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The Effect of Smad3 Knockdown on the Role of Hepatic Stellate Cells Induced by IGFBPrP1

Author: CaoPing
Tutor: LiuLiXin
School: Shanxi Medical
Course: Internal Medicine
Keywords: Hepatic stellate cells Smad3 RNA interference Insulin-like growth factor binding protein -related protein 1 Transforming growth factor β1
CLC: R575.2
Type: Master's thesis
Year: 2010
Downloads: 48
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Abstract

Background: a variety of causes can cause local inflammation of the liver, followed by excessive deposition of extracellular matrix resulting collagen, thus the formation of liver fibrosis. Liver fibrosis is the development of a variety of chronic liver disease to cirrhosis necessary stage and the central link is a reversible disease. In recent years, research on the pathogenesis of hepatic fibrosis has made considerable progress, a large number of studies have shown that hepatic stellate cells (hepatic stellate cells, HSC) is the extracellular matrix (extracellular matrix, ECM) of the main cell source in the liver fibrosis plays a critical role. Liver fibrosis is a multiple cytokines and multiple cell signaling pathways involved in the complex pathological process. Signal transduction pathways involved in liver fibrosis: Smad signaling pathway, JAK / STAT signaling pathway, NF-κB signaling pathway and MAPK signaling pathways, including Smad signaling pathway is to study a clearer signal transduction pathway. Studies have shown that animal liver fibrosis is often accompanied by the HSC progressive increase in the number of Smad3 mRNA expression level was significantly increased, suggesting that the Smad signaling pathway plays a crucial role in the development of liver fibrosis. RNA interference (RNA interference, RNAi) is a the biosphere widespread conservative defensive reaction, discovered in recent years as an important way of post-transcriptional gene silencing by small double-stranded RNA to block the expression of specific genes induced mRNA degradation, so as to achieve the effect of \Insulin-like growth factor binding protein-related protein 1 (insulin-like growth factor binding protein related protein 1, IGFBPrP1) is a relative molecular mass of the 31 KD soluble secreted proteins. Mentor Liu Lixin the study found IGFBPrP1 may be pathogenic factor in the mechanism of liver fibrosis. The IGFBPrP1 the expression of TGFβ1 and collagen expression in the liver tissue of patients with liver fibrosis has a positive correlation. Recombinant IGFBPrP1 can be induced HSC activation and to the activation of HSC increased synthesis of ECM, the anti IGFBPrP1 antibody can reduce liver fibrosis in mouse livers ECM deposition, and a positive correlation between changes in the expression of ECM with Smad3. Clear whether impact of IGFBPrP1 the ECM through Smad3 signaling pathway, and the effects of the IGFBPrP1 expression of HSC TGFβ1 design this topic. Objective: To investigate the first part of the siRNA Smad3 gene expression of hepatic stellate cells Smad3 for Smad3 gene in rats siRNA Smad3 gene expression in hepatic stellate cells. Method: 1. Transfected cells: using small RNA interference (RNAi) technology, will be non-specific with fluorescently labeled siRNA (NC siRNA transfected HSC-T6 cell lines in vitro, with for Smad3 mRNA no homology 21ntsiRNA) and transfection efficiency is detected under a fluorescence microscope. Screening effective siRNA: The experiment was divided into 4 groups: negative control group, two pairs of siRNA group and the control group. The application of real-time quantitative PCR (RT-PCR) and Western blot analysis of the efficiency of siRNA repression of HSC-T6 Smad3 mRNA and protein. The results: 1. For 24h after transfection cells was observed under a fluorescence microscope, visible green fluorescence, indicating successful transfection, the transfection efficiency was approximately 70%. 