CAJ | 학술논문

目的探讨中介素(IMD)对大鼠肾脏缺血再灌注损伤(IRI)的影响,及其过程中一氧化氮合酶(NOS)的作用和机制.方法将健康雄性Wistar大鼠分为4组:假手术组,行右肾切除术,1周后单纯分离左侧肾蒂及肾动脉,而不夹闭肾动脉；肾脏缺血再灌注(IR)组,行右肾切除术,1周后行左肾缺血再灌注手术；IMD基因转染组,右肾切除后左肾行超声微泡介导的IMD-pCDNA3.1(+)质粒转染术,饲养1周,再行左肾缺血再灌注手术；空质粒转染组,右肾切除后左肾行超声微泡介导的pCDNA3.1(+)质粒转染术,饲养1周,再行左肾缺血再灌注手术.大鼠于缺血再灌注术后24 h处死,用免疫组织化学方法检测肾组织IMD表达,取肾组织进行病理学观察,取血清测定尿素氮(BUN)和肌酐(Cr)浓度,检测肾组织中内皮型NOS(eNOS)、诱导型NOS(iNOS)以及神经型NOS(nNOS) mRNA及其蛋白的表达.结果假手术组大鼠肾组织中IMD位于肾小管及间质细胞胞浆内,其表达灰度值为66±35;肾脏IR组大鼠肾小管上皮细胞和间质中IMD表达灰度值为176±48,高于假手术组(P＜0.01)；IMD基因转染组肾组织中IMD表达灰度值为262±68,高于肾脏IR组(P＜0.01)；空质粒转染组IMD表达灰度值为180±51,和肾脏IR组间表达的差异无统计学意义(P＞0.05).与肾脏IR组相比较,IMD基因转染组大鼠肾脏组织病理损伤程度较轻,血清BUN和Cr较低(P＜0.05),eNOS mRNA及eNOS表达升高(P＜0.05),iNOS mRNA及iNOS表达降低(P＜0.05),而两组间nNOSmRNA及nNOS表达的差异无统计学意义(P＞0.05).结论中介素可能通过促进eNOS表达、抑制iNOS表达从而减轻大鼠肾脏IRI.
목적 탐토중개소(IMD)대대서신장결혈재관주손상(IRI)적영향,급기과정중일양화담합매(NOS)적작용화궤제.방법 장건강웅성Wistar대서분위4조:가수술조,행우신절제술,1주후단순분리좌측신체급신동맥,이불협폐신동맥；신장결혈재관주(IR)조,행우신절제술,1주후행좌신결혈재관주수술；IMD기인전염조,우신절제후좌신행초성미포개도적IMD-pCDNA3.1(+)질립전염술,사양1주,재행좌신결혈재관주수술；공질립전염조,우신절제후좌신행초성미포개도적pCDNA3.1(+)질립전염술,사양1주,재행좌신결혈재관주수술.대서우결혈재관주술후24 h처사,용면역조직화학방법검측신조직IMD표체,취신조직진행병이학관찰,취혈청측정뇨소담(BUN)화기항(Cr)농도,검측신조직중내피형NOS(eNOS)、유도형NOS(iNOS)이급신경형NOS(nNOS) mRNA급기단백적표체.결과 가수술조대서신조직중IMD위우신소관급간질세포포장내,기표체회도치위66±35;신장IR조대서신소관상피세포화간질중IMD표체회도치위176±48,고우가수술조(P＜0.01)；IMD기인전염조신조직중IMD표체회도치위262±68,고우신장IR조(P＜0.01)；공질립전염조IMD표체회도치위180±51,화신장IR조간표체적차이무통계학의의(P＞0.05).여신장IR조상비교,IMD기인전염조대서신장조직병리손상정도교경,혈청BUN화Cr교저(P＜0.05),eNOS mRNA급eNOS표체승고(P＜0.05),iNOS mRNA급iNOS표체강저(P＜0.05),이량조간nNOSmRNA급nNOS표체적차이무통계학의의(P＞0.05).결론 중개소가능통과촉진eNOS표체、억제iNOS표체종이감경대서신장IRI.
Objective To observe the effects of intermedin (IMD) on nitric oxide synthetase (NOS) in renal ischemia-reperfusion (IR) rat models and the action mechanism.Methods A total of 24 rats were divided into four groups (n =6 each).Group Ⅰ underwent right nephrectomy one week prior to the exposure of left renal pedicles,but did uot receive any I/R.Group Ⅱ underwent right nephrectomy one week prior to left renal I/R surgery.Group Ⅲ underwent right nephrectomy and left renal IMD-pCDNA3.1 ( + ) transfection by ultrasound-mircobubbles and renal I/R surgeries were performed one week after gene transfection.Group Ⅳ was treated with the same way as group Ⅲ except that empty control vector was transfected.All the animals were killed at the end of 24 h of reperfusion.The expression and site of IMD were determined by using immunohistochemistry.Serum levels of BUN and creatinine were determined.The kidney formaldehyde-fixed and paraffin-embedded sections were stained with HE and PAS by standard methods and then histological changes were analyzed semiquantitatively.The mRNA expression levels of endothelial NOS (eNOS),inducible NOS (iNOS) and neuronal NOS (nNOS) in the kidneys of the four groups were detected by using RT-PCR.The protein expression levels of the three NOS mentioned above in the kidneys were semiquantitatively analyzed by Western blotting.Results IMD was weakly expressed in the plasma of tubulointerstitial cells in sham-operated group; whereas IMD expression in the kidneys subject to I/R was increased.Moreover,as compared with I/R group,IMD expression levels were obviously increased (P＜0.01 ).The degree of morphological changes as well as renal dysfunction in group Ⅲ was obviously lessened as compared with group Ⅱ.The mRNA and protein expression levels of eNOS in group Ⅲ were notably increased as compared with group Ⅱ,while the mRNA and protein expression levels of iNOSin group Ⅲ were obviously reduced as compared with I/R group not transfeeted with IMD (P＜0.05).Meanwhile,there were no significant differences in the mRNA and protein expression levels of nNOS among groups Ⅱ,Ⅲ and Ⅳ.Conclusion IMD gene in the kidneys of rats can promote the expression of eNOS and attenuate over-expression of iNOS in the kidneys following I/R,thus protecting against tubulointerstitial damages and renal dysfunction in rat I/R models.