【作者】 史宁；

【导师】 吴满平；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 高密度脂蛋白(HDL)有抗炎的功能,并可保护肾缺血再灌注(I/R)所造成的损伤。在本实验中,我们研究了高密度脂蛋白的主要蛋白组分,载脂蛋白A-Ⅰ(Apolipoprotein A-Ⅰ,ApoA-Ⅰ)在缺血再灌注损伤中的作用。我们用改良的冷乙醇沉淀法,从血浆F-Ⅳ组分中提取纯化人血浆载脂蛋白A-Ⅰ。用SDS-聚丙烯酰胺凝胶电泳(SDS-polyacrylamide gel electrophoresis,SDS-PAGE)和蛋白免疫印迹法(western blot)鉴定分离得到的蛋白为纯化的ApoA-Ⅰ。对LPS(内毒素)激活的巨噬细胞细胞毒性抑制作用实验和LPS体外结合实验显示,制备得到的ApoA-Ⅰ具有与天然野生型ApoA-Ⅰ同样的生物学活性。在乌拉坦麻醉条件下,夹闭SD大鼠双侧肾蒂,造成肾组织缺血,45分钟后,血管再通,建立肾缺血再灌注(I/R)模型。缺血前30分钟股静脉给予ApoA-Ⅰ(25mg/kg)(给药组)或者生理盐水(缺血再灌注组,I/R组)处理。假手术组(sham组)除不夹闭肾蒂外,其它条件与缺血再灌注组相同。再灌注6小时后,股静脉取血,测定血清肌酐(serum creatinine)水平,血尿素氮(blood urea nitrogen,BUN)水平,血清肿瘤坏死因子α(TNF-α)和白介素1β(IL-1β)的含量。并取一部分大鼠的肾脏,测定组织匀浆中丙二醛(malondialdehyde,MDA)的含量以及超氧化物歧化酶(superoxide dismutase,SOD)和髓过氧化物酶(myeloperoxidase,MPO)的比活性,另取部分大鼠的肾脏做病理切片,观察组织病理变化。同时取大鼠肾脏做免疫组化观察血管内皮细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)和P-选择素(P-selectin)的表达情况。实验结果显示ApoA-Ⅰ能够显著降低I/R大鼠血清中肌酐和尿素氮水平:缺血再灌注组中肌酐和尿素氮水平分别为172.78±19.15μM和18.43±2.96mM,而给药组中肌酐和尿素氮水平分别为114.56±7.30μM(p＜0.01)和11.25±1.94mM(p＜0.01)。同时,ApoA-Ⅰ能够显著降低I/R大鼠血清中TNF-α和IL-1β的水平:缺血再灌注组中TNF-α和IL-1β的浓度分别为10.95±3.63pg/ml,355.00±52.62pg/ml,给药组中TNF-α和IL-1β的浓度分别为6.43±1.43 pg/ml(p＜0.05)和208.75±37.76pg/ml(p＜0.01)。另外,I/R大鼠肾组织MDA含量和MPO活性在给予ApoA-Ⅰ后也分别从34.77±2.48nmol/克湿组织和6.14±0.58U/克湿组织相应地降到23.71±2.03nmol/克湿组织(p＜0.01)和3.03±0.51U/克湿组织(p＜0.01);而I/R大鼠肾组织SOD活性在给予ApoA-Ⅰ后从59.38±15.86U/克湿组织升高到86.40±21.52 U/克湿组织(p＜0.05)。大鼠肾脏免疫组化分析表明,再灌注6小时后,缺血再灌注组血管内皮细胞ICAM-1的表达量明显增加,ApoA-Ⅰ治疗可明显降低血管内皮细胞ICAM-1的表达水平。P-selectin的表达也有同样的变化趋势。同时,组织病理分析也表明ApoA-Ⅰ给药能够减轻缺血再灌注所造成的肾组织损伤和炎症。以上实验结果建议ApoA-Ⅰ在大鼠肾缺血再灌注损伤中有良好的保护作用,具有潜在的临床医用价值。更多还原

【Abstract】 High density lipoprotein(HDL) has the anti-inflammatory function and can protect kidney from ischemia-reperfusion(I/R) injury.In present study,we investigated the function of Apolipoprotein A-Ⅰ(ApoA-Ⅰ),the major apolipoprotein of HDL,in I/R injury.Human plasma precipitateⅣwas utilized to isolate ApoA-Ⅰby cold ethanol precipitation,which is modified by us.Purified ApoA-Ⅰwas identified by SDS-PAGE and Western blot.The study on the detoxification effect of ApoA-Ⅰon LPS-mediated macrophages activation and LPS binding test of ApoA-Ⅰin vitro showed that the prepared ApoA-Ⅰhas the same biological activity as natural wild type ApoA-Ⅰ.We investigated the protective role of ApoA-Ⅰin renal ischemia-reperfusion injury in SD rats anesthetized with urethane.Saline(I/R group) or ApoA-Ⅰ(25 mg/kg) (ApoA-Ⅰgroup) was administered intravenously 30 min before 45min-ischemia by occlusion of the renal pedicles.Rats in sham group were subjected to the same surgical procedures as I/R rats except that the renal pedicles were not occluded.Blood samples were collected at 6 h of reperfusion for determination of serum creatinine, BUN,TNF-αand IL-1βlevels.Kidneys were harvested at 6 h of reperfusion for assay of MDA content and MPO and SOD activity,as well as for observation of histopathological changes and for immunohistochemical analysis of ICAM-1 and P-selectin expression on endothelium.The results showed that administration of ApoA-Ⅰcould significantly reduce serum creatinine and BUN levels in I/R group(172.78±19.15μM vs 114.56±7.30μM,p＜0.05 and 18.43±2.96mM vs 11.25±1.94mM,p＜0.01,respectively),and also reduce serum TNF-αlevels from 10.95±3.63pg/ml to 6.43±1.43pg/ml(p＜0.05), as well as serum IL-1βlevels from 355.00±52.62pg/ml to 208.75±37.76pg/ml (p＜0.01) compared with I/R group.Tissue MDA content and MPO activity in ApoA-I-treated group were significantly reduced compared with I/R group(34.77±2.48nmol/g wet tissue vs 23.71±2.03nmol/g wet tissue,p＜0.01 and 6.14±0.58U/g wet tissue vs 3.03±0.51U/g wet tissue,p＜0.01,respectively),whereas tissue SOD activity in ApoA-I-treated group was significantly increased compared with I/R group(86.40±21.52 U/g wet tissue vs 59.38±15.86U/g wet tissue,p＜0.05).Moreover, ApoA-I treatment suppressed the expression of ICAM-1 on endothelium,and the similar trend could also be observed in the expression of P-selectin.The histopathological analysis of rat kidneys harvested at 6h of rperfusion showed that ApoA-I attenuated I/R-induced renal injury and tissue inflammation.These results suggest that ApoA-I can provide beneficial protective effect on renal ischemia-reperfusion injury in rats,and thus has promising clinical value for the treatment of I/R injury.更多还原