Sodium butyrate induces mouse embryonic stem cells to differentiate into hepatocytes in vitro

AIM:To explore mechanism by which sodium butyrate induces mouse embryonic stem cells (ES) to differentiate into hepatocytes in vitro. METHODS:E14 mouse ES cells were cultivated in a routine way, and then cultivated in suspension to form embryonic bodies (EBs). EBs were transferred into 6-well culture dishes and 3 mmol/L sodium butyrate was added into the culture medium. Morphological changes were investigated by phase contrast microscopy. α-fetoprotein (AFP), albumin (ALB) and cytokeratin 18 (CK18) were examined by immunofluorescence staining. AFP, ALB, α1-antitrypsin (AAT) and TTR mRNA were assayed by RT-PCR. Proportion of ALB positive cells was analyzed by flow cytometry. Periodic acid Schiff (PAS) reaction and indocyanine green (ICG) uptake assay were performed to assess the characteristic hepatocyte function of the differentiated cells. RESULTS:In the presence of sodium butyrate, parts of ES cells differentiated into a population with epithelial morphology similar to mouse hepatocytes. AFP and TTR mRNA expression were observed at 7 d, and ALB and AAT mRNA expressed at 14 d. Hepatocytes specific markers, ALB, AFP and CK18 were positive expression in immunofluorescence staining at 14 d. PAS reaction and ICG uptake were positive for the hepatocyte-like cells. CONCLUSION:Mouse ES cells can be induced into hepatocyte-like cells by sodium butyrate efficiently, and these ES cells-derived hepatocytes possess characteristic hepatocytic function.     

Isolation and identification of human fetal bone-derived mesenchymal stem cells and their differentiation to hepatocyte like cells

AIM: To separate and identify the mesenchymal stem cells (MSCs) from human fetal bone and to study their differentiation to hepatocyte like cells under the action of chemical induction.METHODS: The MSCs from human fetal bone were isolated and purified according to the different growth characteristic of attaching to the wall of cell culture flask.The cell cycle and surface markers of MSCs were identified using flow cytometry.The MSCs were pre-induced by adding DMSO,β-Me and 5-aza for 24 h,then adding the inductive medium of H-DMEM and rh-HGF to induce their differentiation to hepatocyte like cells (HLCs).HLCs were identified by the typical morphological change and the expression of special protein with the method of immunocytochemistry.RESULTS: The MSCs derived from human fetal bone expressed adhesion molecules CD29+,CD44+,but not antigens of hematopoietic CD34,CD45,and not antigens related to GVHD,such as HLA-DR,CD80 and CD86.Exposure of these cells to above-mentioned inductive agents resulted in obvious morphological change and an increase in expression of AFP and ALB.CONCLUSION: The results suggest the existence of plentiful MSCs in human fetal bone.MSCs derived from human fetal bone can easily differentiate to HLCs,and they have a lower immunogenic nature,which may provide the ideal source for tissue engineering (bioartificial liver) for cellular therapeutics.     

Hepatic differentiation of mesenchymal stem cells derived from rats in three-dimensional microenvironment formed by hanging drops

AIM: To establish an optimal differentiated three-dimensional microenvironment formed by hanging drops for the high efficiency of hepatic differentiation from rat mesenchymal stem cells(rMSCs).METHODS: rMSCs were cultured in the hanging drops, which provided a three-dimensional microenvironment, for 21 days in the presence of hepatocyte growth factor(HGF, 20 μg/L). The expression of albumin(ALB), alpha-fetoprotein(AFP) and cytokeratin-18(CK-18) was detected by RT-PCR and immunofluorescence staining at 7th, 14th and 21st days. The secretion of albumin in the culture supernatants was measured by ELISA. RESULTS: rMSCs were aggregated in spheroid with a tubiform medium altitude in the center. In rMSCs cultured in the hanging drops with HGF, the expression of albumin, AFP and CK-18 was all detectable by RT-PCR and immunofluorescence staining at 7th day. The production of albumin in the cells cultured in the hanging drops with HGF was 50.25±5.32, 55.03±7.45 and 54.92±3.18(ng·dish^-1·d^-1) at 7th, 14th and 21st days, respectively, significantly higher than that in the cells in the plate cultivation with or without HGF induction at corresponding time points(P<0.01).CONCLUSION: In the presence of HGF, rMSCs are induced to differentiate into hepatocyte-like cells cultured in the hanging drops, where the three-dimensional spheroidal cultures are promising microenvironment for hepatic transdifferentiation of MSCs.     

