| Expression of AT1amRNA in rat hepatic stellate cells andthestudy on mechanism of hepatic fibrosisAbstractBackground and Objective Liver fibrosis is a common sequence to a variety of liver diseases of varying causes, In liver fibrosis, there is increased hepatic accumulation of extracellular martrix ( ECM ), most notably type I collagen, together with a wide variety of noncollagenous components such as proteoglycans and glycoproteins. There is little doubt that the key event in liver fibrosis is the injury-induced activation of hepatic stellate cells ( HSCs ) with their subsequent transformation to myofibroblastic cells that overproduce the complex array of ECM proteins ultimately deposited in fibrotic liver.Studies on HSCs activation in vitro have shown that this process is regulated by a wide variety of growth factors and cytokines derived from other liver cells ( paracrine factors ) or from HSCs themselves ( autocrine factors ). Angiotensin II ( AngII ) is one of intensive mitogens, whose receptor is including AT1 and AT2. AT1 can be divided into two subtypes:ATia and ATIb. ATia was detected in cardiac muscle, kidney and lung, and considered to play an important role in local tissue and apparatus fibrosis. But there are not any reports on hepatic fibrosis. We for the first time report AT1amRNA expressed on hepatic stellate cells and the effects of angiotensin II and its antagonist on the cells proliferation and synthesis of collagen. The aim of our study is to determine the effect of angiotensin and its receptor AT1a in the mechanism of hepatic fibrosis.-5-Methods l. Rat HSC was prlmary isoIated, cultlvated aud ldentifiedHSCs, isolated from Sprague-Dawley rats by density gradient centrifugation.,were collected and resuspended in Dulbecco's modifie Eagle's mediumcontaining l0 % fetal calf serum.Viability of the cells was evaluated by thetrypan blue exclusion test. The presence of vitamin A in HSCs was assessedby autofluorescence when irradiated at 328 nm. The antibody of desmin wasdetected by immunocytochemistry. 2. Study on expression of ATl,mRNAon HSCs Total RNA was extracted from HSCs and reversed transcripted.The cDNA fragments were arnplified by PCR with special primers, and thePCR products were purified to measure its sequence. The result wascompared with the Data Base of Gene Bank. 3. Study on AngII and AT1.antagonist affected on HSCs growth, Proliferat1on and coIlagenproduction. HSCs were affected with different concentrates of AngII andATl, antagonist: (l). Protract the growth curve of HSCs. (2). Survey thegrowth and proliferation of HSCs by the means of MTT (3). The proliferationof DNA was tested by 3H-TDR release assay. (4).The quantity of collagen..was examined by 'H-proline release assay.Result (2~3) X l0, HSCs were obtained and identified by autofluorescenceand the antibody of desmin, whose viability and purity were respectivelymore than 95% and 90%. ATl,mRNA was detected on HSCs by the mcthodof RT-PCR. PCR products' sequence was similar to the sequence ofATl,cDNA in the GeneBank, and the rate of similarity was 98.6 percent.Stimulated with AngII (its concentration more than 10-7mol/L ), HSCs couldproliferate greatly, and AT1. antagonist (its concentration more than l0-- 6 -6mol/L ) could inhibite the cell growing and multiplying. Moreover, the antagonist at the concentration of lO5mol/L could interdict the effect of AngIL The yield of collagen in HSC got a rise when affected with AngII (its concentration was from 10.8 molIL to 101c mol/L ). But the quantity of collagen dropped greatly when influenced by the antagonist of ATia( 10~ mol/L?I 0~c~ mol/L ).The one way-ANOVA showed that differences above had significant meaning when compared with the control groups (p |
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