| PartⅠInhibitory Effects of siRNA on IGFBPrP1 Gene Expression in Hepatic Stellate CellsObjectiveDesigned and chemically synthesized two pairs small interfering RNAs(siRNAs) specifically knocking down IGFBPrPl gene. To study the inhibitory effects of siRNAs on IGFBPrP1 gene expression and protein expression in hepatic stellate cells. Selected one pair siRNA with the better inhibitory effect on IGFBPrP1 gene expression. To investigate the function of IGFBPrPl protein in hepatic stellate cells.Methods1. Hepatic stellate cells were transfected with FAM-Negative siRNA (10,30,50nmol/L). Fourty-eight hours later, transfection efficiencies were evaluated by fluorescent microscopy.2.30nmol/L IGFBPrP1 siRNA(siRNA1,siRNA2) and negative control siRNA were respectively transfected into HSC-T6 cells. Using FQ-RT-PCR, we measured the expression of IGFBPrP1 gene 48 hours after transfection.3.30nmol/L IGFBPrP1 siRNA (siRNA1,siRNA2) and negative control siRNA were respectively transfected into HSC-T6 cells. Using Western blot method, we measured the expression of IGFBPrP1 protein 48 hours after transfection.Results1. Green fluorescence was discovered under the fluorescence microscope with 10,20 and 30 nmol/L FAM-Negative siRNA was transfected into HSC-T6 cells, and no signals were discovered in control group. The transfection efficiency of 30,50nmol/L FAM-Negative siRNA was remarkably higher than 10nmol/L FAM-Negative siRNA transfection groups (P<0.05). There was no significant difference in transfection efficiency between 30nmol/L and 50nmol/L siRNA groups (P<0.05). There was high death rates in 50nmol/L siRNA group. The rate of transfection was about 70 percent.2.30nmol/L IGFBPrPl siRNA (siRNA1,siRNA2)and negative control siRNA were respectively transfected into HSC-T6 cells.The expression of IGFBPrP1 gene in siRNA1 group were significantly decreased than those in siRNA2 group and negative control group (P<0.01).3.30nmol/L IGFBPrP1 siRNA (siRNA1,siRNA2)and negative control siRNA were respectively transfected into HSC-T6 cells.The expression of IGFBPrP1 protein in siRNA1 group were significantly decreased than those in siRNA2 group and negative control group (P<0.01).Conclusions1. The chemically synthesized siRNA can be transfected into HSC-T6 cells successfully. Better transfection efficiency can be obtained in 30nmol/L siRNA transfected HSC-T6.2. Two pairs chemically synthesized IGFBPrP1 siRNAs (siRNA1,siRNA2) were transfected into HSC-T6 cells.Inhibitory effects of siRNA1 were notable on the expression of IGFBPrP1 mRNA and protein.PartⅡEffect of IGFBPrP1 siRNA on extracellular matrix secretion in the activated hepatic stellate cellsObjectiveTo investigate the effect of inhibiting insulin-like growth factor binding protein related protein1 (IGFBPrP1) by chemically synthesized small interfering RNA (siRNA) on the secretion of extracellular matrix (ECM) in the activated hepatic stellate cells (HSC-T6). MethodsDivide the HSC-T6 cells into three groups as following normal control group (incubated with equal PBS) and negative control group (HSC-T6 treatment with Negative siRNA control or FAM-Negative siRNA) and IGFBPrP1 siRNA group (HSC-T6 treatment with effective IGFBPrPl siRNA). HSC-T6 were treated by IGFBPrP1 siRNA, then the supernatant were collected and cytoplasmic proteins were extracted after 72 hours. The expression levels of IGFBPrP1 and collagenⅠand fibronectin were detected by Western blot.Results1. Compared with that of in normal control group (0.42±0.03) and negative control group (0.40±0.03), the expression level of IGFBPrP1 in IGFBPrP1 siRNA group (0.21±0.04) were significantly decreased (F=48.018, P<0.01).2. Compared