Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells

AIM： To investigate the hepatocytic differentiation of mesenchymal stem cells （MSCs） in co-cultures with fetal liver cells （FLC） and the possibility to expand differentiated hepatocytic cells. METHODS： MSCs were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor （SCF）, hepatocyte growth factor （HGF）, epidermal growth factor （EGF）, and fibroblast growth factor 4 （FGF-4） alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction （RT-PCR） for the liver specific markers cytokeratin-18 （CK-18）, albumin, and alpha-fetoprotein （AFP） was performed in different cell populations. RESULTS- Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential. CONCLUSION： The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also prorides a beneficial environment for expansion and differentiation of FLC.

Comparative study on radiosensitivity of various tumor cells and human normal liver cells

AIM:To investigate the radiation response of various human tumor cells and normal liver cells. METHODS: Cell lines of human hepatoma cells (SMMC-7721), liver cells (L02), melanoma cells (A375) and cervical tumor (HeLa) were irradiated with 60Co γ-rays. Cell survive was documented by a colony assay. Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A. RESULTS: Linear quadratic survival curve was observed in all of four cell lines, and dose-dependent increase in radiation-induced chromatid and isochromatid breaks were observed in GB2B phase. Among these four cell lines, A375 was most sensitive to radiation, while, L02 had the lowest radiosensitivity. For normal liver cells, chromatid breaks were easy to be repaired, isochromatid breaks were difficult to be repaired. CONCLUSION: The results suggest that the y-rays induced chromatid breaks can be possibly used as a good predictor of radiosensitivity, also, unrejoined isochromatid breaks probably tightly related with cell cancerization.

Hepatitis C virus core proteins derived from different quasispecies of genotype 1b inhibit the growth of Chang liver cells

AIM: To investigate the influence of different quasispecies of hepatitis C virus (HCV) genotype 1b core protein on growth of Chang liver cells. METHODS: Three eukaryotic expression plasmids (pEGFP-N1/core) that contained different quasispecies truncated core proteins of HCV genotype 1b were constructed. These were derived from tumor (T) and non- tumor (NT) tissues of a patient infected with HCV and C191 (HCV-J6). The core protein expression plasmids were transiently transfected into Chang liver cells. At different times, the cell cycle and apoptosis was assayed by flow cytometry, and cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) assay. RESULTS: The proportion of S-phase Chang liver cells transfected with pEGFP-N1/core was significantly lower than that of cells transfected with blank plasmid at three different times after transfection (all P < 0.05). The proliferation ratio of cells transfected with pEGFP-N1/corewas significantly lower than that of cells transfected with blank plasmid. Among three different quasispecies, T, NT and C191 core expression cells, there was no significant difference in the proportion of S- and G0/G1-phase cells. The percentage of apoptotic cells was highest for T (T > NT > C191), and apoptosis was increased in cells transfected with pEGFP-N1/core as the transfection time increased (72 h > 48 h > 24 h). CONCLUSION: These results suggest that HCV genotype 1b core protein induces apoptosis, and inhibits cell- cycle progression and proliferation of Chang liver cells. Different quasispecies core proteins of HCV genotype 1b might have some differences in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.

Metabolism of Mequindox in Isolated Rat Liver Cells

Mequindox （MEQ）, 3-methyl-2-quinoxalinacetyl-l,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial agent and feed additive. Its toxicity has been reported to be closely related to its metabolism. To understand the pathways underlying MEQ＇s metabolism more clearly, we studied its metabolism in isolated rat liver cells by using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap （LC-LTQ-Orbitrap） mass spectrometry. The structures of MEQ metabolites and their product ions were readily and reliably characterized on the basis of accurate MS2 spectra and known structure of MEQ. Eleven metabolites were detected in isolated rat liver cells, two of which were detected for the first time in vitro. The major metabolic pathways reported previously for in vitro metabolism of MEQ in rat microsomes were confirmed in this study, including N O group reduction, carbonyl reduction, and methyl monohydroxylation. In addition, we fotmd that acetyl hydroxylation was an important pathway of MEQ metabolism. The results also demonstrate that cellular systems more closely simulate in vivo conditions than do other in vitro systems such as microsomes. Taken together, these data contribute to our understanding of the in vivo metabolism of MEQ.