Protective effects of somatostatin and octreotide on hepatocytes

AIM: To investigate the protective effect of somatostatin (SST) and octreotide (OCT) on rat hepatocytes. METHODS: The primary hepatocytes were pretreated with different concentrations of SST and OCT. The levels of alanine minotransferase (ALT) and aspartate aminotransferase (AST) in culture supernatant were analyzed by the model of ethanol/carbon tetrachloride (CCl4)-induced hepatocyte injury. Additionally, 75 Sprague-Dawley rats were divided into 5 groups at random, including normal control, model control, SST-treated model groups at high, medium and low doses (200 μg·kg-1·d-1, 100 μg·kg-1·d-1 and 50 μg·kg-1·d-1, respectively). Except for the normal controls, all rats were injected with 40% CCl4 subcutaneously for 8 weeks to establish hepatic fibrosis. Meanwhile, rats of SST-treated model groups were given at different doses of SST twice a day in the same way. Thereafter, the liver function and apoptosis index of hepatocytes were detected by standard enzyme method, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. RESULTS: Compared with those of injury model group, the hepatocytes pretreated with SST (10-8-10-6 mol/L) and OCT (10-7-10-5 mol/L) exhibited significantly decreased levels of ALT and AST in the culture supernatant. Furthermore, most indices of liver function including ALT, AST, alkaline phosphatase (ALP), total bilirubin (TBIL) and albumin (ALB) improved obviously in all SST-treated groups, especially in the group treated with low dose of SST. The apoptosis index of hepatocytes in the fibrotic liver was also reduced greatly by the treatment with low dose of SST. CONCLUSION: SST and OCT may protect hepatocytes against CCl4-induced injury, inhibit hepatocyte apoptosis, and improve the liver function. These findings suggest them a potential efficiency in the prevention of hepatic fibrosis.     

Application of ESC-derived hepatic stem cells in therapeutic liver repo pulation

AIM: To study the proliferation, differentiation and the capacity of forming teratomas of ESC-derived hepatic stem cells in mouse pre-treated with retrorsine and 70% partial hepatotomy. METHODS: The ESC-derived hepatic stem cells, labelled with CFDA SE, were transplanted into BALB/c mouse liver. The distribution, incorperation and proliferation of transplanted cells were observed under fluorescent microscopy. Hepatic function was assayed by detecting albumin level in serum. The situation of forming teratomas in vivo was also evaluated.RESULTS: 1 week post-transplantation, some scattered region was green under fluorescent microscopy. The aera of green region increased apparently in 2 weeks, and cord-like structure was observed. Immunofluorescent staining of albumin demonstrated some positve cells, but there was no significant difference for albumin level in serum (P>0.05). No teratoma was formed in the experimental group, while a large teratoma was observed in control group in 6 weeks post-transplantation.CONCLUSION: The ESC-derived hepatic stem cells are normally incorporated into mouse liver parenchymal structure, proliferate and differentiate further in vivo and possess some hepatic functions without forming teratomas.     

