【作者】 周芸；

【导师】 李荣山；

【作者基本信息】 山西医科大学 ， 内科学， 2009， 博士

【摘要】 垂体中叶素Intermedin(IMD)是新发现的内源性心血管、肾脏保护因子,属降钙素基因相关肽(calcitonin gene related peptide, CGRP)超家族成员,对其在肾脏的保护机制尚不清楚。本研究采用分子生物学及细胞生物学手段,对IMD在肾脏缺血再灌注损伤(IRI)中的病理生理意义及其机制进行探讨。分为以下两个部分:一、大鼠肾脏缺血再灌注损伤模型中IMD及其受体的变化目的:制备大鼠肾脏IRI模型,观察IMD及其受体系统的变化,初步探讨IMD在肾脏IRI中的作用。方法:健康雄性Wistar大鼠18只,随机分为假手术组、缺血组和IRI组。双侧夹闭肾动脉45min,再灌注12h制作IRI模型。检测血清尿素氮(BUN)、肌酐(SCr)浓度,并半定量分析肾脏病理组织学变化判断模型成功与否。放射免疫分析法测定血浆、肾组织IMD及血浆肾上腺髓质素(ADM)含量。半定量反转录-聚合酶链反应(RT-PCR)法检测肾组织IMD、ADM及其受体系统CRLR、RAMP1、2、3 mRNA表达;Western blotting方法半定量分析肾组织IMD及其受体CRLR的蛋白表达。结果:⑴与假手术组相比,IRI组大鼠BUN和SCr明显升高(P<0.05);病理组织学显示IRI组大鼠肾小管上皮细胞空泡状变性、刷状缘坏死脱落等,而肾小球无明显改变,半定量评分显示该组与假手术组和缺血组之间存在统计学差异(分别为P<0.05,P<0.01),符合急性肾损伤病理改变,造模成功。⑵大鼠血浆、肾组织IMD含量,IRI组较假手术组升高53.76%、40.01%(P<0.05);较缺血组升高58.34%、42.97%(P<0.05);血浆ADM含量,IRI组较假手术组升高143.22%(P<0.01),较缺血组升高101.96%(P<0.01)。⑶与假手术组相比,IRI组肾组织IMD、ADM、CRLR、RAMP1、RAMP2、RAMP3 mRNA相对含量分别上调了22.1%(P<0.05)、41.1%(P<0.01)、32.8%(P<0.01)、33.7%(P<0.01)、35.9%(P<0.05)、29.4%(P<0.01);IMD和CRLR蛋白表达明显增高,分别增加了80.9%,68.4%(P<0.001)。结论:肾脏IRI模型中血浆和肾组织IMD水平显著增加,同时肾组织IMD及受体系统CRLR/RAMPs的mRNA表达和肾组织IMD、CRLR的蛋白表达均出现与血浆和肾组织IMD水平变化趋势一致的增加,提示IMD参与了肾脏IRI的病理生理过程,可能在保护肾脏方面有重要意义。二、IMD对大鼠肾小管上皮细胞缺氧复氧模型的影响及机制研究目的:以肾小管上皮细胞(NRK-52E)缺氧复氧(H/R)模型模拟在体IRI,通过转染IMD真核表达质粒,探讨IMD细胞保护的分子机制。方法:⑴利用三气培养箱调整氮气压力形成缺氧条件,在缺氧1h,复氧1.5h后检测NRK-52E活细胞计数、细胞存活率和培养液上清乳酸脱氢酶(LDH)含量判断H/R模型成功与否。⑵人工合成IMD目的片段,与pMD19-T Simple载体连接形成克隆载体,再通过双酶切定向克隆技术将其亚克隆至pIRES2-EGFP,构建IMD真核表达载体,命名为pIRES2-EGFP/IMD,测序鉴定。⑶利用Fugene HD转染试剂,将pIRES2-EGFP/IMD表达质粒转染至NRK-52E细胞内,以倒置荧光显微镜和流式细胞仪检测转染效率,选定最优转染条件;并以RT-PCR和Western blotting方法观察其对细胞IMD mRNA和蛋白表达的影响;以G418 300μg/ml加入10%胎牛血清的DMEM/F12完全培养基对细胞进行筛选约2周,获得稳定表达IMD的阳性克隆NRK-52E,Western blotting鉴定筛选后IMD蛋白的表达。⑷对NRK-52E进行H/R实验,分为对照组和6个模型组包括单纯H/R组、空质粒组、IMD质粒组、IMD+PKA抑制剂(H-89)组、环磷酸腺苷(cAMP)类似物(8-Br-cAMP)组和NADPH氧化酶抑制剂(DPI)组,分别检测细胞培养液上清中LDH、cAMP含量,细胞NADPH氧化酶活性、超氧化物歧化酶(SOD)、丙二醛(MDA)和活性氧(ROS)水平,观察IMD对H/R时氧化损伤的影响及分子机制。结果:⑴经缺氧1h,复氧1.5h后,NRK-52E细胞计数量降低,存活率下降,细胞培养液上清中LDH含量显著增加(P<0.05),造模成功。⑵酶切和测序鉴定表明,pMD19-T Simple/IMD克隆质粒和pIRES2-EGFP/IMD真核表达载体构建成功。⑶荧光显微镜和流式细胞仪检测结果显示以质粒3μg: FuGENE HD 12μl配比的转染复合体在48h的转染效率最高,转染效率最高可达71.34±6.24%(P<0.01);半定量RT-PCR和Western blotting结果显示,转重组质粒组IMD mRNA表达量较未转染组增高(P<0.01),蛋白表达也显著增高(P<0.001)。G418筛选后转染阳性的细胞于第3天起逐渐出现克隆样增殖,第5天起未转染的细胞出现批量死亡,以后阳性细胞克隆逐渐增加,至第14天可见融合成片状的阳性克隆细胞,约占总细胞的60~70%。Western blotting结果显示,筛选后IMD蛋白的表达呈现高表达,说明细胞得到稳定表达。⑷给予各种干预因素后,各检测指标的变化如下:①LHD含量:与对照组相比,各模型组LDH显著上升(P﹤0.01);与H/R组相比,IMD质粒组与8-Br-cAMP组LDH上升幅度较小(P﹤0.05)。②cAMP含量:与对照组相比,各模型组cAMP含量明显增加,其中IMD质粒+H-89组和8-Br-cAMP组升高最为显著(P﹤0.01);与H/R组相比,IMD质粒+H-89组和8-Br-cAMP组的cAMP含量上升幅度最显著(P﹤0.01),IMD质粒组也有所上升,DPI组却有所下降(P﹤0.05)。③NADPH氧化酶含量:除DPI组外各模型组NADPH氧化酶含量均明显升高(P﹤0.01),其中8-Br-cAMP组升高幅度最小(P﹤0.05);与H/R组比较,8-Br-cAMP组和DPI组的NADPH氧化酶含量分别下降了33.92%(P﹤0.05)、52.54%(P﹤0.01)。④MDA含量:与对照组相比,模型组中H/R组(P﹤0.01)、空质粒组(P﹤0.01)、IMD质粒+ H-89组(P﹤0.05)和DPI组(P﹤0.05)的MDA含量增加;与H/R组比较,IMD质粒组、IMD质粒+ H-89组、8-Br-cAMP组和DPI组的MDA均有所下降(P﹤0.05)。⑤ROS含量:与对照组相比,模型组中H/R组、空质粒组、IMD质粒+ H-89组的ROS含量增加(P﹤0.05);与H/R组比较,IMD质粒组的ROS含量有所下降(P﹤0.05)。⑥SOD含量:模型组中IMD质粒组、IMD质粒+ H-89组和DPI组的SOD含量有所升高(P﹤0.05),其余各组与对照组和H/R组相比均无明显差别。结论:在肾小管上皮细胞H/R模型中,IMD可通过cAMP/PKA途径增加cAMP水平,抑制NADPH氧化酶活性,减少ROS产生,同时上调SOD,减轻细胞氧化损伤,参与氧化与抗氧化平衡的调节,作为重要的内源性抗氧化剂实现其细胞保护作用。更多还原