Studies of the Kinetochore Proteins of the Regenerating Liver and the Liver Cells of Rats at Different Stages of Development

The kinetochore composition of rat liver cells was studied by indirect immunofluorescence andimmunoblotting using human anti-kinetochore/centromere autoantibodies(ACAs).Besides threemajor antigens(50kD,42 kD and 34 kD),ACAs used in this study could also identify those of 32-30 kD and 20 kD in newborn rat liver cells,90 kD in old rat liver cells,37 kD and 32-30 kD inregenerating liver cells.These results indicate that some kinetochore antigen(s)may be related to cellproliferation or specific for different stages of development.

Malignant Transformation of Human Embryonic Liver Cells Induced by Hepatitis B Virus and Aflatoxin B_1

In order to investigate the effect of hepatitis B virus (HBV) and aflatoxin B 1 (AFB 1) on hepatocarcinogenesis, the human embryonic liver cells infected with HBV were transplanted to nude mice by subcutaneous route and the transplanted mice were divided into 4 groups for study, in which the group A of mice was injected with HBV-infected human embryonic liver cells and followed by injections of AFB 1 once a week (HBV+AFB 1); the group B was treated with HBV as group A, but no AFB 1 was given (HBV +); the group C was injected with normal human embryonic liver cells and AFB 1 was used as group (AFB 1 +) and the group D or control group was injected with normal embryonic liver cells without addition of AFB 1. The experimental results showed that the incidences of tumor formation in different groups were 27.3% (6/22) in group A; 0% (0/13) in group B; 13.3% (2/15) in group C and 0% (0/14) in group D respectively. All the tumors formed were proved to be human hepatocellular carcinoma (HCC) by pathological examinations and the tumor tissues were anthrogenetic as demonstrated by EMA monoclonal antibody. The HBV-X and HBV-S genes could be detected in the tumor tissues by means of slot hybridization and PCR amplification, indicating that the HBV-DNA genes had integrated into DNA of host cells. Thus, we have successfully induced the human HCC through HBV infection and introduction of AFB 1 with a synergistic effect between HBV and AFB 1 in hepatocarcinogenesis.

The role of liver sinusoidal endothelial cells in liver remodeling after injury

Liver transplantation is the optimal treatment for patients with end-stage liver disease,metabolic liver diseases,and hepatic malignancies that are not amenable to resection.Hepatic ischemia-reperfusion injury(IRI)is the main problem in liver transplantation and liver resection,leading to parenchymal cell injury and organ dysfunction.The damage of liver sinusoidal endothelial cells(LSECs)is a critical event in IRI.LSECs work as an important regulating factor of liver regeneration after partial hepatectomy.This review primarily describes the mechanisms of LSECs injury in IRI and explores the roles of LSECs in liver regeneration,and briefly introduces the protective strategies targeting LSECs damaged in IRI.

Apoptosis induced by NaYF_4:Eu^(3+) nanoparticles in liver cells via mitochondria damage dependent pathway

As lanthanide-doped sodium yttrium flouride(NaYF_4)nanoparticles have great potential inbiomedical applications,their biosafety is important and has attracted significant attention.In the present work,three different sized NaYF_4:Eu^(3+)nanoparticles have been prepared.Liver BRL 3 A cell was used as a cell model to evaluate their biological effects.Cell viability and apoptosis assays were used to confirm the cytotoxicity induced by NaYF_4:Eu^(3+)NPs.Apart from the elevated malondialdehyde(MDA),the decrease of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX)and catalase(CAT)activity indicated reactive oxygen species(ROS)generation,which were associated with oxidative damage.The decrease of mitochondrial membrane potential(MMP)value demonstrated the occurrence of mitochondria damage.Then,release of cytochrome c from mitochondria and activation of caspase-3 confirmed that NaYF_4:Eu^(3+)NPs induced apoptosis was mitochondria damage-dependent.

