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The first part of siRNA on hepatic stellate cell inhibition of gene expression IGFBPrP1 purpose design, chemical synthesis of two pairs for insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) gene siRNA, studies on rats IGFBPrP1 inhibition of gene and protein expression role more significant inhibition screened pair IGFBPrP1 siRNA, used for subsequent experiments on rat hepatic stellate cells (HSC-T6) in IGFBPrP1 function studies. Method 1. Application 10,30,50 nmol / L synthetic FAM-Negative siRNA transfected HSC-T6 cells 48h after transfection assessed by fluorescence microscopy transfection efficiency. 2.30nmol / L IGFBPrP1 siRNA (siRNA1, siRNA2) and negative control were transfected into HSC-T6 cells, cultured 48h, using quantitative real-time RT-PCR method for detection of HSC-T6 in IGFBPrPl gene (β-actin as an internal reference ) 3.30nmol / L IGFBPrP1 siRNA (siRNA1, siRNA2) and negative control were transfected into HSC-T6 cells, cultured 48h, detected by Western blot using HSC-T6 in IGFBPrP1 protein (β-actin as an internal reference) Result 1.10 , 30,50 nmol / L of FAM-Negative siRNA transfected cells were seen within the green fluorescence, the fluorescence signal control group, 30nmol / L and 50nmol / L siRNA transfection efficiency was higher than 10nmol / L group (P lt; 0.05); 30nmol / L and 50nmol / L siRNA transfection efficiency group had no significant difference (P gt; 0.05), but 50nmol / L of cell death serious, transfection efficiency of about 70%. 2.30nmol / L IGFBPrP1 siRNA transfected HSC-T6 cells, siRNAl group in HSC IGFBPrP1 gene expression levels were significantly lower than the negative control group and siRNA2 group (P lt; 0.01) 3.30nmol / L IGFBPrP1 siRNA transfected HSC-T6 cells, siRNA1 group, the HSC IGFBPrP1 protein levels were significantly lower than the negative control group and siRNA2 group (P lt; 0.01) Conclusion 1. chemically synthesized siRNA can be successfully transfected HSC-T6, 30nmol / L of siRNA can get a good turn dyeing effect. (2) The two pairs of chemically synthesized siRNA transfected HSC-T6 48h after the mRNA and protein levels, consistent test results, which siRNA1 significant inhibition. The second part IGFBPrP1 siRNA on the activation of hepatic stellate cells secrete extracellular matrix affect the use of chemically synthesized siRNA purposes inhibition of insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) gene expression study its effect on hepatic stellate cells, extracellular matrix expression. Methods rat hepatic stellate cells (HSC-T6) as the research object, namely the establishment of the normal control group (equal volume PBS), negative control group (transfected with Negative siRNA control or FAM-Negative siRNA as a control) and IGFBPrP1 siRNA interference group (the screening of siRNA to inhibit the efficient 30nmol / L transfected HSC-T6). siRNA transfected HSC-T6 72h later, the cells were collected and extracted supernatant cytoplasmic protein, using Western blot, HSC-T6 cytoplasmic proteins and culture supernatant IGFBPrP1 type Ⅰ collagen and fibronectin expression. Results 1. Western blot detection IGFBPrP1 expression: Compared with normal control group (0.42 ± 0.03) and negative control group (0.40 ± 0.03) compared to, IGFBPrP1 siRNA interference group IGFBPrP1 relative protein expression (0.21 ± 0.04) was significantly lower (F = 48.018, P lt; 0.01). 