基质硬度对人肾小管上皮细胞形态和葡萄糖转运蛋白表达的影响
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摘要
肾小管间质纤维化以细胞外基质沉积、瘢痕硬化为特点,是慢性肾脏疾病发展至终末期肾衰竭的共通途径。本研究拟构建体外细胞培养模型探索基质硬度对肾小管上皮细胞形态和功能的影响。采用光催化成胶的聚丙烯酰胺凝胶(PAAgel)制备模拟肾间质纤维化组织硬度的凝胶基质(1~40kPa);接种人肾小管上皮细胞(HK-2)于不同硬度的PAA gel表面,采用免疫荧光染色和共聚焦显微镜考察基质硬度对HK-2形态的影响,并对HK-2细胞上的葡萄糖转运蛋白1 (GLUT1)、葡萄糖转运蛋白2 (GLUT2)和葡萄糖转运蛋白5 (GLUT5)的分布进行定性和半定量考察。研究发现,随着基质硬度增加, HK-2细胞上的GLUT1表达量显著降低, GLUT5在细胞整体的表达量显著下降,而GLUT2的表达和分布未见明显改变。
As the common pathway of chronic renal diseases leading to end-stage renal failure, renal tubulointerstitial fibrosis is characterized by the deposition of extracellular matrix and scar hardening. Our study aimed to construct an in vitro cell culture platform to explore the impact of matrix stiffness on cell morphology and function of renal tubular epithelial cells. Photopolymerized polyacrylamide gels(PAA gel) with varying stiffnesses as model substrates was selected to simulate the matrix stiffness of normal and fibrotic renal tissues with elastic moduli ranging from 1 to 40 kPa. The human renal tubular epithelial cells(HK-2) were seeded on the surface of PAA gels. The impact of matrix stiffness on the morphology of HK-2 were investigated via immunofluorescence staining and confocal microscopy. The expression levels of glucose transporter 1(GLUT1), glucose transporter 2(GLUT2), glucose transporter 5(GLUT5) were semi-quantitatively analyzed. With increasing matrix stiffness, both the levels of GLUT1 and GLUT5 in HK-2 cells were significantly decreased, whereas the expression level and the distribution pattern of GLUT2 in HK-2 remained unchanged with stiffness variation.
As the common pathway of chronic renal diseases leading to end-stage renal failure, renal tubulointerstitial fibrosis is characterized by the deposition of extracellular matrix and scar hardening. Our study aimed to construct an in vitro cell culture platform to explore the impact of matrix stiffness on cell morphology and function of renal tubular epithelial cells. Photopolymerized polyacrylamide gels(PAA gel) with varying stiffnesses as model substrates was selected to simulate the matrix stiffness of normal and fibrotic renal tissues with elastic moduli ranging from 1 to 40 kPa. The human renal tubular epithelial cells(HK-2) were seeded on the surface of PAA gels. The impact of matrix stiffness on the morphology of HK-2 were investigated via immunofluorescence staining and confocal microscopy. The expression levels of glucose transporter 1(GLUT1), glucose transporter 2(GLUT2), glucose transporter 5(GLUT5) were semi-quantitatively analyzed. With increasing matrix stiffness, both the levels of GLUT1 and GLUT5 in HK-2 cells were significantly decreased, whereas the expression level and the distribution pattern of GLUT2 in HK-2 remained unchanged with stiffness variation.
引文
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[29]Mihalko EP,Brown AC.Material strategies for modulating epithelial to mesenchymal transitions[J].ACS Biomater Sci Eng,2018,4:1149-1161.
[2]Ma LJ,Jha S,Ling H,et al.Divergent effects of low versus high dose anti-TGF-beta antibody in puromycin aminonucleoside nephropathy in rats[J].Kidney Int,2004,65:106-115.
[3]Tampe D,Zeisberg M.Potential approaches to reverse or repair renal fibrosis[J].Nat Rev Nephrol,2014,10:226-237.
[4]Zhong Y,Deng Y,Chen Y,et al.Therapeutic use of traditional Chinese herbal medications for chronic kidney diseases[J].Kidney Int,2013,84:1108-1118.
[5]LeBleu VS,Taduri G,O'Connell J,et al.Origin and function of myofibroblasts in kidney fibrosis[J].Nat Med,2013,19:1047-1053.
[6]Hodgkins KS,Schnaper HW.Tubulointerstitial injury and the progression of chronic kidney disease[J].Pediatr Nephrol,2012,27:901-909.
