CAJ | 학술논문

目的观察上调肾脏intermedin( IMD)表达对大鼠肾脏缺血再灌注(I/R)的影响.方法 24只健康雄性Wistar大鼠随机分为假手术组、肾脏I/R组、IMD+I/R组、空质粒+I/R组,每组6只.所有动物于I/R术24h后杀检,取肾组织进行光镜检查,留取血清测定尿素氮(BUN)和血清肌酐(Scr)的浓度.免疫组织化学方法、半定量RT-PCR、Western印迹检测肾组织IMD表达及部位.Western印迹测定内皮素1(ET-1)、肿瘤坏死因子α(TNF-α)蛋白的表达.结果 HE、PAS染色结果显示,I/R组肾小管及间质病理损伤显著重于假手术组,IMD+I/R组小管间质损伤程度较肾脏I/R模型组及空质粒+I/R模型组明显减轻(1.5±0.8比7.6±2.3和7.0±1.8,均P＜0.05].与假手术组[(BUN 3.85±0.21 mmol/L,Scr(48.67±3.61) μmol/L相比,I/R组、IMD+I/R组以及空质粒+I/R组BUN(10.13±2.14) mmol/L,( 7.73±1.03) mmol/L,( 9.77±1.92) mmol/L和Scr(80.33±7.15) μmol/L,(58.50±:3.27)μmol/L,(75.67±7.58) μmol/L均明显升高(均P＜ 0.05),其中IMD+I/R组较I/R组以及空质粒+I/R组BUN和Scr水平显著降低(均P＜ 0.05).免疫组化结果显示,假手术组IMD呈弱阳性表达,主要位于肾小管间质细胞胞质内,I/R组肾组织IMD在肾小管上皮细胞和间质表达较假手术组上调；IMD+I/R组肾组织中IMD表达较I/R组明显上调(P＜0.01).与I/R组及空质粒+I/R组相比IMD+I/R组肾组织IMD mRNA,相对含量分别增加了60.7％、66.1％,蛋白相对含量分别增加了51.4％、55.9％.此外,与I/R组相比,IMD+I/R组肾组织ET-1、TNF-α蛋白表达显著降低(ET-1:0.17±0.02比0.08±0.02；TNF-α:0.35±0.02比0.21±0.04,均P＜0.05).结论在大鼠肾脏I/R前上调IMD表达可能通过抑制ET-1、TNF-α表达保护肾脏结构及功能.
목적 관찰상조신장intermedin( IMD)표체대대서신장결혈재관주(I/R)적영향.방법 24지건강웅성Wistar대서수궤분위가수술조、신장I/R조、IMD+I/R조、공질립+I/R조,매조6지.소유동물우I/R술24h후살검,취신조직진행광경검사,류취혈청측정뇨소담(BUN)화혈청기항(Scr)적농도.면역조직화학방법、반정량RT-PCR、Western인적검측신조직IMD표체급부위.Western인적측정내피소1(ET-1)、종류배사인자α(TNF-α)단백적표체.결과 HE、PAS염색결과현시,I/R조신소관급간질병리손상현저중우가수술조,IMD+I/R조소관간질손상정도교신장I/R모형조급공질립+I/R모형조명현감경(1.5±0.8비7.6±2.3화7.0±1.8,균P＜0.05].여가수술조[(BUN 3.85±0.21 mmol/L,Scr(48.67±3.61) μmol/L상비,I/R조、IMD+I/R조이급공질립+I/R조BUN(10.13±2.14) mmol/L,( 7.73±1.03) mmol/L,( 9.77±1.92) mmol/L화Scr(80.33±7.15) μmol/L,(58.50±:3.27)μmol/L,(75.67±7.58) μmol/L균명현승고(균P＜ 0.05),기중IMD+I/R조교I/R조이급공질립+I/R조BUN화Scr수평현저강저(균P＜ 0.05).면역조화결과현시,가수술조IMD정약양성표체,주요위우신소관간질세포포질내,I/R조신조직IMD재신소관상피세포화간질표체교가수술조상조；IMD+I/R조신조직중IMD표체교I/R조명현상조(P＜0.01).여I/R조급공질립+I/R조상비IMD+I/R조신조직IMD mRNA,상대함량분별증가료60.7％、66.1％,단백상대함량분별증가료51.4％、55.9％.차외,여I/R조상비,IMD+I/R조신조직ET-1、TNF-α단백표체현저강저(ET-1:0.17±0.02비0.08±0.02；TNF-α:0.35±0.02비0.21±0.04,균P＜0.05).결론 재대서신장I/R전상조IMD표체가능통과억제ET-1、TNF-α표체보호신장결구급공능.
Objective To investigate the effect of intermedin (IMD) on renal ischemia/ reperfusion (I/R) injury after the up-regulation of IMD. Methods A total of 24 healthy Wistar male rats were randomly divided into four groups,sham-operated group,I/R group,IMD gene transfection +I/R group and empty plamid +I/R group.All the animals were killed at the end of 24 h of reperfusion.Histological changes and renal function were estimated.The expression and site of IMD were determined by Immunohistochemistry method,semi-quantitative RT-PCR and Western blotting.The protein expressions of endothelin 1 (ET-1),tumor necrosis factor αt (TNF-α) were detected by Western blotting. Results Compared with sham-operated group,tubulointerstitial pathological injury was significant aggravated in I/R group (7.6±2.3) and empty plamid +I/R group (7.0±1.8),and such injury was improved in IMD+I/R group (1.5±0.8) (P＜0.05).Compared with I/R group and empty plamid +I/R group,the renal dysfunction of IMD +I/R group was obviously lessened [BUN:(7.73±1.03) mmol/L vs (10.13±2.14) mmol/L,(9.77±1.92) mmol/L; Scr:(58.50±3.27) μmol/L vs (80.33±7.15) μmol/L, (75.67±7.58) μmol/L,all P＜0.05].IMD expression was weak in the plasma of tubulointerstitial cells in sham-operated group,and was up-regulated in I/R group. Compared with I/R group, immunohistochemical IMD expression increased obviously (262.03±67.89 vs 175.57±48.06,P＜0.01).The mRNA expression of IMD in IMD+I/R group was up-regulated significantly by 60.7％,66.1％ and the protein expression of IMD in IMD+I/R group increased significantly by 51.4％,55.9％ as compared to I/R and empty plasmid +I/R group.Meanwhile,the protein expressions of ET-1 and TNF-αt in IMD+I/R group were obviously lower compared with those in I/R group (ET-1:0.08±0.02 vs 0.17±0.02; TNF-α:0.21±0.04 vs 0.35± 0.02,all P＜0.05). Conclusion IMD gene transfected into kidneys of rats prior to I/R surgery can attenuate the over-expressions of both ET-1 and TNF-o in I/R injured rat kidneys as well as the damages to the structure and function of the kidneys.