The Role And Mechanism Of Differentiation In Hepatoma Cell Line HepG2 Induced By Embryonic Liver Cells At Specific Stages Of Development

Objective: Tumor mortality has leapt to the second in the causes of death by human disease.There are about 55 million people died of liver cancer in China annually,which is 2.21 times the average death rate in the world.The most important type of hepatocellular carcinoma is hepatocellular carcinoma(HCC),accounting for about 90% of primary liver cancer.But it's lack of special treatment for the HCC program.The traditional treatments such as surgery,radiotherapy,Chemotherapy exist the malpractice of high recurrence rate and toxic side effects,which is a poor quality of life for the patient.Therefore,we should re-recognize the causes of tumors and long-lasting cures so as to look for new ways to effectively cure tumors.Studies have shown that embryonic stem cells and tumor cells have many common features: plasticity,immune escape,immortalization,both expressing embryonic characteristic protein.In recent years,a series of basic and clinical studies have shown that tumor cells originated from the tissue stem cells with abnormal value-added and differentiation.A large number of studies have found that the microenvironment of embryonic stem cells acts on tumors,which can inhibit the invasion behavior of tumors and reverse the malignant phenotypes of tumors.According to the mentor Professor Qi Jinsheng that the tumor cells is the "normal stem cells" Stagnating in a differentiation stage,and infinitly proliferating,which is the Cell "back to ancestral" phenomenon.And further suggested that embryonic cells with specific developmental stages can induce tumor cell from the same organization to differentiate(Synchronously and homologously induce differentiation),so as to achieve the purpose of treatment of cancer.The embryonic liver cells of different developmental stages were co-cultured with HepG2 cells.In Pre-experimentItit was confirmed that the embryonic liver cells can induce HepG2 cells differentiate by reconstructing the HNF-4? regulatory loop.Previous studies have shown that the differentiation of hepatocellular carcinoma HepG2 cells induced by embryonic liver cells is the same in different Species,and it is through non-contact communication.Thus,signaling pathways that induced differentiation should be highly conserved and exocrine.The Wnt / ?-catenin signaling pathway is a highly conserved signaling pathway across species,closely related to development,differentiation,and proliferation.Wnt signaling as a key regulator of molecules,play a role by the form of autocrine and paracrine.Wnt / ?-catenin signaling plays a key role in the development and the maintenance of function of the liver and the induction of stem cells into hepatocytes in vitro.So,whether the fetal liver cells by regulating the Wnt / ?-catenin pathway to activate expression of hepatocyte nuclear factor(HNF-4?)induce HCC cell differentiate,it's no related literature.In this study,based on re-acquainting the nature of tumorigenesis,we select liver cancer as the research object,and isolate,purify,identy embryonic liver cells to confirm the differentiation of hepatoma cells HepG2 induced by embryonic hepatocytes at specific stages of development,verifing the "homologous synchronous induce differentiation" theory,and then explore the signaling and molecular mechanisms in the process of the induction of differentiation,which provided experimental basis for the treatment of HCC and new ideas and methods for treatment of cancer.Method:1.The separation purification and identification of Primary embryo liver cellOn the basis of the previous experimental study of the research group,the fetal rats developed at 13.5 d(E13.5 d)is selected for experiments.Using IV collagenase digestion method,mouse embryonic liver tissue is digested and separated into individual embryonic liver cells.repeated repetitive differential adherence method to remove fibroblastsl.HE staining is used to observe themorphology of fetal liver tissue,Immunohistochemistry detect AFP and CK19 expressing in specific development Stages of fetal liver tissue.The expression of CD90.1 and CD133 as stem cell markers in fetal liver cells are detected by flow cytometry.The expression of protein AFP in