| Liver fibrosis is with high incidence in China and could develop into liver cirrosis and even liver cancer,which is seriously harmful for the public health.However,at present,there is no effective and mature treatment strategy for liver fibrosis,mainly because our understanding of the changes of microenvironment in liver fibrosis is still lack of comprehensiveness and profundity,and the intervention of microenvironmental factors for liver fibrosis is still lack of targeting and efficiency.Based on the known pathogenic process and present treatment strategies of liver fibrosis,our research focuses on the interaction between cellular,molecular and physical and chemical factors in the microenvironment of liver fibrosis.The study of liver fibrosis was carried out from two aspects:deepening disease understanding and exploring intervention strategies.Part 1:The expression characteristics,effects and mechanism of Clusterin in chronic hepatic fibrogenesis.Chronic hepatic fibrosis is a chronic liver disease caused by hepatophilic virus,alcohol abuse,drug poisoning,metabolic diseases,autoimmune diseases and other factors.The main pathological changes were abnormal proliferation of connective tissue in the liver and deposition of a large number of extracellular matrix,most of which were accompanied by inflammatory cell infiltration.If liver fibrosis continues to aggravate,it will develop into liver cirrhosis,and even liver cancer,which is seriously harmful to health.At present,the main treatment strategy is to remove the pathogenic factors of the primary disease at early time,but there is no ideal clinical intervention method for those whose pathogenic factors are difficult to remove（such as hepatophilic virus infection and autoimmune diseases）or irreversible hepatic fibrosis.It is mainly due to the lack of in-depth understanding of the changes of hepatic fibrosis microenvironment.It is vital to study and elucidate the interaction between cells,cells and molecules in the microenvironment of hepatic fibrosis in order to control hepatic fibrogenesis.At present,a consensus has emerged that the microenvironmental changes in the occurrence and development of hepatic fibrosis are as follows:degeneration and necrosis of hepatic parenchyma cells,secretion or/and release of inflammatory bodies,recruitment and activation of inflammatory cells.Activated inflammatory cells（especially macrophages）secrete TGFβ1,TNFα,PDGF and other factors,which turn hepatic stellate cells（HSCs）or other fibrogenic cells into myofibroblasts.Myofibroblasts continue to secrete and deposit extracellular matrix,and finally form hepatic fibrosis.Therefore,it is important to attenuate the injury of hepatic parenchyma cells,reduce the inflammatory response and/or weaken the activation of HSCs in order to inhibit the formation of hepatic fibrosis.Clusterin（CLU）,also known as apolipoprotein J,is a secretory protein that widely exists in human tissues and body fluids.At present,CLU has been reported to have a variety of biological functions,such as maintaining intracellular and extracellular protein homeostasis,inhibiting cell death,promoting cell survival,regulating autophagy,etc.CLU is also involved in many pathological processes,e.g.cerebral ischemia injury,Alzheimer’s disease,tumor drug resistance,lung injury repair,lipid deposition,renal fibrosis etc.However,the role of CLU in liver diseases,especially liver fibrosis,is rarely reported.Our previous work revealed that CLU was low expressed in normal liver,but high expressed in liver fibrosis tissue,and was specificly located in fibrosis area,which suggested the correlation between CLU and liver fibrosis.Therefore,we raise the following questions:whether CLU would affect the progress of hepatic fibrosis by regulating the biological behavior of hepatic parenchyma cells,inflammatory cells and/or fibrotic cells?In order to clarify the role of CLU in the occurrence and development of hepatic fibrosis,the following experimental studies were carried out.Here were the results:（1）Five mouse models of were established,including normal liver,chronic hepatic fibrosis,bile duct fibrosis,hepatic steatosis and acute liver injury.It was found that the expression of CLU in chronic hepatic fibrosis and bile duct fibrosis models was significantly higher than that in other models.This suggests the correlation between CLU and fibrosis diseases;（2）Depicts the expression characteristics of CLU in chronic hepatic fibrosis model:with the aggravation of fibrosis,the protein expression of CLU in liver tissue and blood shows an upward trend.However,CLU expression was later than that of HSCs activation;（3）The cell types of CLU expression were described:in normal liver,CLU was highly expressed in individual hepatocytes in the central venous area and portal bile duct cells,weakly expressed in other hepatocytes,not expressed in quiescent HSCs;in fibrotic liver,CLU was highly expressed in injured/necrotic hepatocytes and bile duct cells in fibrotic area,weakly expressed in normal hepatocytes,not expressed in activated HSCs（or myofibroblast）;（4）The mouse model of hepatic fibrosis was established which overexpress CLU in liver by adeno-associated virus（AAV）.It was found that overexpression of CLU in liver could inhibit the activation of HSCs and hepatic fibrosis,promote hepatocyte proliferation and inhibit hepatocyte death in vivo,suggesting that the increased expression of CLU is a feedback regulation of hepatic fibrosis;（5）Hep1-6 cell lines with CLU overexpression and CLU knockdown were constructed in vitro by lentivirus.It was proved that CLU promoted cell survival via NFκB and inhibited TNFα-induced cell apoptosis by inhibiting Caspase8/Caspase3 axis,which was also verified in mouse primary hepatocytes.This reveals the cytological and molecular mechanism that CLU inhibit hepatic fibrosis by regulating the survival and death of hepatocytes.