Analysis Of The Differences In MicroRNA Expression Between Fibrotic And Normal Liver Tissue In Rats And Correlation Between The MicroRNA And Fibrotic Cytokines

Liver cirrhosis is late event of Chronic liver disease, patients with cirrhosis are more susceptible to hepatocarcinoma and liver failure. End-stage liver failure with poor clinical treatment, and bring a huge challenge to people's health and medical financial. At home and abroad, there is a lot of research on liver fibrosis, numerous studies have shown that mechanism of fibrosis is complex, and reversibility. And recent studies have shown that MicroRNAs play an important role in regulation the process of hepatic fibrosis, some MicroRNAs inhibitor could effectively reverse the process. Therefore the studies on MicroRNAs may bring new breakthrough for the clinical treatment of hepatic fibrosis.Part1Constructing the model of liver fibrosis in ratsObject:Make sure the model of liver fibrosis in rats was established.Methods:26male Wistar rats were randomly divided into two groups (A?B), with10rats in A,16rats in B. A was normal control group, given intraperitoneal injection with saline solution (0.5ml/only, twice a week) for12weeks; B was model group of liver fibrosis, from which was randomly selected3as B1group, handle with intraperitoneal injections with Porcine serum (0.5ml/only, twice a week) for4weeks; B2was selected as well as B1, handle with intraperitoneal injections with Porcine serum (0.5ml/only, twice a week) for8weeks; the rest10of B as B3, handle with intraperitoneal injections with Porcine serum (0.5ml/only, twice a week) for12weeks. The experimental animal of B1and B2groups were sacrificed respectively at the end of the4th week and8th week, leaving the liver tissue by HE staining and Masson staining. Dispose of the remaining experimental animal of A and B3groups after finishing the last injection at the end of the12th week. In the group of A and B3, first of all, the liver tissues were collected for HE and MASSON staining, using for observing the change of liver fibrosis on the histology; and then, according to the standard score of liver fibrosis on pathology, we can assess the degree of liver fibrosis in rats; at last, calculating the area of collagen fiber in semi-quantitative way.Results:1.HE and Masson staining suggested that there is no significantly pathological difference between A and B1; in group B2: hepatic cells showed swelling and degeneration, hepatic cord arranged in disorder, connective tissue was hyperplastic, fibrous septa was delicate, and the pseudolobule was could not been seen; in group B3:lobules were disordered, the arrangement of hepatic cord was irregular, hepatic sinuses were distortional, connective tissue was significantly hyperplastic, the fiber septa was formed;2. Compared with the control group A, the degree of hepatic fibrosis and the area of collagen fiber (%) were significantly increased in model group B3, and the difference was significant (P<0.01).Conclusion:We successfully established the model of liver fibrosis in rat through intraperitoneal injection with porcine serum.Part2Analysis of the MicroRNA's expression in fibrotic and normal liver tissue in ratsObject:Study the difference of the MicroRNA's expression between fibrotic and normal liver tissue in rats.Methods:Firstly, extracting the RNA from the liver tissue in three of model group B3and paired control group A, respectively. And then, all of the RNA was tested by deep sequencing technology in Shenzhen Huada Gene. Finally, validated the MicroRNA profiling results by real-time polymerase chain reaction (qPCR).Results:Compared with the MicroRNA's expression in normal liver, there were9kinds of MicroRNA's expression have changed in porcine serum-induced hepatic fibrosis, among which5kinds of MicroRNA (MicroRNA-1, MicroRNA-30b-5p, MicroRNA-144, MicroRNA-451, MicroRNA-674-3p) were downregulated, while4kinds of MicroRNA (MicroRNA-27a,MicroRNA-138, MicroRNA-146b, MicroRNA-342-5p) were upregulated respectively.Conclusion:There were significant differences between fibrotic and normal liver tissue on the expression of MicroRNA in rats, which may be related to the mechanisms of hepatic fibrosis.Part3Observe the correlation between MicroRNA-138and the fibrotic cytokinesObject:To Confirm that the MicroRNA-138is associated with liver fibrosis.Methods:Detecting the expression of MicroRNA-138in the liver tissue of20rats by real-time polymerase chain reaction (qPCR), as well the content of TGF-??CoL-1?TIMP-1?CTGF. And then analyzing the correlation between MicroRNA-138and TGF-??CoL-1?TIMP-1?CTGF by the software of SPSS.Results:1. qPCR test showed that the content of TGF-??CoL-1? TIMP-1?CTGF and MicroRNA-138in the hepatic fibrosis was higher than which in the normal liver tissue, and the difference was significant (P<0.05);2. Statistical analysis show that the correlation coefficients of MicroRNA-138and TGF-??CoL-1?TIMP-1?CTGF were0.818?0.913?0.87?0.82, respectively, and all of the difference were significant (p=0.000<0.01).Conclusion:MicroRNA-138may be play an important role in the occurrence of porcine serum-induced hepatic fibrosis in rat.