【作者】 朱国贞；

【导师】 李荣山；

【作者基本信息】 山西医科大学 ， 内科学， 2010， 博士

【摘要】 背景：Intermedin(IMD)是新近发现的降钙素基因相关肽家族(calcitanin gene-related peptide, CGRP)新成员。既往研究表明IMD对离体心脏缺血再灌注(I/R)损伤具有保护作用,但目前IMD对肾脏I/R的病理生理作用尚不清楚。目的：本实验拟以IMD基因为研究靶点,构建编码大鼠IMD基因的真核表达载体,通过超声微泡基因转染技术将其转染至大鼠肾脏,观察上调IMD基因对大鼠肾脏缺血再灌注(I/R)的作用,并对其作用机制进行探讨。方法：1.编码大鼠IMD基因的真核表达质粒构建与鉴定检索Gene bank中大鼠IMD的cDNA全长,合成克隆质粒pGH-IMD-X1491GIMD并测序鉴定。pGH-IMD-X1491G经HindⅢ、EcoRⅠ双酶切线性化,与真核表达载体pCDNA3.1(+)连接,酶切鉴定。将构建的IMD真核表达质粒命名为IMD-pCDNA 3.1。2.观察上调IMD基因表达对大鼠肾脏I/R的作用健康雄性Wistar大鼠24只,随机分为4组,每组6只。I组：假手术组,行右肾切除术并饲养1周后单纯分离左侧肾蒂及肾动脉关腹。II组：肾脏I/R模型组,行右肾切除术并饲养1周后行左肾I/R术。III组：IMD基因转染+I/R模型组,行右肾切除后稳定10min,左肾施予超声微泡介导的IMD-pCDNA3.1(+)质粒转染术,术后饲养1周再行左肾I/R术。IV组：空白质粒+I/R模型组,行右肾切除后稳定10min,左肾施予超声微泡介导的pCDNA3.1(+)质粒转染术,术后饲养1周再行左肾I/R术。所有行I/R术大鼠缺血45min再灌注24h杀检,留取血清及肾组织。Beckman全自动生化仪检测血清丙氨酸转氨酶(ALT)、天门冬氨基转移酶(AST)、尿素氮(BUN)、肌酐(SCr)浓度；半定量RT-PCR法检测肾组织IMD mRNA表达；Western blotting测定肾组织IMD的蛋白质表达；肾组织切片行HE、PAS两种染色后,半定量分析各组肾脏病理组织学变化。3.上调IMD基因对大鼠肾脏I/R保护作用的机制半定量RT-PCR检测假手术组、肾脏I/R模型组、IMD基因转染+I/R模型组、空白质粒+I/R模型组中肾组织内皮型NOS(eNOS)、诱导型NOS(iNOS)及神经元型NOS(nNOS) mRNA表达,Western blotting半定量分析以上四组大鼠肾组织eNOS、iNOS、nNOS、内皮素-1(ET-1)、肿瘤坏死因子a(TNF-α)蛋白质表达。结果：1.大鼠IMD基因的真核表达质粒构建与鉴定经酶切、测序鉴定,MD-pCDNA3.1(+)真核表达质粒构建成功。2.上调IMD基因表达对大鼠肾脏I/R的作用IMD转基因+I/R模型组较单纯I/R模型组以及空质粒+I/R模型组BUN和Scr水平显著降低(P<0.05)；而各组大鼠ALT、AST水平无显著差异。半定量RT-PCR结果显示：IMD转基因+I/R模型组肾组织IMD mRNA表达较单纯I/R模型组及空质粒+I/R模型组明显上调,相对含量分别增加了60.7%、66.1%；Western Blotting检测结果显示：IMD转基因+I/R模型组肾组织IMD蛋白质表达量较单纯I/R模型组及空质粒+I/R模型组分别增加了51.4%、55.9%。PAS、HE染色显示：单纯I/R模型组、空质粒+I/R模型组除有肾小管上皮细胞空泡变性外,尚有刷状缘脱落、上皮细胞坏死、管型形成、炎细胞浸润；IMD +I/R模型组虽可见上述病理改变,但程度较单纯I/R模型组以及空质粒+I/R模型组明显减轻(P<0.05)。3.上调IMD基因对大鼠肾脏I/R保护作用的机制半定量RT-PCR及Western Blotting检测结果显示,IMD转基因+I/R模型组肾组织eNOS mRNA及其蛋白质表达水平较单纯I/R模型组显著升高、iNOS mRNA及其蛋白质表达水平显著降低(P<0.05)；而nNOS mRNA及其蛋白质在各组间表达量无显著性差异(P>0.05)。此外,与单纯I/R模型组相比,IMD转基因+I/R模型组肾组织ET-1、TNF-α蛋白质表达显著降低(P<0.05)。结论：1.成功构建了以大鼠IMD基因为靶位的真核表达质粒IMD-pCDNA 3.1(+)。2.利用超声微泡技术可将真核表达质粒IMD-pCDNA 3.1(+)成功转染至大鼠肾组织,使其在肾组织局部表达上调,此方法安全有效。3.在大鼠肾脏I/R前上调IMD表达可显著保护肾脏病理结构并减轻肾功能受损。4.大鼠肾脏IMD可通过促进肾脏局部eNOS高表达、抑制iNOS、ET-1以及TNF-α表达,最终发挥对肾脏IRI的保护作用。更多还原

