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The Role of Galectin-1 on Epithelial to Mesenchymal Transition Induced by High Glucose Peritoneal Dialysate in Human Peritoneal Mesothelial Cells

Author: DaiHongQin
Tutor: LiuYingHong
School: Central South University
Course: Internal Medicine
Keywords: galectin-1 High sugar Dialysate Epithelial - mesenchymal transdifferentiation (EMT) siRNA EMT
CLC: R459.5
Type: Master's thesis
Year: 2010
Downloads: 68
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Abstract


Background: Peritoneal Dialysis (Peritoneal dialysis, PD) is one of three ways renal replacement therapy. Ultrafiltration failure (Ultrafiltration Failure, UFF) is the treatment of peritoneal dialysis patients quit one of the most important reason. Numerous studies have demonstrated that high concentrations of glucose peritoneal dialysis solution caused morphological and functional changes in peritoneal cell damage and repair even cells into mesenchymal transdifferentiation (Epithelial-to-mesenchymal Transition, EMT), eventually leading to the peritoneum fibrosis (Peritoneal fibrosis, PF) and ultrafiltration failure occurs. But on the molecular mechanism of peritoneal fibrosis has not yet fully elucidated. Galectin-1 is the first reported mammalian galectin involved in cell adhesion, proliferation, differentiation, apoptosis, inflammation and other physiological and pathological processes, and in the immune system and tumor occurrence and development play an important role. Our previous studies through proteomics methods found that high glucose peritoneal mesothelial cells after galectin-1 expression was significantly increased, suggesting that galectin-1 may play a role in a certain part of peritoneal fibrosis. The latest research report found that galectin-1 pathway inhibited by blocking TGF-β/Smad tubular epithelial cells the expression of collagen Ⅰ, but galectin-1 induced by high glucose dialysate peritoneal mesothelial cells in vitro expression and EMT, EMT, relationships at home and abroad has not been reported. vimentin and zo-1 is a common EMT-related indicators. Vimentin (vimentin) are stromal cells in the middle of the main fiber, it is present in the cells of mesodermal origin, such as fibroblasts, endothelial cells and white blood cells, and with microtubules, actin cytoskeleton together form a network while maintaining cell integrity. Tight junction-associated protein 1 (zonula occluden-1, ZO-1) is composed of tight junctions of the stent, is to maintain the blood vessels and dialysate osmotic pressure gradient on both sides of the correlation structure. Increased expression of Vimentin and zo-1 expression in cells suggested that the loss may occur EMT. RNA interference (RNA interference, RNAi) is a double-stranded RNA (double-stranded RNA, DsRNA) induced homologous mRNA decay, giving rise to post-transcriptional gene silencing (post-transcriptional gene Silencing, PTGS), has been successfully used in gene function and signaling systems downstream molecules relationship studies, there may become antisense, ribozymes after another new means. Objective: To observe the different concentrations of glucose dialysate on peritoneal mesothelial cells (HPMC) galectin-1 and EMT-related indicators vimentin, zo-1 expression, preliminary study galectin-1 relationship with EMT. Methods: A human peritoneal mesothelial cells for the study were randomly divided into four groups: normal control group (5.5mmol / L), 1.5% PDS group, 2.5% PDS group and 4.25% PDS group. Using real-time quantitative PCR (Real-time PCR) determination of galectin-1, vimentin and zo-1mRNA expression levels; Western blot (Westem-blot) method to measure galectin-1, vimentin and zo-1 protein expression. Analysis of galectin-1 and Vimentin, zo-1 expression correlation. Results: (1) normal culture conditions, HPMC express galectin-1, only a small amount of vimentin expression and a certain amount of zo-1. (2) 4.25% PDS stimulation HPMC at different times, galectin-1 protein expression gradually increased, 12h, 24h, 48h galectin-1 protein expression in the control group were 1.09 times, 1.22 times and 1.42 times, were statistically significance (P lt; 0.05). 72h galectin-1 protein expression decreased slightly compared with the previous, 1.37 times for the control group, compared with the control group were significantly different (P lt; 0.05). (3) different concentrations of glucose dialysate stimulated HPMC galectin-1 mRNA and protein expression levels gradually increased in a dose-dependent manner. Galectin-1 mRNA expression in the control group were 1.22 times, 2.34 times and 1.535 times (P lt; 0.05). 1.5% PDS group, 2.5% PDS Group, 4.25% PDS group galectin-1 protein expression in the control group were 1.48 times, 2.01 times and 2.38 times, compared with the control group, were statistically significant (P lt; 0.05). (4) high glucose stimulation HPMC48h PDS after, vimentin mRNA and protein levels were significantly increased in a dose-dependent manner. Compared with the control group, vimentin mRNA expression in the control group were 1.16 times, 1.27 times and 1.69 times (P lt; 0.05). vimentin protein expression in the control group were 1.28 times, 1.51 times and 1.92 times (P lt; 0.05). (5) high glucose stimulation HPMC48h PDS after, zo-1 mRNA and protein levels were significantly reduced in a dose-dependent manner. Compared with the control group, zo-1 mRNA expression in the control group were 77%, 43% (P lt; 0.05) and 20% (P lt; 0.05). zo-1 protein expression in the control group were 88%, 52% (P lt; 0.05) and 40% (P lt; 0.05). (6) galectin-1 mRNA expression of vimentin mRNA expression was positively correlated (r = 0.864, P lt; 0.05), and zo-1 mRNA expression was negatively correlated (r = -0.604, P lt; 0.05); galectin-1 protein levels of protein expression and vimentin expression was positively correlated (r = 0.917, P lt; 0.05); galectin-1 protein levels and zo-1 protein expression was negatively correlated (r = -0.869, P lt; 0.05) Conclusions: (1 ) galectin-1 in human peritoneal mesothelial cells (HPMCs) has expressed high glucose dialysate can increase galectin-1 expression in a dose and time dependent. (2) high glucose dialysate could induce transdifferentiation HPMCs occur. (3) galectin-1 induced by high glucose dialysate HPMCs transdifferentiation related. Objective: galectin-1 siRNA transfection inhibited human peritoneal mesothelial cells galectin-1 expression, detection vimentin, zo-1 expression was observed galectin-1 siRNA can inhibit the high glucose-induced HPMCs dialysate transdifferentiation. Methods: siRNA liposome transfection to HPMC, the HPMC is divided into three groups: normal control group (non-transfected HPMC 5.5mmol / L glucose), high glucose group (non-transfected HPMC 4.25% high glucose stimulation PDS ) and transfection group (galectin-1 siRNA transfection of HPMC 4.25% PDS high glucose stimulation). Using real-time quantitative PCR (Real-timePCR) Determination of galectin-1, vimentin and zo-1mRNA expression levels; Western blot (Westem-blot) method to measure galectin-1, vimentin and z0-1 protein expression. Results: (1) 4.25% PDS high glucose stimulation HPMCs 48h, galectin-1 mRNA and protein expression compared with normal control group were significantly increased (P lt; 0.05). vimentin mRNA and protein expression increased compared with normal control group, zo-1 mRNA and protein expression decreased compared with normal control group, were statistically significant (P lt; 0.05). (2) galectin-1 siRNA was transfected into HPMCs after stimulation with high glucose compared, galectin-1 mRNA and protein expression were inhibited (P lt; 0.05), while vimentin mRNA and protein expression was significantly reduced, zo- 1 mRNA and protein expression was significantly increased, were statistically significant (P lt; 0.05). Conclusion: galectin-1 siRNA can reduce high glucose dialysate peritoneal mesothelial cells induced EMT level.

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