fetal liver cells is detected by immunofluorescence.2.Cell cultureHepG2 cells are cultured in DMEM high glucose medium containing1% non-essential amino acid and 10% newborn bovine serum,0.25% trypsin digestion,inoculated in 100 mL culture flask,and cultured at 37 ?,5% CO2 incubator.3.Establishing model and groupingThe non-contact co-culture system of HepG2 cells and embryonic liver cells is established using the "Transwell" chamber of six-well plates.HepG2 cells were co-cultured with embryonic liver cells in the 13.5 d development stage.Hep G2 cells are cultured in the lower part of the Transwell wells,and the embryogenic liver cells are cultured in the upper part of the membrane of Transwell.To investigate the differentiation of HepG2 cells induced by E13.5 d embryonic liver cells,dividding into four groups:The untreated group(HepG2cells are cultured alone),the co-culture24 h group(HepG2 cells and E13.5 d embryonic hepatocytes are co-cultured for 24 h),co-culture48 h group,co-culture72 h group.To explore the role of the Wnt/?-catenin pathway in the induction of HepG2 cell differentiation.?-catenin inhibitor(XAV-939)is added to co-culture system,the experiment is divided into four groups: simple Hep G2 cell group(con),co-culture group,simple ?-catenin inhibitor group,co-culture group + ?-catenin inhibitor group.Co-cultured 48 h.4.After Hep G2 cells and embryonic liver cells co-cultured,observating and detecting HepG2 cell statusThe morphological changes of HepG2 cells in different groups are observed under microscope with Photographic record.Apoptosis of Hep G2 cells is detected by flow cytometry.The expression of AFP in HepG2 cells is detected by immunofluorescence assay.5.After HepG2 cells and embryonic liver cells co-cultured,detecting the gene transcription level of HCC marker genes afp and c-myc,hepatocyte differentiation control gene hnf-4?,hepatocyte maturation marker gene cyp1b1 and adh1c in HepG2 cellsExtracting total RNA from HepG2 cells by Trizol method with one step.The expression of AFP,c-Myc,CYP1B1,ADH1 C and HNF-4? mRNA is detected by Real-time PCR,?-actin as internal reference.6.After HepG2 cells and embryonic liver cells co-cultured,detecting AFP,HNF-4?,?-catenin protein content in HepG2 cellsCollecting HepG2 cells,the total protein is extracted by RIPA cleavage method.The protein content is determined by Coomassie brilliant blue method.The contents of AFP,HNF-4? and ?-catenin in HepG2 cells are detected by Western blotting.7.After HepG2 cells and embryonic liver cells co-cultured,detecting the nuclear translocation of ?-catenin protein and the expression of HNF-4?protein in HepG2 cellCollecting HepG2 cells,two-step cleavage method is used to extract nucleoprotein and cytoplasmic protein.Coomassie brilliant blue method is used to determine protein content,Western-blotting is used to detect the expression of ?-catenin protein and HNF-4? protein in the nucleus and cytoplasm of HepG2 cells.The distribution of ?-catenin protein and HNF-4?protein in HepG2 cells is detected by immunofluorescence.Result:1.Isolation Purification and identification of primary embryonic liver cellsSelecting of E13.5 d mouse fetuses for experiment,Using ? collagenase digestion method,successfully separate single embryo liver cells,Using repeated repetitive sticking method to purified embryonic liver cells,Culturing embryonic liver cells with DMEM including high sugar,Under a phase contrast microscope embryonic liver cells are round,translucent,Three-dimensional sense,and the high survival rate,long survival time.Expression of AFP and CK19 in fetal liver tissue was detected by immunohistochemistry.The positive expression rates of CD90.1 and CD133 in fetal liver cells were 70.8% and 60.8% respectively by flow cytometry.The protein AFP of embryonic liver cells was detected by immunofluorescence assay,which indicated that the E13.5 d embryonic liver cells were isolated and purified successfully and had the characteristics of stem cells.2.Embryo liver cells induce hepatocarcinoma cells HepG2 differente2.1 After HepG2 cells and embryonic liver cells co-cultured,the observation and detection of Hep G2 cell statusIn this study,a non-contact co-culture system of HepG2 cells and embryonic liver cells was established using the "Transwell" chamber of a six-well