中国血液净化

中国血液净化 ›› 2018, Vol. 17 ›› Issue (05): 293-298.doi: 10.3969/j.issn.1671-4091.2018.05.002

• 临床研究 • 上一篇    下一篇

Klotho 基因过表达对小鼠急性肾损伤肾脏血管内皮生长因子表达的影响

倪文慧1,张颖1,田雨1,杨晶1,李胜开1,尹忠诚1   

  1. 1.  徐州医科大学附属医院肾内科
  • 收稿日期:2018-02-24 修回日期:2018-03-08 出版日期:2018-05-12 发布日期:2018-05-12
  • 通讯作者: 尹忠诚 yzcxyfy@126．com E-mail:yzcxyfy@126.com
  • 基金资助:

    徐州市科技项目(KC15SH046)

The overexpression of Klotho gene on the expression of vascular endothelial growth factor in kidney of acute kidney injury mice

  • Received:2018-02-24 Revised:2018-03-08 Online:2018-05-12 Published:2018-05-12

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摘要： 【摘要】目的通过观察Klotho 基因过表达对肾脏血管内皮生长因子(vascular endothelial growth factor，VEGF)的影响，进一步探讨Klotho 基因对小鼠急性肾损伤(acute kidney injury，AKI)的保护作用机制。方法构建可稳定高效表达Klotho 基因的慢病毒载体，通过病毒转染骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)技术获得高表达Klotho 基因的BMSCs(overexpression of Klotho gene in BMSCs， BMSCs- Klotho) 和对照空载体BMSCs(expression of empty vector in BMSCs，BMSCs-EV)。采用小鼠双侧股肌内注射50%甘油(8 ml/kg)导致横纹肌溶解的方法建立AKI 模型。30 只雄性C57BL/6 小鼠随机分为5 组，每组6 只：正常对照(control,CON)组、PBS 干预(AKI with PBS intervention，AKI + PBS)组、BMSCs- Klotho 干预(AKI with BMSCs- Klotho intervention，AKI+ BMSCs-Klotho)组、BMSCs-EV 干预(AKI with BMSCs-EV intervention，AKI+BMSCs-EV)组和BMSCs 干预(AKI with BMSCs intervention，AKI+BMSCs)组，所有干预均在造模后6h 实施。各组小鼠均在干预治疗3 天后处死。常规生化检测血清肌酐(serum creatinine，Scr)及尿素氮(blood urea nitrogen，BUN)水平，利用免疫组织化学法、蛋白印迹法检测肾脏VEGF 的表达。结果与CON 组比较，AKI+PBS 组、AKI+BMSCs-Klotho 组、AKI+BMSCs-EV 组和AKI+BMSCs 组血清检测Scr(F =370.705, P＜0.001；P＜0.001；P＜0.001；P＜0.001)和BUN(F =307.656, P＜0.001；P＜0.001；P＜0.001；P＜0.001)水平均升高；AKI+BMSCs-Klotho 组上述指标低于AKI + PBS 组、AKI + BMSCs- EV 组及AKI + BMSCs 组(Scr:P＜0.001；P＜0.001；P＜0.001；BUN:P＜0.001；P＜0.001；P＜0.001)。蛋白印迹法与免疫组织化学法均检测到AKI+PBS 组的肾脏VEGF 的表达明显低于CON 组(蛋白印迹：P＜0.001；免疫组化：P＜0.001)。AKI+BMSCs-Klotho 组的肾脏VEGF 表达高于AKI+PBS 组、AKI+BMSCs-EV 组和AKI+BMSCs 组(蛋白印迹：P＜0.001；P＜0.001；P＜0.001；免疫组化：P＜0.001；P＜0.001；P＜0.001)，与CON 组比较无统计学差异(蛋白印迹：P =0.884；免疫组化：P =0.809)。结论Klotho可能通过上调肾脏VEGF 表达对AKI 小鼠起肾脏保护作用。

关键词: Klotho基因, 骨髓间充质干细胞, 急性肾损伤, 血管内皮生长因子

Abstract: 【Abstract】Objective To investigate the overexpression of Klotho gene on the expression of renal vascular endothelial growth factor (VEGF) in order to explore the protective role of Klotho on acute kidney injury (AKI) in mice. Methods Mouse bone marrow mesenchymal stem cells (BMSCs) were infected with the recombinant lentivirus to efficiently and stably express Klotho (BMSCs-Klotho), and those infected with the parent lentivirus was used as the control (BMSCs-EV). AKI model was established by intramuscular injection of 50% glycerol to the interior side of both legs to induce rhabdomyolysis in mice. Thirty male C57BL/6 mice were randomly and equally divided into 5 groups: normal control group (Ctrl group), AKI with PBS intervention (AKI+PBS group), AKI with BMSCs-Klotho intervention (AKI+BMSCs-Klotho group), AKI with BMSCs- EV intervention (AKI+BMSCs-EV group), and AKI with BMSCs intervention (AKI+BMSCs group). Mice were treated with the intervention after AKI for 6 hours, and were sacrificed after the intervention for 3 days. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by routine biochemical methods, and the expression of VEGF in kidney was assayed by immunohistochemistry and immunoblot. Results Scr and BUN levels increased in AKI+PBS group, AKI+BMSCs-Klotho group, AKI+BMSCs-EV group and AKI+ BMSCs group as compared to those in Ctrl group (for Scr, F=370.705, P＜0.001, P＜0.001, P＜0.001 and P＜0.001, respectively; for BUN, F=307.656, P＜0.001, P＜0.001, P＜0.001 and P＜0.001, respectively). Scr and BUN levels were lower in AKI+BMSCs-Klotho group than in AKI+PBS group, AKI+BMSCs-EV group and AKI+BMSCs group (for Scr, P＜0.001, P＜0.001 and P＜0.001, respectively; for BUN, P＜0.001, P＜0.001 and P＜0.001, respectively). The expression of VEGF in kidney as assayed by immunoblot and immunohistochemistry was significantly lower in AKI+PBS group than in Ctrl groups (P＜0.001). The expression of VEGF in kidney was significantly higher in AKI+BMSCs-Klotho group than in AKI+PBS group, AKI+BMSCs-EV group and AKI+BMSCs group (for immunoblot, P＜0.001, P＜0.001 and P＜0.001, respectively; for immunohistochemistry, P＜0.001, P＜0.001 and P＜0.001, respectively), but had no statistical significance between AKI+BMSCs-Klotho group and Ctrl group (for immunoblot, P=0.884; for immunohistochemistry, P= 0.809). Conclusion Klotho has a protective effect on AKI through up-regulation of VEGF in kidney in mice.

Key words: Klotho gene, Bone marrow mesenchymal stem cells, Acute kidney injury, Vascular endothelial growth factor