48h after transfection, RT-PCR and Western blot analysis show siRNA1, siRNA2 transfection group of Smad3 mRNA inhibition rate of 32% and 41%, respectively, siRNA2 transfected group Smad3 protein expression level of inhibition rate was 61.5%, compared with the negative control group, the difference was statistically significant (P lt; 0.01) inhibition rate of 7.5%, while the siRNAl Smad3 protein expression levels of transfected group, no significant difference compared with the negative control group. The prompt siRNA2 Smad3 expression better inhibition efficiency, after the experiments were conducted with siRNA2. Conclusion: Smad3 by siRNA can effectively inhibit Smad3 mRNA and protein expression in HSC-T6 cells lines. The second part IGFBPrP1 silent for a the Smad3 hepatic stellate cells secrete extracellular matrix purpose: clear the insulin-like growth factor binding protein-related protein 1 (IGFBPrPl) whether the secretion of extracellular matrix by Smad3 ring HSC. Method: 1. Choose vitro cultured hepatic stellate cells (HSC-T6) were established as a research object, 30μg / L IGFBPrPl treatment group and the control group (adding an equal amount of phosphate buffer solution (PBS)), the intervention factor treatment 48h cell culture supernatants were collected by Western blot assay by IGFBPrP1 stimulate HSC of the supernatant CollagenI and FN expression. . Cultured in vitro hepatic stellate cells (HSC-T6) as the object of study, namely the establishment of the negative control group (transfected with NC siRNA), Smad3 RNAi group (transfection of Smad3 siRNA2) and of Smad3 RNAi IGFBPrPl group (Smad3 siRNA2 turn 24h after transfection, plus 30μg / L IGFBPrP1 role 48h) and IGFBPrP1 group (culture medium plus 30μg/LIGFBPrPl at 48h). Groups of total cellular protein were extracted from the culture supernatant was collected (?) Vestern blot assay groups Smad3, Collagen I and FN expression. Results: Western blot results show IGFBPrP1 role in HSC after Collagen I and FN expression compared with blank control group was significantly higher (0.74 ± 0.09 vs 0.53 ± 0.07; 0.63 ± 0.07 vs 0.46 ± 0.07, P lt; 0.01) , a statistically significant difference. 2. Smad3 RNAi group Smad3 expression compared to the negative control group was significantly lower (0.14 ± 0.02 vs 0.33 ± 0.06, P lt; 0.01). Smad3 RNA interference, another set of experiments first HSC-T6 then joined IGFBPrP1 were stimulated expression of Collagen I and FN over IGFBPrP1 group was significantly lower (0.42 ± 0.05 vs 0.74 ± 0.09; 0.34 ± 0.06 vs 0.63 ± 0.07, P lt; 0.01). Conclusion: IGFBPrP1 important components of the ECM can be increased secretion of Collagen I and FN, the role of one of the mechanisms through Smad3 signaling pathway. Third part IGFBPrP1 on hepatic stellate cells TGFβ1 Expression Preliminary purpose: To observe the insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) effects of transforming growth factor β1 (transforming growth factor beta 1, TGFβ1) expression of HSC to determining IGFBPrP1 be taken as one of the causes of TGFβ1. Methods: select vitro hepatic stellate cells (HSC-T6) were established as a research object, IGFBPrP1 10μg / L 20μg / L, 30μg / L treatment group and the control group (adding an equal amount of phosphate buffered solution (PBS). ), intervention factor for 24h the cells were collected after climbing film and culture supernatant using immunocytochemistry staining HSC in TGFβ1 expression changes; enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants of TGFβ1 content. Western blot assay by 30μg / L IGFBPrP1 stimulate HSC culture supernatant of TGFβ1 expression. Results: Immunocytochemistry results show: brownish yellow or brown particle deposition, TGFβ1 positive expression were seen in the cytoplasm of cells. Image analysis showed IGFBPrP1 10μg / L group (10.90 ± 0.35), 20μg / L group (12.09 ± 0.54), 30μg / L group (11.89 ± 0.32) of TGFβ1 expression than those of the control group (7.71 ± 0.63) was significantly enhanced, and dose-dependent manner within a certain range. ELISA showed: increased of TGFβ1 in the blank control group has been a certain amount of secretion, but 20μg / L group (169.59 ± 24.87), 30μg / L group (188.34 ± 32.24) of TGFβ1 content is still blank control group (136.16 ± 19.52) , the difference was statistically significant (P lt; 0.05). Western blot analysis showed: IGFBPrP1 group (0.46 ± 0.05) TGFβ1 expression than the control group (0.30 ± 0.02) increased, the difference was statistically significant (P lt; 0.05). Conclusion: IGFBPrP1 HSC synthesis and secretion of TGFβ1 vitro can be increased, and gradually increased with the increase of IGFBPrP1 dose TGFβ1 expression in a certain dose range.

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