中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2013年
3期
162-167
,共6页
贾黎静%苏晓清%杨冬%李琛%殷洪山
賈黎靜%囌曉清%楊鼕%李琛%慇洪山
가려정%소효청%양동%리침%은홍산
糖尿病%外周血管疾病%内皮,血管%整合酶相互作用分子1
糖尿病%外週血管疾病%內皮,血管%整閤酶相互作用分子1
당뇨병%외주혈관질병%내피,혈관%정합매상호작용분자1
Diabetes mellitus%Peripheral vascular diseases%Endothelium,vascular%Integrase interactor 1
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.
目的 檢測整閤酶相互作用分子1(INI1)基因在糖尿病大鼠下肢缺血性疾病血管中的錶達,探討其對血管內皮細胞影響的分子機製.方法 以8週齡的雄性SD大鼠為研究對象,通過尾靜脈註射鏈脲佐菌素(STZ)的方法穫得16隻糖尿病大鼠模型,以完全隨機分組方法將大鼠分為2組(每組8隻):實驗組部分結扎雙側股外動脈製作下肢缺血模型,對照組僅進行糖尿病造模,不作其他處理.以實時熒光定量逆轉錄聚閤酶鏈反應(RT-PCR)和Western blotting技術檢測2組下肢動脈壁INI1的mRNA和蛋白錶達情況.閤成針對INI1的小榦擾RNA(siRNA-INI1),併轉染入人臍靜脈血管內皮細胞株HUVEC-12,對照組分彆為無關序列轉染及脂質體對照；應用噻唑藍(MTT)比色法、Transwell侵襲小室實驗、RT-PCR、Western blotting等技術檢測轉染INI1-siRNA前後HUVEC-12細胞遷移及血管生成能力的變化,進一步檢測遷移相關基因細胞間黏附分子1(ICAM-1)、基質金屬蛋白酶2(MMP-2)、基質金屬蛋白酶抑製物1(TIMP-1)和血管生成相關基因血管內皮生長因子(VEGF)、堿性成纖維細胞生長因子(bFGF)、血管緊張素2(Ang-2)的變化情況.以t檢驗與方差分析做組間數據對比.結果 糖尿病下肢缺血大鼠的動脈壁內INI1 mRNA和蛋白錶達均較對照組明顯增加(分彆為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)；轉染後與脂質體及無關序列對照組相比,INI1-siRNA組HUVEC-12細胞INI1 mRNA及蛋白錶達均顯著降低(INI1mRNA分彆為0.26±0.01、0.75 ±0.08、0.80 ±0.07,INI1蛋白分彆為0.11 ±0.02、0.79 ±0.12、0.82±0.14,F =36.49、24.17,均P＜0.01)；INI1-siRNA組遷移能力明顯增彊(分彆為66 ±5、35±3、37 ±5,F=19.39,P ＜0.01).與兩對照組相比INI1-siRNA組HUVEC-12中ICAM-1、MMP-2、VEGF、bFGF和Ang-2的mRNA和蛋白錶達明顯增彊(均P＜0.05),TIMP-1的mRNA和蛋白錶達顯著降低(F=36.48、237.91,P＜0.05).結論 INI1可抑製血管內皮細胞的遷移和血管生成能力,該效應可能是通過調控細胞中ICAM-1、MMP-2、TIMP-I、VEGF、bFGF和Ang-2的錶達實現的.
목적 검측정합매상호작용분자1(INI1)기인재당뇨병대서하지결혈성질병혈관중적표체,탐토기대혈관내피세포영향적분자궤제.방법 이8주령적웅성SD대서위연구대상,통과미정맥주사련뇨좌균소(STZ)적방법획득16지당뇨병대서모형,이완전수궤분조방법장대서분위2조(매조8지):실험조부분결찰쌍측고외동맥제작하지결혈모형,대조조부진행당뇨병조모,불작기타처리.이실시형광정량역전록취합매련반응(RT-PCR)화Western blotting기술검측2조하지동맥벽INI1적mRNA화단백표체정황.합성침대INI1적소간우RNA(siRNA-INI1),병전염입인제정맥혈관내피세포주HUVEC-12,대조조분별위무관서렬전염급지질체대조；응용새서람(MTT)비색법、Transwell침습소실실험、RT-PCR、Western blotting등기술검측전염INI1-siRNA전후HUVEC-12세포천이급혈관생성능력적변화,진일보검측천이상관기인세포간점부분자1(ICAM-1)、기질금속단백매2(MMP-2)、기질금속단백매억제물1(TIMP-1)화혈관생성상관기인혈관내피생장인자(VEGF)、감성성섬유세포생장인자(bFGF)、혈관긴장소2(Ang-2)적변화정황.이t검험여방차분석주조간수거대비.결과 당뇨병하지결혈대서적동맥벽내INI1 mRNA화단백표체균교대조조명현증가(분별위0.83±0.07、0.21±0.03화0.92±0.08、0.31 ±0.03,t=23.03、20.19,균P＜0.01)；전염후여지질체급무관서렬대조조상비,INI1-siRNA조HUVEC-12세포INI1 mRNA급단백표체균현저강저(INI1mRNA분별위0.26±0.01、0.75 ±0.08、0.80 ±0.07,INI1단백분별위0.11 ±0.02、0.79 ±0.12、0.82±0.14,F =36.49、24.17,균P＜0.01)；INI1-siRNA조천이능력명현증강(분별위66 ±5、35±3、37 ±5,F=19.39,P ＜0.01).여량대조조상비INI1-siRNA조HUVEC-12중ICAM-1、MMP-2、VEGF、bFGF화Ang-2적mRNA화단백표체명현증강(균P＜0.05),TIMP-1적mRNA화단백표체현저강저(F=36.48、237.91,P＜0.05).결론 INI1가억제혈관내피세포적천이화혈관생성능력,해효응가능시통과조공세포중ICAM-1、MMP-2、TIMP-I、VEGF、bFGF화Ang-2적표체실현적.
Objective 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.