Differentiation of rat bone marrow mesenchymal stem cells expressing hepatocyte growth factor into hepatocyte-like cells

AIM:To observe the hepatic differentiated efficiency of rat bone marrow mesenchymal stem cells (rMSCs) expressing hepatocyte growth factor (HGF) in three-dimensional microenvironment formed by hanging drop. METHODS:rMSCs were isolated and cultured in vitro, and flow cytometry was used to detect the expression of CD44, CD90, CD34 and CD45. Recombinant retrovirus carrying cDNA of human HGF (pLNCX2-hHGF) was constructed and infected with rMSCs (hHGF-rMSCs). hHGF expression in hHGF-rMSCs was detected by RT-PCR, immunofluorescence staining and ELISA. hHGF-rMSCs were cultured in hanging drop for 21 days. The expression of albumin (ALB),cytokeratin-18 (AFP) and alpha fetoprotein (CK-18) were detected by RT-qPCR and immunofluorescence staining in the 7th, 14th and 21st day, respectively. The secretion of albumin in cultured supernatant was measured by ELISA. RESULTS:CD44 and CD90 were highly expressed in the third generation of rMSCs, but CD34 and CD45 were hardly expressed. The expression of hHGF at mRNA and protein level were all detectable in the hHGF-rMSCs, and the secretion in the cultured supernatant was about (123.71±8.81) μg/L in a period of 21 days. In the hHGF-rMSCs, ALB, AFP and CK-18 were highly expressed at mRNA level from the 7th to the 21st day, and there were significant differences compared with rMSCs at the same time point (P<0.01). The results of immunofluorescence staining showed that the protein expression of AFP was negative on day 7 and 14, and positive on day 21; while the protein expression of ALB and CK-18 was positive on day 7 and lasted until day 21. ALB was positively observed in the culture supernatant of hHGF-rMSCs from 7th to 21st day measured by ELISA, and there was significant difference between the hHGF-rMSCs and rMSCs (P<0.01). CONCLUSION:hHGF transduced-rMSCs can be induced to differentiate into hepatocyte-like cells after cultured in hanging drop which provides a three-dimensional microenvironment. All these results might help to provide new seed cells for cell therapy of clinical liver diseases and in vitro bioartificial liver.     

Non-viral vector-mediated high efficiency expression of manganese superoxide dismutase gene in mouse liver

AIM: To investigate the transfection efficiency of mouse liver with non-viral vector containing manganese superoxide dismutase (Mn-SOD) gene. METHODS: The eukaryotic expression vector, gWiz/Mn-SOD, encoding human manganese superoxide dismutase was constructed. The plasmids of gWiz/Mn-SOD were mixed with cationic lipids, followed by injection into mice via branch of superior mesenteric vein, to induce Mn-SOD over-expression in murine liver detected by RT-PCR, Western blotting, SOD activity and immunohistochemical staining. RESULTS: gWiz/Mn-SOD transfection resulted in the obvious expression of exogenous Mn-SOD mRNA and protein in hepatic tissues at 8 hours after injection, and elevated mitochondria SOD activity 8.4 times in transfected hepatocytes than that in non-transfected cells at 72 hours after injection. It was showed that nearly 70% of mouse hepatocytes was obviously Mn-SOD positive after transfection. CONCLUSION: High expression efficiency of Mn-SOD gene in mouse liver is achieved safely, by injection of gWiz/Mn-SOD and cationic lipid mixture into branch of superior mesenteric vein.     

miRNA-122 promotes mouse embryonic stem cell-derived hepatic precursor cells to differentiate into hepatocytes