plate.The morphological images of HepG2 cells were observed by phase contrast microscope.Compared with normal group(Cell grew strongly and superimposedly,with high density,polygonal or shuttle type shape,cytoplasm translucent,multiple nuclei and irregular shape Co-cultured for 24 h HepG2 cells proliferated slowly,Co-cultured for 48 h,HepG2 cells showed significant growth inhibition,which morphology tended to normal hepatocyte-like hexagons,and most of them became mononuclear cells.Co-cultured for 72 h,HepG2 cells were retracted,turned round,decreased in brightness,fall off and eventually disintegrated.Detected by flow cytometry.Compared with the normal group,early apoptosis and necrotic were detected in HepG2 cells after co-culture.Compared with the normal group,the apoptosis rate of HepG2 cells increased after co-cultured by 48 h.The expression of AFP in HepG2 cells was detected by immunofluorescence.Compared with normal group,the expression of AFP in Hep G2 cells was significantly decreased after co-cultured by 48 h.2.2 After HepG2 cells and embryonic liver cells co-cultured,the changes of afp,c-myc,cyp1b1 and adh1 c gene transcription level in HepG2 cellsAfter Hep G2 cells were co-cultured with E13.5 d embryonic liver cells,Compared with the con group,the relative expression of AFP and c-MycmRNA in Hep G2 cells significantly decreased in 24 h and 48 h groups.The relative expression of HNF-4? mRNA significantly increased in the 24 h and48 h groups.The relative expression of CYP1B1 and ADH1 C mRNA was not significantly different in the 24 h group,but significantly increased in the 48 h group.After co-culture,the expression of cancerous genes in Hep G2 cells decreased and the expression of differentiated genes increased.After 48 h of co-culture,the expression of mature genes in HepG2 cells increased.After HepG2 cells were co-cultured with embryonic liver cells for 72 h,a large number of cells died and were not detected.2.3 After HepG2 cells and 13.5 d embryonic liver cells co-cultured 48 h,the changes of AFP,HNF-4?,?-catenin protein content in HepG2 cells.After Hep G2 cells were co-cultured with E13.5 d embryonic liver cells for 48 h,compared with the normal group,the AFP protein content of HepG2 cells in co-culture group,inhibitor group and co-culture+inhibitor group all decreased significantly,the HNF-4? protein content of HepG2 cells in the co-culture group increased significantly,There was no significant difference in inhibitor group and co-culture+ inhibition group,The ?-catenin protein content of HepG2 cells in the co-culture group had no significant changes,and it in the inhibitor group and co-culture+inhibitor group significantly decreased.Due to the inhibition of ?-catenin expression,HNF-4? protein expression did not change significantly after co-culture process,which affected the affort of differentiation.The changes of the expression of AFP,HNF-4? and ?-catenin protein in HepG2 cells were detected by immunofluorescence were consistent with that by Western-blotting assay.3.E13.5d Embryonic Liver Cells Induce Differentiation of Hepatocellular Carcinoma Cell HepG2 by Adjusting Wnt/?-catenin PathwayWestern-blotting and immunofluorescence assay showed that HepG2 cells co-cultured with E13.5 d embryonic liver cells for 48 h,the nuclear translocation of ?-catenin occurred in HepG2 cells,and the expression of HNF-4? protein in Hep G2 nucleus significantly increased.After ?-catenininhibitor was added,the expression of ?-catenin significantly reduced,and the expression of HNF-4? protein didn't change significantly,indicating that the increased expression of HNF-4? protein related to the nuclear translocation of?-catenin.Conclusion:1.Specific developmental stages were isolated and purified and successfully cultured,identified with stem cell characteristics.2.The microenvironment of the mouse embryonic liver cells in the Specific developmental stages can induce hepatocarcinoma cell line Hep G2 to differentiate,lossing the ability of infinite proliferation in a non-contact manner.3.The Wnt/?-catenin signaling pathway is involved in the course that the mouse embryonic liver cells induce human hepatoma cell Hep G2 to differentiate though the nuclear translocation of ?-catenin regulates the expression of HNF-4? to promote the differentiation of HCC cells.