IGFBP7对脓毒症小鼠肾小管上皮细胞分裂周期的影响及其机制研究

Urine IGFBP7 increased obviously in acute kidney injury(AKI) patients and was considered to be a marker for AKI, however, the reason and mechanism were still uncovered.Our pre-experiment had suggested IGFBP7 expession was increased in renal tubular epithelial cells of sepsis mice,which was positive corelated to serum creatinine, and renal cell division cycle was ceased at stage G0/G1 simutaneously. As renal tubule epithelium injury is common in septic AKI. So our hypothesis is that IGFBP7 might control kidney epithelium cell cycle in septic AKI and play an important role in kidney epithelium injury and repair. We plan to observe the relationship of IGFBP7 level and kindey tubule epithelium cell cycle in different stage of sepsis mice. And then silence IGFBP7 gene in human kidney tubule epithelium cell line by siRNA technology. After pre-conditioned with inhibitor of BRAF-MEK-ERK, kidney tubule epithelium is stimulated with LPS or serum of sepsis patients. Epithelium cell cycle and related proteins were obsereved. Finally,the correlation between blood/urine IGFBP7 levels and clinical index for AKI was also detected.Our objective is identifying the role of IGFBP7 in cell division cycle of idney tubule epithelium and epithelium repair ,and the clinical value for predicting the develepment and prognosis of AKI.

尿液IGFBP7可作为急性肾损伤 (AKI) 早期诊断最重要的标记物之一，但机制不清。项目组前期研究发现脓毒症AKI小鼠肾小管上皮细胞IGFBP7表达明显升高，且细胞分裂周期停滞于G0-G1。另有研究显示，IGFBP7可促进肿瘤细胞的衰老和凋亡。我们推测IGFBP7可能在脓毒症AKI时通过调控肾小管上皮细胞分裂周期，从而影响肾小管上皮细胞的损伤修复。本研究拟观察实验小鼠在脓毒症不同时期IGFBP7与肾小管上皮细胞分裂周期之间的关系；培养人肾小管上皮细胞，利用siRNA使其IGFBP7表达静止，予BRAF-MEK-ERK通路抑制物预处理，再予LPS刺激，观察肾小管上皮细胞分裂相关蛋白水平的变化；检测脓毒症患者血、尿样IGFBP7水平与AKI临床指标相关性。通过以上研究，在分子、细胞、动物及临床层面探索IGFBP7影响人肾小管上皮细胞分裂周期及损伤修复的机制，及对AKI的预警和预后作用。

上皮间充质转化(epimesenchymal transition, EMT)过程导致肾小管上皮细胞纤维化，在急性肾损伤(acute kidney injury, AKI)发展过程中具有重要意义。IGFBP7作为一种早期的生物标志物被用于识别有AKI风险的患者，并且IGFBP7在AKI后患者尿液中明显升高。然而，IGFBP7在AKI中的作用仍不明确。本研究旨在探讨IGFBP7在脂多糖(LPS) 体外诱导的HK-2细胞和体内盲肠结扎穿刺(CLP)脓毒症诱导的AKI的作用和机制。本课题组通过体外实验结合体内实验证明：1）IGFBP7通过ERK1/2信号调节脓毒症诱导的AKI；2）IGFBP7通过ERK1/2信号调节脓毒病诱导的表皮间质转化。.我们发现AKI患者和LPS(1, 2 and 5 g/ml）体外诱导的HK-2细胞的IGFBP7表达增加。LPS诱导的HK-2细胞在G1-G0期出现细胞周期阻滞和细胞凋亡，ERK1/2激活与ERK1/2通路蛋白的表达改变，包括被IGFBP7敲低抑制的细胞周期蛋白 D1、P21、Bax和Bcl-2。此外，IGFBP7过表达可显著诱导HK-2细胞在G1-G0期细胞周期阻滞和细胞凋亡，并被ERK1/2信号抑制剂PD98509抑制。在HK2细胞中LPS诱导IGFBP7蛋白表达存在剂量依赖并能促进EMT发生和激活ERK1/2,随后 EMT相关蛋白质如E-钙黏素, -SMA, 波形蛋白, Snail, TGF-1, CTGF被调控,并且EMT相关蛋白质被ERK1/2信号传导抑制剂PD98059抑制。此外,IGFBP7过表达显著增加-SMA、波形蛋白、Snail、TGF-1 、 CTGF 表达，减少HK2细胞钙粘蛋白表达，这些IGFBP7过表达的影响被PD98059抑制。IGFBP7的下调能有效抑制CLP诱导小鼠肾纤维化、肌酐(Cr)、血尿素氮(BUN)和白蛋白(ALB)浓度、细胞凋亡和ERK1/2信号通路活化。结果表明，抑制IGFBP7可有效保护肾小管上皮细胞免受体外LPS或体内CLP诱导的EMT的侵袭，缓解肾损伤的严重程度，这可能与ERK1/2信号通路下调有关。