Experimental Study Of The Association Between Polymorphisms Of The Insulin Growth Factor-1 Promoter And Vasculopathy In Rat With Portal Hypertension

OBJECTIVE1.To construction of a stable,reliable,consistent of natural evolution of rat model withcirrhotic portal hypertension;to study liver lesions and vasculopathy induced by portalhypertension.2.To study expression change of insulin-like growth factor 1 (IGF-1) in cirrhotic portalhypertension in rats;to explore mechanism of IGF-1 in portal hypertensive vascularlesions.3.To study promoter polymorphism on IGF- 1 gene and its effect of IGF- 1 gene expression;to study the association between polymorphisms of the insulin growth factor-1 promoterand vasculopathy in rat with portal hypertension.METHODS1.To duplicate model of vasculopathy with portal hypertension by intraperitoneal injectionof CCL4;to detect liver lesion and vasculopathy by HE staining,Masson trichrome staining,transmission electron microscopy,CT scanning;to understand hemodynamicchange by portal venous pressure measurements.2 To detect mRNA and protein level of IGF- 1 in rat liver tissue by RT-Real Time-PCR andimmunohistochemistry;to detect serum IGF protein levels ELISA;and respectivelycompared the results with normal rats.3.To detect rats IGF-1 gene promoter by application of sequence-specific primer PCR anddissymmetry PCR of cirrhosis rat;to test IGF-1 gene promoter polymorphism byapplication of gene chip technology.RESULTS1.After intraperitoneal injection of CCL4,hepatic lobules structure can be seen disorders,reconstruction,fibrous tissue hyperplasia,accompanied by intrahepatic extensiveformation of false lobules by HE staining;vascular endothelial cells of portal veinhyperplasia,smooth muscle cells hypertrophy,fibroblasts and collagen fibers increase;thevolume of rat liver between liver cirrhosis group and control group is separately 9741±2715mm3 and 12898±3081 mm3 by CT scanning,the difference is statisticallysignificant;portal pressure between liver cirrhosis group and normal by manometry in ratsis 7.6±2.6mmHg and 12.9±3.7 mmHg respectively,difference between the two groupswas significant.2.IGF-1 protein expression of two groups of liver tissue by immunohistochemicaldetection is 0.40±0.13 and 0.68±0.19,and the difference has statistical significance;IGF-1 mRNA of two groups of liver is 3.95±2.26 and 6.12±3.14 By RT-Real Time-PCRDetection,and the difference has statistical significance;serum IGF protein is 687±137ng/ ml and 919±183 ng/ ml Measured by ELISA,and the difference has statisticalsignificance;there is a negative correlation between liver cells IGF-1 concentrations orserum IGF-1 concentration as well as liver cells IGF-1 mRNA concentration and portalvein pressure By the linear analysis.3.IGF-1 protein content caused different activity of gene promoter by -10 nucleotide polymorphism are 0.44±0.13 (AI) and 0.34±0.10 (AI) by sequence-specific primer PCRand asymmetric PCR and gene chip technology of liver cirrhosis in rats of liver cirrhosis,the difference has statistical significance;portal vein pressure were 13.0±3.5 (mmHg)and 9.6±2.6 (mmHg) caused by -35 nucleotide polymorphism,the difference between thetwo groups has statistical significanceCONCLUTIONS1.It is a stable model similarly to human natural course of disease established by CCL4which characterized with vasculopathy in portal hypertension;it is an ideal model forstudying portal portal hypertension which has a significant change of vein hemodynamics.2.The level of IGF-1 protein and gene transcription continues to drop according todeterioration of liver cirrhosis in rat model with cirrhotic portal hypertension.IGF-1 is aprotective factor in cirrhotic portal hypertension.3.Variation of IGF-1 gene promoter region on -10,-35 base locus can lead to activitychanges of IGF-1 gene promoter,which affect gene expression changes of IGF-1,therebyaffect the vasculopathy in portal hypertension.Polymorphism of IGF-1 gene promoterleads to individual reaction in course of cirrhotic portal hypertension.