Influence of adriamycin on changes in Nanog, Oct-4, Sox2, ARID1 and Wnt5b expression in liver cancer stem cells

AIM: To determine the influence of Adriamycin (ADM) on the changes in Nanog, Oct4, Sox2, as well as, in ARID1 and Wnt5b expression in liver cancer stem cells.

Passage of bone-marrow-derived liver stem cells in a proliferating culture system

AIM： To explore the feasibility of passage of bone- marrow-derived liver stem cells （BDLSCs） in culture systems that contain cholestatic serum.METHODS： Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction （RT-PCR） and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.RESULTS： The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units （H-CFUs） were formed. H-CFUs expressed markers of embryonic hepatocytes （alpha-fetoprotein, albumin and cytokeratin 8/18）, biliary cells （cytokeratin 19）, hepatocyte functional proteins （transthyretin and cytochrome P450-261）, and hepatocyte nuclear factors 1α and -3β）. They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.CONCLUSION： BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Role of liver stem cells in hepatocarcinogenesis

Liver cancer is an aggressive disease with a high mortality rate. Management of liver cancer is strongly dependent on the tumor stage and underlying liver disease. Unfortunately, most cases are discovered when the cancer is already advanced, missing the opportunity for surgical resection. Thus, an improved understanding of the mechanisms responsible for liver cancer initiation and progression will facilitate the detection of more reliable tumor markers and the development of new small molecules for targeted therapy of liver cancer. Recently, there is increasing evidence for the "cancer stem cell hypothesis", which postulates that liver cancer originates from the malignant transformation of liver stem/progenitor cells(liver cancer stem cells). This cancer stem cell model has important significance for understanding the basic biology of liver cancer and has profound importance for the development of new strategies for cancer prevention and treatment. In this review, we highlight recent advances in the role of liver stem cells in hepatocarcinogenesis. Our review of the literature shows that identification of the cellular origin and the signaling pathways involved is challenging issues in liver cancer with pivotal implications in therapeutic perspectives. Although the dedifferentiation of mature hepatocytes/cholangiocytes in hepatocarcinogenesis cannot be excluded, neoplastic transformation of a stem cell subpopulation more easily explains hepatocarcinogenesis. Elimination of liver cancer stem cells in liver cancer could result in the degeneration of downstream cells, which makes them potential targets for liver cancer therapies. Therefore, liver stem cells could represent a new target for therapeutic approaches to liver cancer in the near future.

Effects of ethanol on liver sinusoidal endothelial cells-fenestrae of rats

Important advances have been made in research into the mechanism of alcoholic liver disease (ALD) over the past few years,but the role of liver sinusoidal endothelial cell (LSEC) in ALD has not been elucidated adequately. This study was undertaken to investigate the effect of ethanol on fenestrae of LSECs in rats. METHODS: A rat model of alcoholic liver disease was established by means of direct intragastric instillation of ethanol. Fifty-five rats of experimental (35 rats) and control (20) groups were sacrificed at the end of 4,8,12 weeks respectively, and also at the end of 12-week abstinence. After heart perfusion, the liver tissue was fixed and stained with hematoxylin and eosin for observation of serial changes of LSEC-fenestrae under a transmission electron microscope. RESULTS: Normal LESC was flat with a nucleus and organelles arranged regularly. The distal cytoplasm displayed as a lamina with many fenestrae, lacking the basement membrane(BM) underneath the endothelium. At the end of 4-week alcohol feeding, the number of fenestrae decreased at the distal cytoplasm in some LSECs, without the formation of the BM underneath the endothelium. At the end of 8 weeks, the number of fenestrae decreased significantly or even disappeared. The BM began to develop incompletely underneath the endothelium, while the active fibroblast appeared. At the end of 12 weeks, the number of fenestrae decreased more significantly and the complete BM could even be seen. But the changes were mostly limited in the single or adjoining sinus, and fibrosis was scarcely formed. At the end of 12-week abstinence, defenestration and formation of the endothelial BM lightened significantly. CONCLUSIONS:Defenestration and formation of the BM in LSECs develop gradually with the chronic stimulation of ethanol. Hepatic sinusoidal capillarization and fibrosis will be seen if their state is more serious. These early changes, i. e., limited and regional defenestration and capillarization may be the basis of alcoholic peri-fibrosis. This kind of he- patic fibrosis is reversible after removal of etiological factors.

