国际麻醉学与复苏杂志
國際痳醉學與複囌雜誌
국제마취학여복소잡지
INTERNATIONAL JOURNAL OF ANESTHESIOLOGY AND RESUSCITATION
2012年
1期
1-5,21
,共6页
过氧化物酶增殖物激活受体-γ1%脑缺血/再灌注%基因转染
過氧化物酶增殖物激活受體-γ1%腦缺血/再灌註%基因轉染
과양화물매증식물격활수체-γ1%뇌결혈/재관주%기인전염
PPAR-γ1%Cerebral ischemia/reperfusion injury%Gene transfection
目的 研究通过脑室内途径腺病毒载体转染过氧化物酶体增殖物激活受体-γ1(peroxisome proliferater-activated receptor-γ/1,PPAR-γ1)到脑的可行性,如果可行则进一步观察其对缺血/再灌注(ischemia/reperfusion,I/R)脑是否具有保护作用. 方法 选择90只成年雄性SD大鼠,按随机数字表法分成5组(每组18只).第1组脑室内注入生理盐水0.1 ml,第2组注入不含PPAR-γ1基因的空腺病毒载体0.1 ml,第3组注入表达PPAR-γ1的腺病毒载体(adenovirus vector encoding PPAR-γ1,Adv-PPAR-γ1)0.1 ml,第4组注入腺病毒介导的增强型绿色荧光蛋白(adv encoding enhanced green fluorescence protein,Adv-EGFP)0.1 ml,第5组吡格列酮5 mg/kg灌胃qd3d.3d后建立脑I/R模型.第1组为假手术组,不阻断大鼠大脑中动脉.第2、3、5组阻断大脑中动脉90 min再灌注24 h.采集脑组织标本,第4组免疫荧光显微镜下观察腺病毒表达情况；1、2、3、5组进行2,3,5三苯基氯化四氮唑(triphenyltetrazolium chloride,TTC)染色法测量脑梗死体积、伊文氏蓝法测定血脑屏障通透性、干-湿重法测定脑含水量、光镜、电镜下进行病理形态学观察；脑组织髓过氧化物酶(myeloperoxidase,MPO)活性测定；用Western Blot方法检测缺血区脑组织白介素-1β(IL-1β)、细胞黏附分子-1(inter-cellular adhesion mdecule-1,ICAM-1)、水通道蛋白-4(aquaporin protein,AQP-4)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白的表达. 结果 脑室内途径腺病毒载体转染Adv-EGFP,免疫荧光反应阳性.脑I/R后,脑梗死体积(42.3±2.6)％、血脑屏障通透性(0.094 5±0.009 5)、脑组织含水量(87.4±4.7)％、MPO活性明显增高(0.213±0.044),IL-1β(0.84±0.05)、ICAM-1(0.85±0.07)、AQP-4(0.84±0.06)和MMP-9(0.80±-0.03)蛋白的表达增加.脑室内注射Adv-PPAR-γ1或吡格列酮灌胃均能降低脑梗死体积[(35.7±2.5)％、(32.2±5.9)％]、血脑屏障通透性(0.077 4±0.008 8、0.073 7±0.006 4)、脑组织含水量[(82.9±4.7)％、(84.5±5.5)％]、MPO活性(0.151±0.018、0.166±0.026),抑制IL-13(0.71 ±0.04、0.75±0.05)、ICAM-1(0.69±0.03、0.79±0.07)、AQP-4(0.77±0.04、0.78±0.06)和MMP-9(0.76±0.06、0.77±0.05)的上调. 结论 通过脑室内注射腺病毒载体转染PPAR-γ1到脑的方法可行,且对I/R脑具有保护作用.
