【作者】 孙蕾；

【导师】 黄文彦；

【作者基本信息】 上海交通大学 ， 儿科学， 2015， 硕士

【摘要】 急性肾损伤(Acute Kidney Injury,AKI)为临床常见危重症,指多种原因引起的肾小球滤过率(Glomerular Filtration Rate,GFR)突然下降,临床表现为氮质血症、水电解质和酸碱平衡紊乱以及全身各系统症状。尽管人类对AKI的诊断治疗水平尤其是肾脏替代技术有了长足进步,但AKI死亡率仍高达5%-10%,伴肾外器官衰竭患者可达50%-70%。此外,AKI是引发慢性肾功能不全常见原因之一。急慢性肾损伤已经成为威胁人类健康的重要疾病。因此,积极寻找AKI发生及发展机制,对AKI进行早期诊断及治疗一直是全球肾脏病研究者的热门课题。肾脏急性缺血缺氧再灌注损伤(Acute Ischemia-reperfusion Kidney Injury,AIKI)是临床最常见的AKI病因之一,其主要病理特征为急性肾小管坏死。研究表明,急性肾小管坏死发生后细胞周期循环在肾小管修复过程中起着极其重要的作用。业已证明,补体应答基因-32(Response Gene to Complement 32,RGC-32)为细胞周期关键调控分子,促进细胞周期循环参与细胞增生。本研究第一部分在建立AIKI动物模型基础上,通过实时荧光定量PCR、Western blotting以及免疫组织化学等方法观察了RGC-32在AIKI大鼠肾组织中的动态表达变化及分布,并初步了解其在AIKI大鼠肾组织中的表达规律及其意义。研究发现,大鼠肾缺血再灌注损伤24h后,RGC-32在肾组织表达水平出现明显下调并持续至缺血再灌注后72h,术后1周其表达量逐渐上升至基本正常水平,提示RGC-32可能在急性肾损伤过程中发挥作用。随后,为明确RGC-32在大鼠AIKI过程中的可能机制,本研究第二部分通过体外肾小管上皮细胞损伤模型,利用基因转染技术干预RGC-32表达,进一步观察了RGC-32对肾小管上皮细胞损伤及修复的影响。第一部分:RGC-32在急性肾损伤大鼠肾组织中的表达及意义【目的】:通过建立大鼠AIKI模型,观察RGC-32在AIKI大鼠肾组织中表达变化与分布规律,了解RGC-32在AIKI中的意义。【方法】:采用国际公认的双侧肾蒂夹闭法建立AIKI模型,SD大鼠随机分组:(1)模型组(AIKI组)(n=64):分离大鼠双侧肾蒂,持续阻断45min。并分别于肾脏缺血再灌注后2h、6h、24h、48h、72h、1w、2w、4w分别处死大鼠8只。通过腹主动脉采血检测血清肌酐(Serum Creatinine,Scr)水平。留取肾脏组织,通过实时荧光定量PCR、Western blotting以及免疫组织化学法明确肾组织损伤程度及RGC-32表达变化与分布规律。(2)假手术组(sham组)(n=64):仅分离双侧肾蒂而不进行肾蒂阻断,其余操作同AIKI组。【结果】:(1)通过免疫组织化学方法发现:正常大鼠肾组织中RGC-32主要表达于近端小管、远端小管及集合管上皮细胞,肾小球仅有微弱表达;RGC-32在肾小管间质及肾脏血管无表达。(2)AIKI组缺血再灌注后2h、6h、24h、48h、72h、1w、2w、4w RGC-32蛋白相对表达水平分别为0.0168±0.0029,0.0156±0.0021,0.0065±0.0013,0.0075±0.0013,0.0096±0.0014,0.0132±0.0016,0.0169±0.0014,0.0179±0.0022。其中缺血再灌注后24h、48h、72h两组间RGC-32蛋白表达水平差异具有显著统计学意义(P<0.01)。RGC-32 m RNA表达水平变化与免疫组化结果相符。RGC-32蛋白表达与肾小管-间质损伤评分存在显著负相关(r=-0.514,P<0.01)。【结论】:(1)首次发现RGC-32蛋白在正常SD大鼠肾组织中主要表达于肾小管上皮细胞胞浆,肾小球系膜细胞可有微弱表达,肾血管及肾小管间质无表达。(2)大鼠肾脏缺血再灌注后,RGC-32于再灌注后24h-72h表达水平明显降低。RGC-32可能在AKI发生发展过程中发挥重要作用。第二部分:RGC-32通过细胞周期参与肾小管上皮细胞损伤及修复过程【目的】:通过体外肾小管损伤模型,研究RGC-32对肾小管上皮细胞周期影响以及参与肾小管上皮细胞损伤修复过程的作用。【方法】:(1)体外培养NRK-52E细胞,TNF-α(10ng/ml)干预制备体外肾小管上皮细胞损伤模型并鉴定。(2)采用瞬时转染技术分别转染RGC-32真核表达载体和RGC-32 si RNA,制备RGC-32高表达组及RGC-32敲低组,验证转染效果。(3)通过流式细胞仪,检测RGC-32高表达组及RGC-32敲低组细胞周期变化。(4)Western blotting方法检测各组细胞损伤标志物及细胞外基质表达。【结果】:(1)与对照组相比,RGC-32敲低组G2/M期比例明显增多(P<0.05);RGC-32高表达组S期比例明显增加(与空白质粒对照组比较P<0.05)且不影响G2/M期。(2)与对照组相比,RGC-32敲低组细胞损伤标志物NGAL、KIM-1及细胞外基质α-SMA、Fibronectin表达明显增高。【结论】:RGC-32可能通过调控细胞周期G2/M期在肾小管上皮细胞损伤修复过程中发挥重要作用,其确切机制有待进一步阐明。更多还原

