRNA transfection(66 ±5,35 ±3,37 ±5,respectively; F =19.39,P ＜ 0.01).Compared with the control groups,the mRNA and protein expression of ICMA-1,MMP-2,VEGF,bFGF and Ang-2 were significantly increased while the expression of TIMP-1 was reduced in INI1-siRNA transfected group(all P ＜ 0.05).Conclusion INI1 could significantly suppress the angiogenesis and endothelial migration of the vascular endothelium,and these effects might be implemented through the regulation of ICMA-1,MMP-2,VEGF,bFGF,Ang-2 and TIMP-1.