[7]Nangaku M.Mechanisms of tubulointerstitial injury in the kidney:final common pathways to end-stage renal failure[J].Intern Med,2004,43:9-17.
[8]Zeisberg M,Neilson EG.Biomarkers for epithelialmesenchymal transitions[J].J Clin Invest,2009,119:1429-1437.
[9]Shao K,Ding N,Huang S,et al.Smart nanodevice combined tumor-specific vector with cellular microenvironment-triggered property for highly effective antiglioma therapy[J].ACSNano,2014,8:1191-1203.
[10]Li X,Qu B,Jin X,et al.Design,synthesis and biological evaluation for docetaxel-loaded brain targeting liposome with“lock-in”function[J].J Drug Target,2014,22:251-261.
[11]Sabino-Silva R,Mori RC,David-Silva A,et al.The Na+/glucose cotransporters:from genes to therapy[J].Braz JMed Biol Res,2010,43:1019-1026.
[12]Wright EM,Loo DDF,Hirayama BA,et al.Surprising versatility of Na+-glucose cotransporters:SLC5[J].Physiology,2004,19:370-376.
[13]Liu X,Li W,Liang Z,et al.Prednisolone-glucose derivative conjugate:synthesis,biodistribution and pharmacodynamics evaluation[J].Arch Pharm,2012,345:925-933.
[14]Mueckler M,Thorens B.The SLC2(GLUT)family of membrane transporters[J].Mol Aspects Med,2013,34:121-138.
[15]Sugawara-Yokoo M,Suzuki T,Matsuzaki T,et al.Presence of fructose transporter GLUT5 in the S3 proximal tubules in the rat kidney[J].Kidney Int,1999,56:1022-1028.
[16]Liu F,Mih JD,Shea BS,et al.Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression[J].J Cell Biol,2010,190:693-706.
[17]Georges PC,Hui JJ,Gombos Z,et al.Increased stiffness of the rat liver precedes matrix deposition:implications for fibrosis[J].Am J Physiol Gastrointest Liver Physiol,2007,293:G1147-G1154.
[18]Pebworth MP,Cismas SA,Asuri P.A novel 2.5D culture platform to investigate the role of stiffness gradients on adhesion-independent cell migration[J].PLoS One,2014,9:e110453.
[19]Wang Y,Gong T,Zhang ZR,et al.Matrix stiffness differentially regulates cellular uptake behavior of nanoparticles in two breast cancer cell lines[J].ACS Appl Mater Interfaces,2017,9:25915-25928.
[20]Leight JL,Wozniak MA,Chen S,et al.Matrix rigidity regulates a switch between TGF-beta 1-induced apoptosis and epithelial-mesenchymal transition[J].Mol Biol Cell,2012,23:781-791.
[21]Stroka KM,Aranda-Espinoza H.Effects of morphology vs cell-cell interactions on endothelial cell stiffness[J].Cell Mol Bioeng,2011,4:9-27.
[22]Wang Y,Qin S,Gong T,et al.Matrix stiffness regulates cell uptake of nanoparticles in 2D and 3D cultures of breast cancer cells[J].Acta Pharm Sin(药学学报),2017,52:1324-1330.
[23]Arda K,Ciledag N,Aktas E,et al.Quantitative assessment of normal soft-tissue elasticity using shear-wave ultrasound elastography[J].Am J Roentgenol,2011,197:532-536.
[24]Derieppe M,Delmas Y,Gennisson JL,et al.Detection of intrarenal microstructural changes with supersonic shear wave elastography in rats[J].Eur Radiol,2012,22:243-250.
[25]Sommerer C,Scharf M,Seitz C,et al.Assessment of renal allograft fibrosis by transient elastography[J].Transpl Int,2013,26:545-551.
[26]Mitra SK,Hanson DA,Schlaepfer DD.Focal adhesion kinase:in command and control of cell motility[J].Nat Rev Mol Cell Biol,2005,6:56-68.
[27]Wang N,Butler JP,Ingber DE.Mechanotransduction across the cell-surface and through the cytoskeleton[J].Science,1993,260:1124-1127.
[28]Wei SC,Fattet L,Tsai JH,et al.Matrix stiffness drives epithelial mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway[J].Nat Cell Biol,2015,17:678-688.
[29]Mihalko EP,Brown AC.Material strategies for modulating epithelial to mesenchymal transitions[J].ACS Biomater Sci Eng,2018,4:1149-1161.