沉默PAX2基因对肾间质纤维化大鼠的影响

李丽; 吴玉斌; 徐海生; 王长山

doi:10.7499/j.issn.1008-8830.2016.06.016

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中国当代儿科杂志 ›› 2016, Vol. 18 ›› Issue (6) : 551-557. DOI: 10.7499/j.issn.1008-8830.2016.06.016

论著·实验研究

沉默PAX2基因对肾间质纤维化大鼠的影响

李丽¹, 吴玉斌², 徐海生¹, 王长山³

作者信息 +

Influence of PAX2 gene silencing on renal interstitial fibrosis in rats

LI Li¹, WU Yu-Bin², XU Hai-Sheng¹, WANG Chang-Shan³

Author information +

文章历史 +

摘要

目的探讨体内沉默PAX2基因对肾间质纤维化大鼠肾小管上皮细胞转分化(EMT)的影响。方法 64只Wistar大鼠麻醉后,单侧输尿管结扎法制作肾间质纤维化大鼠模型,随机分为阴性对照组和PAX2基因沉默组,每组32只。将200 μL NC-siRNA-in vivo jetPEI^TM混合液转染阴性对照组大鼠,将200 μL PAX2-siRNA-in vivo jetPEI^TM混合液转染PAX2基因沉默组大鼠,各组分别于转染后3、5、7、14 d分为4个亚组,每组8只。留取各组肾组织标本,应用Real-Time PCR及Western blot法检测肾皮质PAX2 mRNA及其蛋白的沉默情况,以及E-钙粘连素(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)mRNA和蛋白表达情况。结果 PAX2基因沉默组PAX2 mRNA和蛋白表达量较阴性对照组均降低 (P < 0.05)。随着梗阻时间的延长,两组E-cadhelin mRNA和蛋白表达量逐渐下降,α-SMA mRNA和蛋白表达量逐渐升高;在转染14 d时,PAX2基因沉默组E-cadhelin mRNA和蛋白相对表达量明显高于阴性对照组 (P < 0.05),而α-SMA mRNA和蛋白相对表达量明显低于阴性对照组 (P < 0.05)。结论沉默PAX2基因在肾间质纤维化晚期大鼠中可明显抑制肾小管EMT进程,可能对肾间质纤维化有治疗作用。

Abstract

Objective To investigate the influence of silencing PAX2 gene in vivo on epithelial-mesenchymal transition (EMT) of renal tubular cells in rats with renal interstitial fibrosis. Methods A total of 64 Wistar rats were anaesthetized, and unilateral ureteral obstruction (UUO) was performed to establish a rat model of renal interstitial fibrosis. The 64 rats were randomly divided into negative control and PAX2 gene silencing groups (n = 32 each). The rats in the control group were transfected with 200 μL NC-siRNA-in vivo jetPEI^TM solution. Those in the PAX2 gene silencing group were transfected with 200 μL PAX2-siRNA-in vivo jetPEI^TM solution. Each group was further divided into 4 subgroups based on the post-transfection time (3, 5, 7 and 14 days after transfection), with 8 rats in each subgroup. Renal tissue samples were harvested in each group. Real-time PCR and Western blot were used to measure the mRNA and protein expression of PAX2 in the renal cortex, as well as the mRNA and protein expression of E-cadherin and α-SMA. Results Compared with the control group, the PAX2 gene silencing group showed significantly lower mRNA and protein expression of PAX2 (P<0.05). In the two groups, the mRNA and protein expression levels of E-cadherin were gradually reduced over the time of obstruction, while those of α-SMA gradually increased. At 14 days after transfection, the PAX2 gene silencing group had significantly higher mRNA and protein expression of E-cadherin but lower mRNA and protein expression of α-SMA compared with the control group (P<0.05). Conclusions PAX2 gene silencing can significantly inhibit the process of EMT of renal tubular cells in rats with advanced fibrosis, suggesting that PAX2 gene silencing may have a therapeutic effect on renal interstitial fibrosis.