【Abstract】 BackgroundIntermedin (IMD) is a newly discovered peptide that belongs to calcitonin gene-related peptide(CGRP). It has been proved that IMD has protective effects on ischemia/reperfusion (I/R) in isolated rat hearts, whereas patho-physiology function of IMD in renal I/R is not clear.ObjectiveIn the present study, we aim to design and construct eukaryotic expression vector encoding rat IMD gene and to transfect it into kidney tissue with ultrasound-mircobubbles in order to observe effects of IMD on renal I/R in rat models and to investigate the mechanisms of IMD protective function in renal I/R.Methods1. Construction and identification of eukaryotic expression plasmid encoding rat IMD geneFirstly, full-length rat IMD gene in gene bank was retrieved. The cloning plasmid named as pGH-IMD-X1491G was synthetized and then sequencing analysis was performed. Secondly, pGH-IMD-X1491G was linearrizated by HindⅢand EcoRI enzyme digestion and then was ligated to pCDNA3.1(+) eukaryotic expression vector. Finally, the recombinant plasmid designated as IMD-pCDNA 3.1 was confirmed by restrictive enzyme digestion.2. Effects of IMD in renal I/R rat modelsA total of 24 rats were divided into four groups each consisting of six animals. GroupⅠunderwent right nephrectomy one week prior to the exposure of left renal pedicles, but did not receive any ischemia/reperfusion. GroupⅡunderwent right nephrectomy one week prior to left renal I/R surgeries. GroupⅢunderwent 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 animals were treated 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 before the sera and kidney tissues were kept for detection. Serum levels of ALT, AST, BUN and creatinine were detected by Beckman automatic biochemistry analyzer. The mRNA expressions of IMD in the kidneys were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of IMD in the kidneys were detected by Western blotting. Kidney formaldehyde-fixed paraffin sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) by standard methods and then histological changes were analyzed semiquantitatively.3. Researches into the mechanism of IMD protective function of rat renal I/RThe mRNA expressions of endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in the kidneys of the four groups were determined by RT-PCR. The protein expressions of eNOS, iNOS, nNOS, ET-1 and TNF-a in the kidneys were semiquantitatively analyzed by Western blotting.Results1. Construction and identification of eukaryotic expression plasmid encoding rat IMD geneRestriction enzyme digestion and sequencing analysis showed that eukaryotic expression plasmid of IMD-pCDNA3.1(+) had been constructed successfully.2. Effects of IMD in renal I/R rat modelsThe serum levels of BUN and Cr in rats of group III were obviously lower than those of groupⅡand groupⅣ(P<0.05), while the differences of ALT and AST levels among the four groups were not evident. Compared with group II and group IV, the mRNA expressions of IMD in kidneys of group III were up-regulated significantly by 60.7%,66.1% and the protein expressions of IMD in kidneys increased significantly by 51.4%、55.9%. I/R animals in groupⅡ&IV exhibited serious tubular cellular vacuolization and necrosis, loss of brush border, cast formation and tubular dilatation, which were significantly lessened in the ultrasound-mediated IMD gene transfer group. When compared with IMD-treated I/R group, rats in groupⅡandⅣproduced a significant increase in total severity score (P<0.05)3. Researches into the mechanism of IMD protective function of rat renal I/RCompared with those in groupⅡ, we noticed higher mRNA and protein expressions of eNOS in groupⅢ, while the mRNA and protein expressions of iNOS in groupⅢwere obviously lower compared with those in group of I/R without IMD gene treatment (P<0.05). Meanwhile, there were no significant differences in the mRNA and protein expressions of nNOS among groupⅡ,ⅢandⅣ. Moreover, the protein expressions of ET-1 and TNF-αin groupⅢwere obviously lower compared with those in group of I/R without IMD gene treatment (P<0.05)Conclusions1. Eukaryotic expression plasmid of IMD-pCDNA3.1(+) was constructed successfully.2. It is safe and effective to transfect IMD-pCDNA3.1(+) eukaryotic expression plasmid into kidney tissue with ultrasound-mircobubbles as it could lead to the up-regulation of IMD in the local site of kidney tissue. 3. Up-regulation of IMD prior to renal I/R could protect kidney against tubulointerstitial damage and renal dysfunction in I/R rat models.4. IMD gene in kidneys of rats can promote the expressions of eNOS and attenuate over-expressions of iNOS, ET-1 and TNF-αfollowing I/R, thus protect kidneys against severe damages in I/R rat models.更多还原