Epithelial cells with hepatobiliary phenotype:Is it another stem cell candidate for healthy adult human liver?

AIM： To investigate the presence and role of liver epithelial cells in the healthy human adult liver. METHODS： Fifteen days after human hepatocyte primary culture, epithelial like cells emerged and started proliferating. Cell colonies were isolated and sub- cultured for more than 160 d under specific culture conditions. Cells were analyzed for each passage using immunofluorescence, flow cytometry and reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： Flow cytometry analysis demonstrated that liver epithelial cells expressed common markers for hepatic and stem cells such as CD90, CD44 and CD29 but were negative for CD34 and CDl17. Using immunofluorescence we demonstrated that liver epithelial cells expressed not only immature （α-fetoprotein） but also differentiated hepatocyte （albumin and CK-18） and biliary markers （CK-7 and 19）, whereas they were negative for OV-6. RT-PCR analysis confirmed immunofluorescence data and revealed that liver epithelial cells did not express mature hepatocyte markers such as CYP2B6, CYP3A4 and tyrosine amino-transferase. Purified liver epithelial cells were transplanted into SCID mice. One month after transplantation, albumin positive cell foci were detected in the recipient mouse parenchyma. CONCLUSION： According to their immature and bipotential phenotype, liver epithelial cells might represent a pool of precursors in the healthy human adult liver other than oval cells.

Structural and functional aspects of the liver and liver sinusoidal cells in relation to colon carcinoma metastasis

Nowadays, liver metastasis remains difficult to cure. When tumor cells escape and arrive in the liver sinusoids, they encounter the local defense mechanism specific to the liver. The sinusoidal cells have been widely described in physiologic conditions and in relation to metastasis during the past 30 years. This paper provides an “overview” of how these cells function in health and in diseases such as

An efficient method of sorting liver stem cells by using immuno-magnetic microbeads

AIM： To develop a method to isolate liver stem cells fast and efficiently. METHODS： Fetal mouse liver cells were characterized by cell surface antigens （c-Kit and CD45/TER119） using flow cytometry. The candidate liver stem cells were sorted by using immuno-magnetic microbeads and identified by -lone-forming culture, RT-PCR and immunofluorescence says. RESULTS： The c-Kit-（CD45/TER119）- cell population with 97.9% of purity were purified by immuno-magnetic microbeads at one time. The yield of this separation was about 6% of the total sorting cells and the cell viability was above 98%. When cultured in vitro these cells had high clone-forming and self-renewing ability and expressed markers of hepatocytes and bile duct cells. Functionally mature hepatocytes were observed after 21 d of culture. CONCLUSION： This method offers an excellent tool for the enrichment of liver stem cells with high purity and viability, which could be used for further studies. It is fast, efficient, simple and not expensive.

Usefulness of liver infiltrating CD86-positive mononuclear cells for diagnosis of autoimmune hepatitis