【Abstract】 Part 1: The Expression and Significance of Response Gene to Complement 32 in Rat with Acute Kidney InjuryObjective: To investigate the expression of Response Gene to Complement 32(RGC-32) in rat with acute kidney injury(AKI), and to explore the role of RGC-32 in renal injury induced by ischemia reperfusion.Methods: Sprague Dawley rats were randomly divided into 2 groups, including sham operation group(n=64) and acute ischemia reperfusion injury(IRI) group(n=64). Rats were sacrificed following reperfusion 2h, 6h, 24 h, 48 h, 72 h, 1week(w), 2w and 4w. Serum samples were taken from aorta abdominalis for Serum creatinine(Scr) measurement at different reperfusion time points. The distribution and expression of RGC-32 in renal tissue were observed by means of immunohistochemistry. The mean density of the images detected by Image-Pro Plus 6 was designated as the representative RGC-32 expression levels. Meanwhile, RGC-32 m RNA expression was measured by q PCR.Results:(1) The location of RGC-32 in normal renal tissues: RGC-32 majorly expressed in cytoplasm of epithelial cells of proximal tubular, distal renal tubular and collecting ducts, and expressed slightly in glomerular, while RGC-32 did not expressed in renal interstitium and the vessels of kidney.(2) The expression levels of RGC-32 measured by immunohistochemistry at different reperfusion time were 0.0168±0.0029, 0.0156±0.0021, 0.0065±0.0013, 0.0075±0.0013, 0.0096±0.0014, 0.0132±0.0016, 0.0169±0.0014, 0.0179±0.0022, respectively. Compared with the sham operation group, the level of RGC-32 expression in IRI group was significant lower at 24 h,48h,72 h after IRI(P<0.05). The expression levels of RGC-32 m RNA at different reperfusion time also measured by q PCR were corroborated the immunohistochemistry finding. And the expression of RGC-32 was correlated with renal tubule-interstitial injury score(r= -0.514,P<0.01).Conclusions:(1) We firstly found that RGC-32 protein located in renal tubular in renal tissues of rat, and expressed slightly in glomerular, while RGC-32 did not express in renal interstitium and the vessels of kidney.(2) Our data showed that the RGC-32 expression in AKI rat decreased significantly reduces with different reperfusion time and performs a time-dependent manner.RGC-32 may play an important role in the pathogenesis of AKI following ischemia-reperfusion injury in rat.Part 2: Response Gene to Complement 32 acts as a novel cell cycle factor on renal tubular epithelial cells repairObjective.: This study aimed to evaluate the cell cycle influence of response gene to complement-32(RGC-32) in processing renal tubular epithelial cells injury and repair. Methods.:We cultured NRK-52E cells in vitro and treated with TNF-α, and then determinate the RGC-32 expression levels of cell injury. Then we made the NRK-52E cells expressed in either high or low expression by transient transfection, and determinate the cell cycle distribution, and the expression and significance of fibrosis factors.Results : First, when the NRK-52E cells treated with TNF-α, the cells were injured by TNF-α. NGAL and RGC-32 expression was significantly increased. Second, RGC-32 regulates the cell cycle distribution of the NRK-52E cells by controlling G2/M checkpoint in cell cycle. Our data showed that the cell number of G2/M phases increased dramatically, indicating that the low RGC-32 expression group induced G2/M arrest. Third, the low RGC-32 expression group NRK-52E cells whose G2/M phases were prevented, had significantly increased the expression of fibrosis factors.Conclusion: In this study, we indicated that in vitro RGC-32 probably has an important impact on the repair process of renal tubular epithelial cells by regulating G2/M phase checkpoint. However, the exact mechanism needs to be further elucidated.更多还原