AIM：To investigate whether miRNA-122 (miR-122) promotes the differentiation of mouse embryonic stem cell (ESC)-derived hepatic precursor cells (HPCs) into hepatocytes. METHODS：Mouse ESCs were initially induced to differentiate into HPCs by stimulating with fibroblast growth factor 4 (FGF-4), sodium butyrate and dexamethasone (Dex) sequentially. Then a recombinant adenovirus expressing vector pAV.Ex1d-CMV>miR-122/IRES/eGFP was constructed by Gateway technology and transfected into the mouse ESC-derived HPCs 9 d after induction, so as to gain the cells with stable high expression of miR-122. The morphological changes of transfected cells were observed under inverted phase-contrast microscope. The liver-specific gene expression levels were detected by real-time RT-PCR. The liver-specific protein expression levels were also detected by immunofluorescence method. The liver functions were assessed by indocyanine green (ICG) uptake experiment, glycogen staining and urea synthesis function test. RESULTS：The mouse ESCs were successfully induced into HPCs by stimulating with FGF-4, sodium butyrate and Dex sequentially. At 6 d after transfection of miR-122, the morphology of the cells was closer to the mature hepatocytes. The mRNA levels of liver-specific genes such as albumin (ALB), transthyretion, α1-antitrypsin, glucose-6-phosphatase, cytokeration 8, cholesterol 7α-hydroxylase and cytochrome P450 3A4 were up-regulated. The expression levels of liver-specific proteins such as ALB and cytokeratin 18 were increased, while alpha-fetoprotein was decreased. The results of ICG uptake experiment, glycogen staining and urea synthesis function test indicated that the hepatocyte functions were strengthened as compared with control group. CONCLUSION：The combination of FGF-4, sodium butyrate and Dex successfully induces the mouse ESCs into HPCs. Over-expression of miR-122 effectively promotes the differentiation and maturation of mouse HPCs.     

Treatment of hepatocellular carcinoma in vitro and in vivo with yCD/TK double suicide gene driven by AFP promoter

AIM: To study the effect and mechanism of yeast cytosine deaminase/ thymidine kinase (yCD/TK) double suicide gene driven by alpha fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) in vitro and in vivo. METHODS: The expression plasmid with yCD/TK double suicide gene, which was driven by AFP promoter, was constructed. HepG2 (AFP positive) and SMMC7721 (AFP negative) human HCC cell lines were both transfected with the above-mentioned expression plasmid through cationic liposome. The cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The cell proliferation and cell cycle phase were evaluated by MTT test and flow cytometry respectively. The effect of double suicide gene on HCC xenografts in nude mice was observed through measuring the tumor size and the number of apoptosis cells. RESULTS: The double suicide gene was expressed selectively on HepG2 cells, rather than on SMMC7721 cells. The 5-FC and/or GCV inhibited effectively the proliferation of HepG2 cells in a dose-dependent manner, but had no influence on SMMC7721 cells. The inhibitory effect on HepG2 cells among different treatments was GCV+5-FC>5-FC>GCV. In vivo, the treatments inhibited markedly the growth of HepG2 cell xenografts in nude mice, transfected with yCD/TK gene. More apoptotic cells were found in HepG2 xenografts after the treatment. However, the growth of SMMC7721 cell xenografts could not be inhibited by this double suicide gene therapy, and few apoptotic cells were found. CONCLUSION: yCD/TK double suicide gene driven by AFP promoter has a significant efficacy in treatment of AFP positive HCC. Cell apoptosis may be an important mechanism of yCD/TK double suicide gene-inhibiting the growth of HCC.     

Quantitative analysis of RNA levels from single hepatocytes in vivo: combined use of real-time RT-PCR and laser microdissection

AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed.To better understand cellular functions,the isolation of single cells and subsequent quantification of the expressed genes is essential.METHODS: Normal liver tissue was obtained from operation,snap-frozen in liquid nitrogen and sectioned in crystat.Individual hepatocytes were microdissected.RNA was extracted,then reverse transcribed and amplified using real-time quantitative polymerase chain reaction (PCR).RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide.In this way,cells were collected,RNA was extracted,reverse transcribed to cDNA and used for analysis of RNA expression by real-time quantitative PCR.The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells.CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real-time PCR.These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.     