目的 研究通過腦室內途徑腺病毒載體轉染過氧化物酶體增殖物激活受體-γ1(peroxisome proliferater-activated receptor-γ/1,PPAR-γ1)到腦的可行性,如果可行則進一步觀察其對缺血/再灌註(ischemia/reperfusion,I/R)腦是否具有保護作用. 方法 選擇90隻成年雄性SD大鼠,按隨機數字錶法分成5組(每組18隻).第1組腦室內註入生理鹽水0.1 ml,第2組註入不含PPAR-γ1基因的空腺病毒載體0.1 ml,第3組註入錶達PPAR-γ1的腺病毒載體(adenovirus vector encoding PPAR-γ1,Adv-PPAR-γ1)0.1 ml,第4組註入腺病毒介導的增彊型綠色熒光蛋白(adv encoding enhanced green fluorescence protein,Adv-EGFP)0.1 ml,第5組吡格列酮5 mg/kg灌胃qd3d.3d後建立腦I/R模型.第1組為假手術組,不阻斷大鼠大腦中動脈.第2、3、5組阻斷大腦中動脈90 min再灌註24 h.採集腦組織標本,第4組免疫熒光顯微鏡下觀察腺病毒錶達情況；1、2、3、5組進行2,3,5三苯基氯化四氮唑(triphenyltetrazolium chloride,TTC)染色法測量腦梗死體積、伊文氏藍法測定血腦屏障通透性、榦-濕重法測定腦含水量、光鏡、電鏡下進行病理形態學觀察；腦組織髓過氧化物酶(myeloperoxidase,MPO)活性測定；用Western Blot方法檢測缺血區腦組織白介素-1β(IL-1β)、細胞黏附分子-1(inter-cellular adhesion mdecule-1,ICAM-1)、水通道蛋白-4(aquaporin protein,AQP-4)、基質金屬蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白的錶達. 結果 腦室內途徑腺病毒載體轉染Adv-EGFP,免疫熒光反應暘性.腦I/R後,腦梗死體積(42.3±2.6)％、血腦屏障通透性(0.094 5±0.009 5)、腦組織含水量(87.4±4.7)％、MPO活性明顯增高(0.213±0.044),IL-1β(0.84±0.05)、ICAM-1(0.85±0.07)、AQP-4(0.84±0.06)和MMP-9(0.80±-0.03)蛋白的錶達增加.腦室內註射Adv-PPAR-γ1或吡格列酮灌胃均能降低腦梗死體積[(35.7±2.5)％、(32.2±5.9)％]、血腦屏障通透性(0.077 4±0.008 8、0.073 7±0.006 4)、腦組織含水量[(82.9±4.7)％、(84.5±5.5)％]、MPO活性(0.151±0.018、0.166±0.026),抑製IL-13(0.71 ±0.04、0.75±0.05)、ICAM-1(0.69±0.03、0.79±0.07)、AQP-4(0.77±0.04、0.78±0.06)和MMP-9(0.76±0.06、0.77±0.05)的上調. 結論 通過腦室內註射腺病毒載體轉染PPAR-γ1到腦的方法可行,且對I/R腦具有保護作用.
목적 연구통과뇌실내도경선병독재체전염과양화물매체증식물격활수체-γ1(peroxisome proliferater-activated receptor-γ/1,PPAR-γ1)도뇌적가행성,여과가행칙진일보관찰기대결혈/재관주(ischemia/reperfusion,I/R)뇌시부구유보호작용. 방법 선택90지성년웅성SD대서,안수궤수자표법분성5조(매조18지).제1조뇌실내주입생리염수0.1 ml,제2조주입불함PPAR-γ1기인적공선병독재체0.1 ml,제3조주입표체PPAR-γ1적선병독재체(adenovirus vector encoding PPAR-γ1,Adv-PPAR-γ1)0.1 ml,제4조주입선병독개도적증강형록색형광단백(adv encoding enhanced green fluorescence protein,Adv-EGFP)0.1 ml,제5조필격렬동5 mg/kg관위qd3d.3d후건립뇌I/R모형.제1조위가수술조,불조단대서대뇌중동맥.제2、3、5조조단대뇌중동맥90 min재관주24 h.채집뇌조직표본,제4조면역형광현미경하관찰선병독표체정황；1、2、3、5조진행2,3,5삼분기록화사담서(triphenyltetrazolium chloride,TTC)염색법측량뇌경사체적、이문씨람법측정혈뇌병장통투성、간-습중법측정뇌함수량、광경、전경하진행병리형태학관찰；뇌조직수과양화물매(myeloperoxidase,MPO)활성측정；용Western Blot방법검측결혈구뇌조직백개소-1β(IL-1β)、세포점부분자-1(inter-cellular adhesion mdecule-1,ICAM-1)、수통도단백-4(aquaporin protein,AQP-4)、기질금속단백매-9(matrix metalloproteinase-9,MMP-9)단백적표체. 결과 뇌실내도경선병독재체전염Adv-EGFP,면역형광반응양성.뇌I/R후,뇌경사체적(42.3±2.6)％、혈뇌병장통투성(0.094 5±0.009 5)、뇌조직함수량(87.4±4.7)％、MPO활성명현증고(0.213±0.044),IL-1β(0.84±0.05)、ICAM-1(0.85±0.07)、AQP-4(0.84±0.06)화MMP-9(0.80±-0.03)단백적표체증가.뇌실내주사Adv-PPAR-γ1혹필격렬동관위균능강저뇌경사체적[(35.7±2.5)％、(32.2±5.9)％]、혈뇌병장통투성(0.077 4±0.008 8、0.073 7±0.006 4)、뇌조직함수량[(82.9±4.7)％、(84.5±5.5)％]、MPO활성(0.151±0.018、0.166±0.026),억제IL-13(0.71 ±0.04、0.75±0.05)、ICAM-1(0.69±0.03、0.79±0.07)、AQP-4(0.77±0.04、0.78±0.06)화MMP-9(0.76±0.06、0.77±0.05)적상조. 결론 통과뇌실내주사선병독재체전염PPAR-γ1도뇌적방법가행,차대I/R뇌구유보호작용.