导出引用

李丽, 吴玉斌, 徐海生, 王长山. 沉默PAX2基因对肾间质纤维化大鼠的影响[J]. 中国当代儿科杂志. 2016, 18(6): 551-557 https://doi.org/10.7499/j.issn.1008-8830.2016.06.016

LI Li, WU Yu-Bin, XU Hai-Sheng, WANG Chang-Shan. Influence of PAX2 gene silencing on renal interstitial fibrosis in rats[J]. Chinese Journal of Contemporary Pediatrics. 2016, 18(6): 551-557 https://doi.org/10.7499/j.issn.1008-8830.2016.06.016

参考文献

[1] Hewitson TD. Fibrosis in the kidney: is a problem shared a problem halved?[J]. Fibrogenesis Tissue Repair, 2012, 5(Suppl 1): S14.
[2] Picard N, Baum O, Vogetseder A, et al. Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat[J]. Histochem Cell Biol, 2008, 130(1): 141-155.
[3] Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis[J]. J Am Soc Nephrol, 2010, 21(2): 212-222.
[4] Kim MK, Maeng YI, Sung WJ, et al. The differential expression of TGF-β1, ILK and wnt signaling inducing epithelial to mesenchymal transition in human renal fibrogenesis: an immunohistochemical study[J]. Int J Clin Exp Pathol, 2013, 6(9): 1747-1758.
[5] Xiong H, Hong J, Du W, et al. Roles of STAT3 and ZEB1 proteins in E-cadherin down-regulation and human colorectal cancer epithelial-mesenchymal transition[J]. J Biol Chem, 2012, 287(8): 5819-5832.
[6] Satish L, Gallo PH, Baratz ME, et al. Reversal of TGF-β1 stimulation of α-smooth muscle actin and extracellular matrix components by cyclic AMP in Dupuytren's-derived fibroblasts[J]. BMC Musculoskelet Disord, 2011, 12: 113.
[7] Li L, Wu Y, Zhang W. PAX2 re-expression in renal tubular epithelial cells and correlation with renal interstitial fibrosis of rats with obstructive nephropathy[J]. Ren Fail, 2010, 32(5): 603-611.
[8] André ND, Silva VA, Ariza CB, et al. In vivo knockdown of CXCR4 using jetPEI/CXCR4 shRNA nanoparticles inhibits the pulmonary metastatic potential of B16 F10 melanoma cells[J]. Mol Med Rep, 2015, 12(6): 8320-8326.
[9] Liu H, Wang Y, Wang M, et al. Fluorinated poly(propylenimine) dendrimers as gene vectors[J]. Biomaterials, 2014, 35(20): 5407-5413.
[10] Li L, Wu Y, Wang C, et al. Inhibition of PAX2 gene expression by siRNA (polyethylenimine) in experimental model of obstructive nephropathy[J]. Ren Fail, 2012, 34(10): 1288-1296.
[11] Lee TY, Chang CI, Lee D, et al. RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA[J]. Mol Cells, 2013, 35(4): 320-326.
[12] Sharma R, Sanchez-Ferras O, Bouchard M. Pax genes in renal development, disease and regeneration[J]. Semin Cell Dev Biol, 2015, 44: 97-106.
[13] Zhou TB. Signaling pathways of PAX2 and its role in renal interstitial fibrosis and glomerulosclerosis[J]. J Recept Signal Transduct Res, 2012, 32(6): 298-303.
[14] Li L, Wu Y, Yang Y. Paired box 2 induces epithelial-mesenchymal transition in normal renal tubular epithelial cells of rats[J]. Mol Med Rep, 2013, 7(5): 1549-1554.
[15] Yan X, Zhang Y, Zhang H, et al. Amphiphilic polyethylenimine (PEI) as highly efficient non-viral gene carrier[J]. Org Biomol Chem, 2014, 12(12): 1975-1982.
[16] Liang W, Gong H, Yin D, et al. High-molecular-weight polyethyleneimine conjuncted pluronic for gene transfer agents[J]. Chem Pharm Bull (Tokyo), 2011, 59(9): 1094-1101.
[17] Liu C, Liu F, Feng L, et al. The targeted co-delivery of DNA and doxorubicin to tumor cells via multifunctional PEI-PEG based nanoparticles[J]. Biomaterials, 2013, 34(10): 2547-2564.
[18] Huang X, Hartley AV, Yin Y, et al. AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications[J]. J Virol Methods, 2013, 193(2): 270-277.
[19] Shao W, Paul A, Abbasi S, et al. A novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient siRNA delivery in breast cancer therapy: preparation and in vitro analysis[J]. Int J Nanomedicine, 2012, 7: 1575-1586.
[20] Thakur S, Viswanadhapalli S, Kopp JB, et al. Activation of AMP-activated protein kinase prevents TGF-β1-induced epithelial-mesenchymal transition and myofibroblast activation [J]. Am J Pathol, 2015, 185(8): 2168-2180.
[21] Winbanks CE, Darby IA, Kelynack KJ, et al. Explanting is an ex vivo model of renal epithelial-mesenchymal transition[J]. J Biomed Biotechnol, 2011, 2011: 212819.