2. Western blot detection of collagen type Ⅰ expression: IGFBPrP1 siRNA interference group Ⅰ collagen relative protein expression (0.63 ± 0.03) compared with normal control group (0.61 ± 0.03) and negative control group (0.35 ± 0.05) was significantly reduced (F = 81.246, P lt; 0.01). 3. Western blot detection of the expression of fibronectin: fibronectin interference group relative protein expression (0.43 ± 0.04) compared with normal control group (0.76 ± 0.07) and negative control group (0.74 ± 0.05) was significantly lower (F = 43.850 , P lt; 0.01). Conclusion 1. IGFBPrP1 siRNA can specifically inhibit IGFBPrP1 in HSC-T6 expression. 2. IGFBPrP1 regulate HSC-T6 in Collagen Ⅰ and FN synthesis and secretion function, thereby leading to liver fibrosis. The third part IGFBPrP1 with TGFβ1 Relationship of Objective TGFβ1 on HSC-T6 in IGFBPrP1 and extracellular matrix expression; through the application of siRNA blocking HSC-T6 in IGFBPrP1 gene expression observed TGFβ1 to its extracellular matrix effects, to clarify IGFBPrP1 in liver fibrosis position. Method 1. Choose rat hepatic stellate cells (HSC-T6) as the research object, namely the establishment of the normal control group (equal volume PBS) and TGFβ1 treatment group (4ng/ml added to a final concentration of TGFβ1). TGFβ1 for 24h, using the Western blot assay HSC-T6 cytosolic protein in the culture supernatant IGFBPrP1 and Collagen Ⅰ and FN expression changes, and make IGFBPrP1 with Collagen Ⅰ and FN correlation analysis. (2) the rat hepatic stellate cells (HSC-T6) were divided into three groups: negative control group (transfected with Negative siRNA control or FAM-Negative siRNA as a control), siRNA interference group (will be screened high inhibition efficiency siRNA to 30nmol / L transfected HSC-T6) and siRNA TGFβ1 group (will inhibit the efficient transfection of siRNA HSC-T6, then add TGFβ1 stimulation of the HSC-T6). TGFβ1 for 24h, using the Western blot assay] HSC-T6 cytosolic protein in the culture supernatant IGFBPrP1 and TGFβ1, Collagen Ⅰ and FN expression changes. Results 1.TGFβ1 on HSC-T6 in IGFBPrP1, Collagen Ⅰ and FN expression: TGFβ1 treated IGFBPrP1, Collagen Ⅰ and FN relative protein expression compared with the normal control group was significantly increased (IGFBPrP1: 0.58 ± 0.09 vs 0.40 ± 0.07, t = 3.178, P lt; 0.05; Collagen Ⅰ: 0.79 ± 0.08 vs 0.58 ± 0.08, t = 3.649, P lt; 0.05; FN: 0.84 ± 0.09 vs 0.66 ± 0.06, t = 3.451, P lt; 0.05). IGFBPrP1 expression changes with Collagen Ⅰ and FN expression was positively correlated (r values ??were 0.787,0.814, P lt; 0.05). 2. IGFBPrP1 siRNA against TGFβ1 stimulated HSC-T6 in IGFBPrP1, Collagen Ⅰ, FN and TGFβ1 expression: siRNA interference group IGFBPrP1, Collagen Ⅰ, FN and TGFβ1 relative protein expression compared with the negative control group was significantly lower (IGFBPrP1: 0.22 ± 0.06 vs 0.40 ± 0.03, F = 19.384, P lt; 0.01; Collagen Ⅰ: 0.36 ± 0.12 vs 0.61 ± 0.09, F = 14.711, P lt; 0.05; FN: 0.44 ± O.10 vs 0.75 ± 0.11, F = 25.591, P lt; 0.05; TGFβ1: 0.17 ± 0.03 vs 0.23 ± 0.02, F = 7.321, P lt; 0.05); siRNA TGFβ1 group Collagen Ⅰ and FN expression compared to the negative control group (Collagen Ⅰ: 0.79 ± 0.12; FN: 0.90 ± 0.06) increased significantly (P lt; 0.05), IGFBPrP1 and TGFβ1 expression did not change significantly (P gt; 0.05); siRNA interference group and siRNA TGFβ1 group, siRNA TGFβ1 group IGFBPrP1, Collagen Ⅰ and FN expression was significantly increased (P values ??were lt; 0.01), TGFβ1 expression no significant change (P gt; 0.05). Conclusion IGFBPrP1 with hepatic fibrosis induced cytokine TGFβ1 in the strongest development of liver fibrosis may mutually reinforcing.
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