【Abstract】 Being a newly-found endogenous cardio-renal-protective substance, Intermedin (IMD) belongs to the calcitonin gene-related peptide (CGRP) superfamily; its renal-protection mechanism is still unknown. Applying the methods of both cytobiology and molecular biology, this study is designed to explore the physiological and pathological significance of IMD in renal ischemia-reperfusion injury(IRI). This study includes two parts:1. The changes of IMD and its receptors in rat renal ischemia reperfusion injuryObjective The rat models of renal ischemia reperfusion injury(IRI) were established to observe the changes of IMD and its receptors after IRI, and explore the functions of IMD in renal IRI. Methods Eighteen male Wistar rats were randomly divided into sham-operated,ischemia and IRI groups. IRI rat models were made by clamping both renal arteries for 45min and reperfusing for 12 hours. IRI models were evaluated through measuring blood urea nitrogen(BUN) and serum creatinine(SCr) concentration, and through semi-quantitated anaylsis of renal histological changes. The contents of IMD and ADM in plasma and renal tissue were assessed by radioimmunoassay (RIA). The mRNA expressions of IMD, ADM, CRLR and RAMPs in the kidneys were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR). The changes of protein expressions of IMD and CRLR in the kidneys were detected by Western blotting.Results⑴Compared with the sham ones, BUN and SCr in IRI rats increased significantly(P<0.05). Kidneys pathologic changes of IRI rat models were significant, as shown in tubule vacuolar degeneration or necrosis, loss of brush border; while there was little change in glomerulus. Semi-quantitated analysis suggests there was significant difference between IRI groups and the other two groups. (the sham-operated groups, P<0.05 and the ischemia groups, P<0.01). The results go with the changes of acute renal injury, so models are successfully established.⑵Compared with sham-operated and ischemia groups, the IMD contents of serum and renal in IRI group increased by 53.76%, 40.01% and 58.34%, 42.97% (P<0.05), and serum ADM showed a marked increase by 143.22%, 101.96% (P<0.05).⑶Compared with sham-operated group, the mRNA expressions of IMD, ADM, CRLR, RAMP1, RAMP2, RAMP3 in kidneys of IRI group were all up-regulated significantly by 22.1%(P<0.05, 41.1%(P<0.01), 32.8%(P<0.01), 33.7%(P<0.01), 35.9%(P<0.05), 29.4%(P<0.01), and the protein expressions of IMD and CRLR in kidneys increased significantly by 80.9%, 68.4%(P<0.001).Conclusion In IRI group, levels of IMD in blood plasma and kidney increased significantly; and consistently there appeared marked increases of the mRNA expressions of IMD and of its receptors, and the protein expressions of IMD and CRLR in kidneys. These results