Establishment Of Dynamic Hepatic Fibrosis Model In Mice And Study Of Extracellular Matrix Transcriptome

Objective: China is a region with chronic liver disease,especially chronic hepatitis B.The development of chronic hepatitis caused by liver fibrosis is the only way for chronic hepatitis B to develop into liver cirrhosis and liver cancer.Liver fibrosis occurs in almost all patients with chronic liver injury[5].Early detection and treatment of liver fibrosis is the key to the treatment of chronic liver disease.However,in addition to the etiological treatment,our current clinical treatment of liver fibrosis lacks other methods that can prevent its progression or reverse liver fibrosis;it also requires more efficient and sensitive detection in the diagnosis and assessment of liver fibrosis and its prognosis[6,7].The extracellular matrix(ECM)is a macromolecule with many biological activities.In the course of hepatic fibrosis,persistent chronic inflammation stimulates activation of stellate cells(HSCs),which in turn secretes large amounts of ECM proteins,matrix-degrading enzymes and their inhibitors,triggering the deposition and remodeling of ECM,once both Balance tends to ECM deposition,which leads to the development of fibrosis(52,53).Therefore,ECM is the most important protein component in the development of liver fibrosis(54-56).An in-depth understanding of the changes in the specific composition of ECM during the development of fibrosis will be of great significance for the development of new strategies for the diagnosis and treatment of fibrosis(29).Purpose:To establish the dynamic liver fibrosis model,application of transcriptomics technology for the preliminary screening and comparison of ECM protein changes in mouse liver fibrosis dynamic model,exploring the key ECM proteins during the development of liver fibrosis,and finding new markers for the diagnosis of hepatic fibrosis and potential new treatments Target.Method: This study starts with the extracellular matrix transcriptomics.First,a dynamic hepatic fibrosis model was created: Experimental animal healthymice were randomly divided into two groups,including the experimental group and the normal group.The normal control group was fed normally and the model group was injected intraperitoneally with carbon tetrachloride(CCl4 in olive oil,1:1 dilution)at 1mg/g twice a week.After 4 weeks and 10 weeks of CCL4 treatment and 2 weeks after stopping the injection of carbon tetrachloride,the mice were randomly treated.The livers of the mice were immediately removed,and the liver size was measured by a ruler;then,a portion of the liver tissues was quickly taken as a neutral buffer.The formalin fixative was fixed,embedded in paraffin,and stained with sirius scarlet;another part of the liver tissue was preserved with liquid nitrogen and used for m RNA gene chip screening.Afterwards,transcriptome analysis of RNA microarrays was performed on different stages of liver tissue samples.Extracellular matrix m RNA was screened out and compared between groups.Differential genes were compared,and then differential genes were analyzed by GO analysis and KEGG database.For the enrichment of a particular item,we can screen out the genes of interest from the differential genes for subsequent studies.Results: Part I: In the liver fibrosis model of each group,the results of Sirius red collagen staining showed that there were no collagen staining in the normal hepatic hilus and other normal blood vessel walls.No staining was found in other parts of the liver;CCL4 4w group mice intrahepatic collagen Sedimentation was mainly located between the portal area and the portals.Fibrous hyperplasia between the portal veins was evident in the lobules,and bridging fibers were partially formed.Collagen was further increased between the portals of the CCL4 10 w group,and bridging fibers were formed between most of the portals.The hepatic lobules have been completely divided by the surrounding bridging fibers,and pseudolobules have formed;the collagen fibers between the portal areas of the CCL4 2w AFD group have been significantly reduced compared with the CCL410 W group,and the bridging fibers have been ablated.Histopathological sections showed that each model was successfully prepared and could be used in the next transcriptomic study.Part II: Differential genes m RNA were screened in the stromal group.We compared the differential genes that were screened in the CCL4 TO the normal group,there are total194 differential genes.In the phase up-regulation genes are total of 126,includeCol1a1,Adam10.The down-regulation genes are total of 68,include Loxl4,Lamc2.In the CCL4 10 W to CCL4 4W compared group,extracellular matrix transcriptomics screened the most differential genes,a total of 56 of which,The number of differentially up-regulated genes was27,include Col1a1,Col1a2 and Col6a3.MMP14,MMP24 were down-regulated.At CCL4 2W AFD to CCL4 10 W group,the number of differentially up-regulated genes was104,and the number of down-regulated genes was 82,especially the collegens types of Col1a1,Col1a2,Col3a1,Col4a2 and the matrix metalloproteins of MMP2.CCL4 2W AFD after induction of drug discontinuation Compared with the normal group,the total number of differentially screened genes was 123,with a total number of up-regulated genes of 67 as the collgens like Col1a1,and a total of 56 down-regulated genes as Col1a2.The MMPs of the MMP9 still highly expression.Conclusions: Differential genes m RNA were screened in the stromal group.The protein may be the weight of the development of liver fibrosis.