（6）In hepatic stellate cell line HSC-T6 and mouse primary hepatic stellate cells,and recombinant CLU protein was added to the culture system.We found that CLU did not affect the proliferation of HSCs,but decreased the activation level of HSCs.The inhibitory effect of CLU on the activation of HSCs was mediated by TGFβ/Smad pathway.This reveals the cytological and molecular mechanism that CLU inhibits HSCs activation and hepatic fibrosis via hepatocyte paracrine.Conclusion:the high expression of CLU in damaged hepatocytes during development of hepatic fibrosis,on one hand,indirectly inhibits the progress of hepatic fibrosis by inhibiting hepatocyte death,promoting hepatocyte survival and restoring liver function,on the other hand,the paracrine CLU of hepatocytes inhibits the activation of HSCs and directly inhibits the progress of hepatic fibrosis.The results enriches the microenvironmental understanding of self-regulation of hepatic fibrosis.Part 2:The effect and mechanism of physiological hypoxia on stemness maintenance and bidirectional differentiation of induced hepatic stem cells.Huge numbers of patients suffer liver fibrosis in China,if not diagnosed and treated in time,they will gradually develop into liver failure.Traditionally,irreversible liver failure can only be treated by"liver transplantation".However,the cost of"liver transplantation"is high,and the population of liver failure in China is huge,and the source of donors is extremely scarce,which greatly hinders the development and application of"liver transplantation"technology.In recent years,cell therapy technology developed rapidly.Orthotopic hepatocyte transplantation（OLT）is the alternative to"liver transplantation"because of its low cost,easy operation and low risk.However,hepatocytes also have the problem of insufficient source of donor liver.In addition,the efficient and high activity isolation technology of hepatocytes is not mature,and the expansion ability of hepatocytes in vitro is limited.In recent years,lineage reprogramming technology has developed rapidly.It greatly broadens the source of cell donors,showing great potential in the field of cell therapy.Inducible liver stem cell（induced hepatic stem cells,iHepSCs）is the first established liver stem cell line in the world based on lineage reprogramming technology.It can be stably amplified in vitro and can differentiate into mature hepatocytes and bile duct cells under specific induction conditions in vitro.Colonization into the model of type I hereditary tyrosinemia（HT1）can partly restore liver function,and the homing cells in mouse DDC model（bile duct injury）can differentiate into bile duct cells,which provides a great application prospect for the cell therapy of chronic liver disease.However,the efficiency of iHepSCs differentiation into hepatocytes and bile duct cells is still low,and the efficiency of colonization into the mouse liver is also low,which may be due to the imperfect culture and induction conditions of iHepSCs.In recent years,it has been considered that the adult stem cells located in a specific stem cell nest（niche）,and niche factors can regulate the biological behavior of stem cells,in which oxygen content is a key factor affecting the stemness,proliferation and differentiation of stem cells.Physiological hypoxia refers to the normal physiological state,which means that the oxygen content in the body is significantly lower than that in the atmosphere（21%）.Recent evidence has shown that physiological hypoxic environment has a significant regulatory effect on the stemness maintenance,expansion culture and committed differentiation of mesenchymal stem cells,neural stem cells,hematopoietic stem cells and inducible stem cells,thus showing a better culture effect.However,it is still unknown whether physiological hypoxia could enhance stemness property and improve the efficiency of bidirectional differentiation of iHepSCs.The liver receives double blood supply,and is an oxygen abundant organ.The oxygen content in the portal area（adult liver stem cells residing）is about 9-11%.Therefore,we selected the 10%oxygen content culture condition to simulate the physiological hypoxic environment,in order to explore the effects of physiological hypoxia on the stemness property,proliferation,hepatic and bile differentiation of iHepSCs.The results showed that:（1）Physiological hypoxic culture did not significantly change the morphological characteristics and chromosome karyotype of iHepSCs;（2）Physiological hypoxia could enhance the expression of iHepSCs stem markers CK19,Sox9,EpCAM and Lgr5 and enhance the ability of colony formation;（3）Physiological hypoxia significantly decreased the level of intracellular ROS,and induce the expression of HIF1αand HIF2α;（4）Physiological hypoxia promoted the proliferation of iHepSCs by promoting G1/S transition of cell cycle through p53-p21 axis;（5）Hypoxia promoted the efficiency of biliary differentiation of iHepSCs in Matrigel.However,the efficiency of hepatic differentiation of iHepSCs was inhibited.;（6）Short-term hypoxic preconditioning for 24 hours could significantly improve the efficiency of hepatic differentiation of iHepSCs.Conclusion:physiological hypoxic environment enhances the stemness propety of iHepSCs and increases the amplification rate of iHepSCs in vitro.Hypoxic culture improves the efficiency of biliary differentiation,while hypoxic short-term pretreatment improves the efficiency of liver differentiation.This is helpful to explore the intervention strategy of stem cell biological behavior regulation based on microenvironment factors,and provides an important reference for optimizing the suitable culture conditions of applied stem cells. |
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