Lipid-induced p62dok up-regulation enhances hepatic gluconeogenesis

AIM:To investigate the potential role of p62^dok in the regulation of hepatic gluconeogenesis. METHODS:The expression of p62^dok, insulin signaling transduction, and hepatic gluconeogenesis were investigated in the liver tissues of mice treated with high-fat diet(HFD) and in cultured mouse hepatocytes treated with free fatty acid(FFA). The experiments of gene silencing and overexpression were conducted to observe the effects of p62^dok on insulin signal transduction and hepatic gluconeogenesis in cultured mouse hepatocytes. Western blotting was used to detect the protein levels and the phosphorylation statue. RESULTS:The increased p62^dok levels were found in the liver tissues from HFD-treated mice and FFA-treated hepatocytes. Meanwhile, phosphorylation of Akt and forkhead box O1 protein(FoxO1) was were decreased and the expression of glucose-6-phosphatase(G6Pase) and phosphoenolpyruvate carboxykinase(PEPCK) was increased. Silencing of p62^dok in cultured hepatocytes treated with FFA induced the increase in phosphorylation of Akt and FoxO1, and decrease in the protein levels of G6Pase and PEPCK. CONCLUSION:Up-regulation of p62^dok induced by HFD or FFA enhances hepatic gluconeogenesis via inhibiting insulin signal transduction.     

Relationship between single nucleotide polymorphism -229C/T in P1 promoter of furin gene and functions of hepatocytes in patients with liver cirrhosis

AIM: To study the influences of P1 promoter activity of furin gene on the functions of hepatocytes in patients with liver cirrhosis. METHODS: The patients with liver cirrhosis of 180 cases were recruited. The single nucleotide polymorphism (SNP -229 C/T) in P1 promoter of furin gene was genotyped using competitively differentiated polymerase chain reaction. The relationships between the promoter activity based on genotyping and the serum levels of liver enzymes, total bilirubin, albumin and prothrombin were observed. RESULTS: The distribution frequencies of allele C and T were 75.3% (271/360) and 24.7% (89/360). Those of genotypes CC, CT and TT were 62.2% (112/180), 26.1% (47/180) and 11.7% (21/180), respectively. The distribution frequencies of the genotypes were not related to the serum levels of major liver enzymes, albumin, total bilirubin and prothrombin, except for alkaline phosphatase and γ-glutamyl transferase. CONCLUSION: The activity of furin promoter exerts no effects on the main functions of hepatocytes, suggesting that furin may be a new therapeutic target for HBV infection.     

Comparison of the effects on differentiation of mouse embryonic stem cells into insulin-secreting cells among three cell culture protocols

AIM：To compare the effects of three different cell culture protocols：embryonic body (EB) formation，EB formation-monolayer and monolayer on differentiation of mouse embryonic stem (ES) cells into insulin-secreting cells.METHODS：E14.1 mouse ES cells were treated with GLP-1,betacellulin，activin A,bFGF and nicotinamide by using EB formation,EB formation-monolayer and monolayer culture protocol respectively for 30 days,then insulin expression was examined by RT-PCR,DTZ-staining and immunohistochemistry.The percentage of insulin-secreting cells was evaluated by flow cytometry.RESULTS：DTZ-staining positive cells and insulin immunohistochemical staining positive cells were observed in the differentiated cells for all the three groups.mRNAs of insulin and some other islet-related genes were detected,insulin expression was the strongest in EB formation-monolayer,and the weakest was in monolayer.The percentage of insulin-positive cells of the differentiated cells in the EB formation-monolayer group was higher than that in the EB formation group (P＜0.01),the latter was higher than that in the monolayer group (P＜0.01).CONCLUSION：Among the three cell culture protocols,EB formation-monolayer is the most effective approach in the induction of mouse ES cells to differentiate into insulin-secreting cells.     

Construction of a recombinant adenovirus carrying IL- 12 -IRES-CK gene and its expression in hepatocyte

AIM: To construct a bicistronic recombinant adenovirus carrying creatine kinase (CK) and human interleukin 12 (hIL-12) gene, and then detect the expression of both genes in vitro and metabolic product of CK in vivo. METHODS: Two PCR products, CK and hIL- 12 genes linked by IRES, were inserted into adenoviral vector. Adenovirus particles carrying CK-IRES-IL- 12 gene was generated through homologous recombination, packaging and propagation. Rabbit hepatocytes were isolated and transfected with adenovirus particles. CK and IL-12 gene expression was confirmed by Western blotting and ELISA, respectively.RESULTS: The expression of CK and human IL-12 in hepatocyte were confirmed in vitro. Metabolic product of CK, phosphocreatine (PCr), could be detected in liver by non-invasive phosphorus-31 magnetic resonance spectroscopy (³¹P MRS) in vivo.CONCLUSION: In vivo detection of ectopic expression of CK gene in liver can be achieved by non-invasive ³¹P MRS through adenovirus transduction. The bicistronic recombinant adenovirus Ad5-hIL-12-IRES-CKb carrying CK and hIL- 12 provides a useful tool for the further study of using a reporter gene expression (CK) dynamically, non-invasively monitor a therapeutic gene expression (IL-12) in liver.     