with that of in normal control group (0.61±0.03) and negative control group (0.35±0.05), the content of collagenⅠin IGFBPrP1 siRNA group (0.63±0.03) were markedly reduced (F=81.246, P<0.01).3. Compared with that of in normal control group (0.76±0.07) and negative control group (0.74±0.05), the change level of fibronectin in IGFBPrP1 siRNA group (0.43±0.04) were significantly decreased (F=43.850, P<0.01)Conclusions1. siRNA targeting IGFBPrP1 specifically inhibits the expression of IGFBPrP1 in HSC-T6.2. IGFBPrP1 can regulate the synthesis and secretion of collagenⅠand fibronectin in HSC-T6, then lead to the formation of liver fibrosis.PartⅢPrimary study on relationship between IGFBPrP1 and TGFβ1ObjectiveTo investigate the effect of TGFβ1 on the expression of IGFBPrP1 and Collagen and FN in HSC-T6. And to observe the changes of biological character (including collagenⅠand FN and TGFβ1) affected by TGFβ1 after siRNA knocking down IGFBPrP1. The purpose of the study is to clear the role of IGFBPrP1 in liver fibrosis.Methods1. HSC-T6 were cultured in vitro and established the normal control group (incubated with equal PBS) and TGFβ1 treatment group (TGFβ1 treatment with 4ng/ml).HSC-T6 were treated by TGFβ1 for 24h, then the expression levels of IGFBPrP1 and collagenⅠand fibronectin were detected by Western blot. The relationship of IGFBPrP1 with CollagenⅠand FN was analyzed.2. Divide the HSC-T6 cells into three groups as following negative control group (HSC-T6 treatment with Negative siRNA control or FAM-Negative siRNA) and IGFBPrP1 siRNA group (HSC-T6 treatment with effective IGFBPrP1 siRNA) and IGFBPrP 1 siRNA+TGFβ1 group (HSC-T6 were treated by effective IGFBPrP1 siRNA, then incubated with 4ng/ml TGFβ1). HSC-T6 were treated by TGFβ1 for 24h, then the expression levels of IGFBPrP1 and TGFβ1 and collagenⅠand fibronectin were detected by Western blot. Results1. Effects of TGFβ1 on the expression of IGFBPrP1 and collagenⅠand fibronectin in HSC: The expression levels of IGFBPrPl and collagenⅠand fibronectin in TGFβ1 group were significantly increased than those in normal control group (IGFBPrP1:0.58±0.09 vs 0.40±0.07, t=3.178, P<0.05; CollagenⅠ:0.79±0.08 vs 0.58±0.08, t=3.649, P <0.05; FN:0.84±0.09 vs 0.66±0.06,t=3.451, P<0.05). There was a significantly positive correlation between IGFBPrPl and CollagenⅠor FN in HSC-T6 treated TGF-β1 (r =0.787,0.814, P<0.05)2. Effects of siRNA knocking down IGFBPrP1 on the expression of IGFBPrP1 and collagenⅠand fibronectin and TGFβ1 in HSC induced by TGFβ1:The expression levels of IGFBPrP1 and collagenⅠand fibronectin and TGFβ1 in IGFBPrP1 siRNA group were significantly decreased than those in negative control group (IGFBPrP1:0.22±0.06 vs 0.40±0.03, F=19.384, P<0.01; CollagenⅠ:0.36±0.12 vs 0.61±0.09, F=14.711, P <0.05; FN:0.44±0.10 vs 0.75±0.11, F=25.591,P<0.05; TGFβ1:0.17±0.03 vs 0.23±0.02, F=7.321, P<0.05). The expression levels of collagenⅠand fibronectin in the TGFβ1-stimulating HSC treated with IGFBPrP1 siRNA were significantly increased than those in negative control HSC without IGFBPrP1 siRNA. (Collagen I:0.79±0.12; FN: 0.90±0.06, P<0.05). And the expression levels of IGFBPrP1 and TGFβ1 were no significant difference. (P>0.05). Compared with the IGFBPrP1 siRNA group, the expression levels of IGFBPrP1 and collagenⅠand fibronectin were significantly increased (P<0.01). And the expression of TGFβ1 were no significant difference. (P> 0.05).ConclusionThe generation of IGFBPrPl and TGFβ1 that has been considered as the most strong factor to liver fibrosis maybe a causal relationship in the development of hepatic fibrogenesis. |
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