Objective To explore the feasibility of PPAR-γ1 gene transfection via intracerebroventricular injection and possible protective effect against cerebral ischemia reperfusion injury.Methods Ninety adult male SD rats were randomly divided into 6 groups(n=18).In group Ⅰ,the rats were intracerebroventricularly injected with normal saline 0.1 ml.In group Ⅱ,the rats were injected with empty adenoviral(Adv)vector 0.1 ml.In group Ⅲ,the rats were injected with 0.1 ml Adv-PPAR-γ1 and the group Ⅳ received Adv-EGFP 0.1 ml.The group Ⅴ was intragastrically administered with Pioglitazone.After 3 days,middle cerebral artery occlusion model was established.The group I was sham-operation group.In group Ⅱ-Ⅴ middle cerebral artery was obstructed for 90 min followed by reperfusion for 24 h.Twenty-four hours after operation,samples were collected.The expression of green fluorescent protein in Group Ⅳ was observed with fluorescence microscope to assess the adenovirus-mediated transfection.The cerebral infarction volume was measured by triphenyltetrazolium chloride(TTC)staining.The permeability of blood-brain barrier was determined by Evan's blue.Brain water was calculated by wet-dry weight method.The histopathology was observed under light microscope and electron microscope.The activity of myeloperoxidase(MPO)was tested.The expressions of IL-1β,inter-cellular adhesion mdecule-1(ICAM-1),aquaporin protein(AQP-4)and matrix metalloproteinase-9(MMP-9)protein were determined by Western Blotting.Results After the animals were intracerebroventricularly administered Adv-EGFP plasmid,that the fluorescence for EGFP was positive in brain tissue suggested the successful transfection of viral gene.In response to I/R injury,the permeability of blood-brain barrier(0.094 5±0.009 5),brain water(87.4±4.7),and the activity of MPO were dramatically increased(0.213±0.044)as well as the cerebral infarction volume(42.3±2.6).The expressions of IL-1β(0.84±0.05),ICAM-1(0.85±0.07),AQP-4(0.84±0.06)and MMP-9(0.80±0.03)protein were up-regulated.Either intracerebroventricularly injection with Adv-PPAR-γ1 or intragastric administration with Pioglitazone could reduce the cerebral infarction volume(35.7±2.5,32.2±5.9),the permeability of blood-brain barrier(0.077 4±0.008 8,0.073 7±0.006 4),brain water(82.9±4.7,84.5±5.5),the activity of MPO(0.151±0.018,0.166±0.026)and the expression of IL-1β(0.71±0.04,0.75±0.05),ICAM-1(0.69±0.03,0.79±0.07),AQP-4(0.77±0.04,0.78±0.06)and MMP-9 (0.76±0.06,0.77±0.05)protein.Conclusions Transfection of via cerebral ventricle It was feasible to transfect the PPAR-γ1 gene into brain tissue via intracerebroventricular injection of the adenoviral vector and demonstrated the protective effect against cerebral ischemia reperfusion injury.