suggest that IMD may have participated in the process of renal IRI, and may have important physiological and pathological significance in terms of kidney protection.2. The influence of IMD exerted on the hypoxia/reoxygenation model of rat renal tubular epithelial cells and its mechanismObjective The hypoxia/reoxygenation(H/R) injury models of rat renal tubular epithelial cells (NRK-52E), which were transfected the eukaryotic expression vector of rat IMD, were established to simulate IRI in vivo, to investigate the molecule mechanism of IMD cytoprotection.Methods⑴To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 1h and then with reoxygenation for 1.5h. The models were evaluated by detecting living cell count, cell viability and the activity of lactate dehydrogenase (LDH) in the culture medium.⑵The full length IMD cDNA, which was synthesized artificially, was connected with pMD19-T Simple vector to form a cloned vector. This cloned vector was sub-cloned into pIRES2-EGFP to construct an eukaryotic expression vector, which is named pIRES2-EGFP/IMD. Restriction enzyme digestion and sequencing analysis were adopted to test the recombinant plasmids.⑶NRK-52E cells were transfected by transfection complex comprising optimal proportion of pIRES2-EGFP/IMD plasmid and Fugene HD reagents. Transfection efficiency was tested by observation for EGFP made by fluorescent microscope and flow cytometery (FCM) after 48h. Total RNA and protein was extracted from EGFP positive NRK-52E cells. IMD mRNA and protein expression level was evaluated by RT-PCR and Western-blotting. The cell groups with high transfection efficiency were screened for two weeks or so by incubating with medium containing G418(300μg/ml), and the positive cloning NRK-52E cells of stable expression IMD obtained. The expression of IMD protein in IMD plasmid group after screening was evaluated by Western-blotting.⑷The NRK-52E cells were divided into 7 groups. One of them was control group; the other six model groups were exposed to H/R condition, following the intervention of single H/R, primitive vector, highly expressed IMD vector, NADPH oxidase inhibitor(DPI), cyclic adenosine monophosphate(cAMP) analogue (8-Br-cAMP), PKA inhibitor(H-89), respectively. The contents of LDH and cAMP in the culture medium and the levels of NADPH oxidase, superoxide dismutase(SOD), malondialdehyde(MDA) and reactive oxygen species(ROS) in cells were detected to observe the influence of IMD on H/R injury and its molecule mechanism.Results⑴After hypoxia 1h and reoxygenation 1.5h, cell count and cell viability decreased significantly and the activity of LDH increased significantly (P<0.05); the model was established successfully.⑵Restriction enzyme digestion and sequencing analysis showed that pMD19-T Simple/IMD cloning vector and pIRES2-EGFP/IMD eukaryotic expression vector had been constructed successfully.