Construction of shRNA eukaryotic expression vectors targeting PERK gene and effect of PERK gene knockdown on apoptosis in endoplasmic reticulum stress-induced L02 hepatocytes

AIM: To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting the gene of RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase ( PERK ), and to observe the effect of PERK gene knockdown on apoptosis of human normal hepatic L02 cells treated with thapsigargin. METHODS: Three shRNA expression vectors targeting PERK gene, named PERK1-shRNA, PERK2-shRNA and PERK3-shRNA, and one non-homologous negative control expression vector (HK-shRNA) were constructed based on the nucleotide sequence of PERK and the criteria of designing small interfering RNA (siRNA), and were identified by enzyme digestion and DNA sequencing analysis. After L02 hepatocytes were transfected with the plasmids, the PERK expression was determined by RT-PCR and Western blotting, and the plasmid with the best inhibitory effect on PERK expression was screened. The cell viability and apoptotic rate of L02 hepatocytes transfected with PERK-shRNA under endoplasmic reticulum stress (ERS) were measured by the methods of MTT and flow cytometry,respectively. RESULTS: Four shRNA expression vectors were constructed. The mRNA and protein expression levels of PERK gene decreased significantly in L02 hepatocytes transfected with PERK1-shRNA, PERK2-shRNA and PERK3-shRNA as compared with those in control cells (P<0.05). The interfering effect of PERK1-shRNA on PERK gene expression was the best. PERK knockdown by PERK1-shRNA increased the viability and inhibited the apoptosis of L02 cells under ERS.CONCLUSION: The shRNA expression vectors targeting PERK gene are constructed, and PERK gene knockdown may inhibit apoptosis in L02 hepatocytes under ERS.     

Mesenchymal stem cells contribute to the formation of liver fibrosis

AIM: Myofibroblasts play key roles in the formation of liver fibrosis. It has been reported that bone marrow mesenchymal stem cells can differentiate into myofibroblasts, and migrate to the damaged organs to participate the fibrotic process. Therefore, this study was designed to investigate the possible function and mechanism of bone marrow mesenchymal stem cells in developing liver fibrosis. METHODS: A mesenchymal stem cell line Ap8c3 was tagged with enhanced green fluorescent protein(eGFP)(Ap8c3-eGFP). The rat model of liver fibrosis was established by bile duct ligation(BDL) or injection of pig serum(IPS). Ap8c3-eGFP was intravenously injected into BDL or IPS-induced liver fibrotic rats. The eGFP positive(eGFP⁺) cells and expression of α-smooth muscle actin(α-SMA) in these eGFP⁺ cells in rat livers and bone marrow(BM) were detected. RESULTS: Intravenous engraftment of Ap8c3- eGFP resulted in homing to fibrotic livers as well as BM. Co-expression of α-SMA by eGFP⁺ cells was found in liver sections, and eGFP⁺ cells were found mainly in fibrotic septum in fibrotic livers. Ap8c3-eGFP was observed to differentiate into myofibroblasts, which specifically expressed α-SMA after homing to bone marrow.CONCLUSION: Differentiation of mesenchymal stem cells into myofibroblasts plays important roles in the formation of liver fibrosis. BM-derived myofibroblasts can migrate to damaged livers and participate in the formation of liver fibrosis.     

Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM: To explore the role of DNA methylation of microRNA-30a-5p(miR-30a-5p) promoter region in hepatic injury. METHODS: Four-week-old normal mice and cystathionine β-synthase (CBS) single gene knockout mice were used and divided into normal (CBS^+/+, n=12) group and single gene knockout (CBS^+/-, n=12) group, and the mice were fed with high methionine diet for 8 weeks. HL-7702 hepatic cells were routinely cultured in vitro and divided into control group, homocysteine (Hcy) group and Hcy+5-azacytidne (AZC) group. Serum Hcy, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer. The levels of ALT and AST in the cells culture medium were determined by the microplate method. Hepatic injury in the mice were observed with HE staining. Cell viability staining was used to measure the viability of hepatocytes. RT-qPCR was used to detect the expression of miR-30a-5p in the liver tissues and hepatocytes. The correlation between the expression of miR-30a-5p and serum ALT and AST levels was analyzed by Pearson correlation analysis. DNA methylation level of miR-30a-5p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR (nMS-PCR). RESULTS: Compared with the CBS^+/+ mice, the serum levels of Hcy, ALT and AST in the CBS^+/- mice were significantly increased (P < 0.05). HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution. The expression level of miR-30a-5p in the liver tissues was decreased (P < 0.01). Besides, the expression level of miR-30a-5p in the mice was negatively correlated with serum ALT and AST levels (r²=0.4557, P=0.0003, r²=0.4626, P=0.0003), and the DNA methylation of miR-30a-5p promoter region was increased (P < 0.01). In the HL-7702 cells, compared with control group,the ALT and AST levels were increased in Hcy group (P < 0.05, P < 0.01), and the cell viability was remarkablely decreased. DNA methylation of miR-30a-5p promoter region was increased (P < 0.01), which decreased after treated the cells with AZC (P < 0.05), while the expression level of miR-30a-5p in the cells was increased (P < 0.05). CONCLUSION: Hypermethylation of miR-30a-5p promoter region may play an important role in hepatic injury.     

Relationship between TGF β1 expression and apoptosis in hepatocytes during acute hemorrhagic necrotic pancreatitis

AIM: To observe the expression of TGF β1 in hepatocytes during acute hemorrhagic and necrotic pancreatitis (AHNP) and to study the relationship between TGF β1 and apoptosis in hepatocytes. METHODS: AHNP was induced in 40 rats weighting 260-280 g by intraductal administration of 5% sodium taurocholate. The pathologic morphologic changes of liver and pancreas were observed under light microscope. The hepatocyte apoptosis was examined through TdT (terminal deoxynucleotidyl transferase) mediated dUTP nick end labeling (TUNEL) and the expression of TGF β1 in hepatocytes was analyzed through immunohistochemistry. RESULTS: The liver injuries were found at 3 h after the inducement. These changes were aggravated with the development of the disease. The apoptotic hepatocytes were found after 3 h (P<0.05), and TGF β1 expressed in liver cells was observed at the same time (P<0.05). Both became more and more obvious with the development. CONCLUSION: AHNP can induce TGF β1 expression and apoptosis in hepatocytes, TGF β1 expression is correlation with hepatocyte apoptosis.     

Roles of four liver cells in regulation of PA-plasmin system during liver fibrogenesis in rats

AIM: To investigate the roles of hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs) and endothelial cells (ECs) in the regulation of PA-plasmin system during liver fibrogenesis in rats. METHODS: Experimental liver fibrosis was induced in rats by injection of carbon tetrachloride (CCl₄) twice a week for 12 weeks. Four kinds of liver cells were separated from the normal and fibrotic livers of the rats. The expression levels of urokinase plasminogen activator (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) in liver cells were determined by Northern and Western blotting. RESULTS: The expression of PAI-1 and uPAR was markedly increased in HSCs during liver fibrosis in rats as compared to those in the ECs. CONCLUSION: HSCs and ECs may play very important roles in the regulation of PA-plasmin system during liver fibrogenesis in rats. The activated HSCs are main cells to secrete PAI-1 and uPAR.