Biological Role Of Extracellular Matrix In Liver Physiological And Pathological Tissue And Its Application Research

Part I Background:Liver transplantation is the only effective treatment for end-stage liver disease and one of the most effective diagnosis and treatment for various benign and malignant liver tumors.However,due to the shortage of liver source,as well as the high price in China,most patients cannot get the best diagnosis and treatment.Therefore,an alternative product for liver transplantation is urgently needed,which can fill the gap of liver source.In recent years,a lot of progress has been made in liver tissue engineering.However,under the current development level of tissue engineering current,artificial liver still lacks a microenvironment similar to that of the native tissue and cannot restore the fine threedimensional structure of the tissue.Thus,it is necessary to design a novel matrix material and a new preparation process,to realize the liver preparation process closer to real liver tissue.Methods:First,in order to create a microenvironment similar to that of the native tissue,liver extracellular matrix(LECM)was extracted from the liver of Bama pigs,and gelatin methacrylate(Gel MA)was added to develop liver-specific bio-ink(LECM/ Gel MA bioink)to achieve the solidification of the hydrogel.The mechanical and biological properties of the developed liver-specific bioink were characterized.Subsequently,the liver specific bioink was coated with human induced liver cells(HIHEP cells)to form cell-loaded bioink.The liver microtissues were prepared using digital light processing(DLP)based bio-printing technology,and the biological metabolism and synthesis function of liver microtissues were verified by liver function indexes.Finally,mass spectrometry was used to analyze the composition of LECM,to better understand the underlying mechanism how the LECM promotes the growth of Hi HEP cells.Results:By eluting the cellular components in pig liver,the LECM produced by us not only completely removed the cellular components,but also retained the intact ECM components.We mixed LECM and Gel MA in proportion to obtain liver specific bioink.Through careful characterization of mechanical properties,swelling property and cytocompatibility,it was found that LECM/ Gel MA not only had excellent mechanical properties in vitro,but also could better promote the growth of hi HEP cells in vitro.Subsequently,in the process of preparing liver microtissues using DLP based bio-printing technology,we found that liver ECM could improve the printing performance and cell viability of Gel MA bioink.In the microtissue,the cells were well spread and have better hepatocellular specific functions(albumin secretion and urea).The results of the mass spectrometry showed that the large biological proteins in LECM,such as promoting cell growth and adhesion,may be involved in the growth and adhesion of liver cells.Conclusion:Our finding provides a promising cell-loaded bioink based on LECM for the fabrication of liver microtissue.Based on the liver-specific bioink,we designed a novel liver microtissue with an internal cogel-like structure that mimics liver lobules.In vitro experiments indicated that this microtissue has partial liver function.Our research results can provide a brandnew insight for the preparation of liver tissue engineering products and become a potential liver tissue engineering product to help restore liver function.Part IIBackground: Hepatocellular Carcinoma(HCC)is one of the cancer types with the highest mortality rate in the world.The main reason is that HCC is highly heterogeneous,so frequent follow-up and accurate prognostic system are needed to evaluate the efficacy of clinical treatment.However,the existing clinical evaluation system,which is based on the traditional gross classification,cannot meet the needs of accurate tumor treatment.Therefore,a novel scoring system combining current tumor evaluation methods with intra-tumoral heterogeneity is urgently needed to help predict the prognosis of HCC.Methods: We analyzed the transcriptome data of 371 TCGA patients with HCC and mapped changes in ECM genes during HCC progression.Based on this,a prediction model for HCC was established by LASSO regression and validated in HCC datasets of GEO,ICGC and SRRSH dataset.Finally,we explored the relationship between ECM genes and tumor microenvironment,as well as tumor mutation genes through analyzing immunosuppressor genes.We also explore the correlation between ECM genes and hypoxia-related genes,and correlation analysis between ECM genes and TCGA exon data.Results: We found that ECM changes dynamically during the development of HCC.A prediction model of HCC prognosis based on 18 ECM related genes was constructed and validated in GEO,ICGC and the HCC dataset of SRRSH set.It was found that the prediction model based on ECM characteristics could better predict overall survival(OS)and disease-free Survival(DFS)of HCC patients.In addition,we found a clear correlation between ECM-related genes and the tumor microenvironment through the analysis of immunosuppressor genes and the correlation analysis of hypoxia scores.At the same time,through TCGA exon data,we found that ECM genes had a strong correlation with TP53 mutation.Conclusion: Based on bioinformatic analysis,we constructed a risk score for ECM that can predict the prognosis of patients with HCC.At the same time,we also found that the ECM proteins secreted by the tumor affects the immune signal,hypoxia state and TP53 mutation in the tumor microenvironment.The correlation between TP53 mutation and ECM-related molecules may provide a new perspective for combination targeted therapy of HCC.Part IIIBackground:TP53,which encodes p53 protein,is a tumor suppressor gene,but it is also the most frequently mutated gene in human tumors,whose mutation often influences tumor suppressor activity and promotes tumor metastasis.However,ECM remodeling occurs in the process of tumor metastasis,and the changes of ECM are closely related to the malignant biological behavior of tumors.Methods: Based on the results of the second part of the study,we first compared the correlation between TP53 mutation and the expression of ADAM9 through the HCC dataset in the TCGA database,and explored the changes in the transcriptional expression of ADAM9 at the cellular level through the knockdown/overexpression of TP53.Results: Based on the TCGA HCC data set,we found that TP53 mutation could affect the expression level of ADAM9 in HCC,and this change was most obvious in the TP53 GOF mutation(p < 0.05).By overexpression/knockdown to change the TP53 level of PLC-8024(TP53 GOF)and Hep G2(TP53 WT)cells,we found that ADAM9 was significantly correlated with the level of TP53 GOF,but the level of TP53 WT could not affect the transcriptional expression level of ADAM9.Conclusion: Our work found that TP53 mutation can reshape the tumor microenvironment by changing the expression level of ADAM9,thus promoting tumor progression.Since the treatment of TP53 mutation has proven to be challenging in clinical practice at present,targeting the downstream of TP53 mutation might be a valuable solution for effective treatment of HCC.