⑶NRK-52E cells was transfected with the optimal transfection proportion of pIRES2-EGFP/IMD to Fugene HD reagent as 3μg: 12μl, transfection efficiency evaluated by fluorescence microscope and FCM showed that the transfection efficiency increased with the prolongation of time and reached its peak 71.34±6.24% at 48h(P<0.01). The result of RT-PCR and Western blotting showed the expression levels of IMD mRNA and protein in IMD plasmid group were increased significantly (P<0.01 and P<0.001, respectively). The positive transfected cells after G418 screening were found clone-like proliferation on the third day, and the untransfected cells died in large bulk on the fifth day. Then the positive transfected cells increased gradually; and the positive cloning cells blending into slices were found on the fourteenth day, which occupied 60-70% of the total cell number. The result of Western blotting showed the expression of IMD protein in IMD plasmid group after screening increased significantly, which indicate the IMD expression of the cell is stable.⑷In NRK-52E cells following the seven treatments, the changes of detected index were shown:①Contrasted with the control group, the level of LDH of all model groups increased significantly (P<0.01); but the extent of increase in IMD plasmid group and 8-Br-cAMP group were smaller than that in H/R group(P﹤0.05).②Contrasted with the control group, the level of cAMP of all model groups also increased significantly(P<0.01), and IMD plasmid + H-89 group and 8-Br-cAMP group were exceptionally noticeable; when compared with H/R group, the two groups were the most remarkably increased groups(P﹤0.01), IMD plasmid group increased mildly and DPI group decreased(P﹤0.05).③Contrasted with the control group, the activity of NADPH oxidase of all model groups increased significantly except DPI group(P<0.01), but the extent of increase in 8-Br-cAMP group was the smallest. Compared with H/R group, the levels of NADPH oxidase of 8-Br-cAMP group and DPI group decreased by 33.92%(P﹤0.05) and 52.54%(P﹤0.01), respectively.④The content of MDA increased in H/R group (P﹤0.01), primitive plasmid group (P﹤0.01), IMD plasmid + H-89 group (P﹤0.05) and DPI group (P﹤0.01), but decreased in IMD plasmid group, IMD plasmid + H-89 group, 8-Br-cAMP group and DPI group(P﹤0.05) compared with H/R group.⑤The content of ROS increased in H/R group, primitive plasmid group, IMD plasmid + H-89 group (P﹤0.05), but decreased in IMD plasmid group compared with H/R group(P﹤0.05).⑥The content of SOD increased in IMD plasmid group, IMD plasmid + H-89 group and DPI group(P﹤0.05), but it changed little in the other four groups.Conclusion In H/R model of NRK-52E, IMD could increase the level of cAMP via cAMP/PKA access, inhibit the activity of NADPH oxidase, decrease the production of ROS, upgrade SOD, alleviate the cell oxidative damage, regulate the oxidative and anti-oxidative balance. All these suggest that IMD, as an endogenous antioxidant, plays a protective role in cell protection during H/R-induced oxidative injury.更多还原