AIM： Although the pathogenic mechanism underlying autoimmune hepatitis （AIH） remains unclear, the immune system is thought to be critical for the progression of the disease. Cellular immune responses may be linked to the hepatocellular damage in AIH. Recently, much attention has been focused on the critical functions of costimulatory molecules expressed on mononuclear cells in the generation of effective T cell-mediated immune responses. Analysis of costimulatory molecule expressed on mononuclear cells from the patients with AIH may give us insight into the pathogenic mechanism of hepatocellular damage in AIH. METHODS： Peripheral blood mononuclear cells （PBMC） were taken from the patients with AIH （34 cases） and healthy controls （25 cases）. Uver infiltrating mononuclear cells （LIMCs） were taken from the patients with AIH （18 cases）, the patient with chronic hepatitis C （CH-C） （13 cases） and the patients with fatty liver （2 cases）. Using flow cytometry, the cells were analyzed for the expression of costimulatory molecules, such as CD80, CD86, and CD152 （CTLA-4）. The results were compared with clinical data such as the level of gammaglobulin, histological grade, presence or absence of corticosteroids administration and the response to corticosteroids. RESULTS： The levels of CD80＋, CD86＋ and CD152＋ PBMC were significantly reduced in the patients with AIH as compared with healthy controls. By contrast, those cells were significantly higher in LIMC than in PBMC of the patients with AIH. Especially, the level of CD86＋ LIMC showed a marked increase irrespective of the degree of disease activity in the patients with AIH,although CD86＋ cells were rarely present in PBMC. The levels of CD86＋ cells were present in significantly higher frequency in patients with AIH than in the patients with CH-C. Furthermore, the patients with AIH with high levels of CD86＋ LIMC showed good responses to corticosteroids, whereas 2 cases of AIH with low levels of CD86＋ LIMC did not respond well. CONCLUSION： These results suggest that LIMC overexpressing costimulatory molecules such as CD80 and CD86 appears to play a role in the pathogenesis of AIH. Especially, CD86 molecule expressed on the LIMC may be useful for the diagnosis of AIH and for the prediction of the therapeutic effects of corticosteroids on AIH.

Regulators of liver cancer stem cells

Hepatocellular carcinoma(HCC)is a leading cause of cancer deaths.It is often detected at a stage when there are few therapeutic options.Liver cancer stem cells(LCSCs)are highly tumorigenic and resistant to chemotherapy and radiation therapy.Their presence in HCC is a major reason why HCC is difficult to treat.The development of LCSCs is regulated by a variety of factors.This review summarizes recent advances on the factors that regulate the development of LCSCs.Due to the importance of LCSCs in the development of HCC,a better understanding of how LCSCs are regulated will help to improve the treatments for HCC patients.

Interaction between Colon Cancer Cells and Human Liver Sinusoidal Endothelial Cells Promotes Liver Metastasis of Tumor Cells

OBJECTIVE To investigate the effect of co-culture between colon cancer cells （SW1116） and human liver sinusoidal endothelial cells （HLSECs） on cancer cell metastasis, and to provide a novel model for studying the mechanism of colon cancer liver metastasis. METHODS HLSECs and SW1116 were co-cultured for 21 rounds in vitro. Transwell migration, gelatin-zymography, CCK-8 proliferation and colony formation assays were used to examine the invasion, proliferation, and colony forming ability of cancer cells. Assays were carried out to examine tumor growth ability and liver metastasis. The associated molecular change was examined by western blotting. RESULTS After 21 selection rounds, colon cancer cells SWl 1161）21 displayed a clear boundary. Compared with the 5W1116 cells, SW1116P21 cells had a greater invasive ability, cell proliferation and colony formation in soft agar. A gelatin-zymography assay showed that the ability of SW1116P21 cells to secrete matrix metalloproteinase-2/9 was significantly greater than that of SWl116 cells. Additionally, the capacity for subcutaneous tumor formation of SW1116P21 was significantly increased. It was found that mice injected with SW1116P21 cells developed significantly more visually observable liver nodules than mice injected with SW1116 cells. Western blotting showed increased vimentin expression and decreased E-cadherin expression in the SW1116P21 cells, compared with the SWl 116 cells. CONCLUSION The interaction between SW1116 and HLSECs may promote tumor cell invasion, proliferation and colony formation in vitro, and tumor formation and liver metastasis in vivo. An epithelial-mesenchymal transition occurs in SWl 116P21 cells, which contributes to the change in the characteristics of tumor cells.

Efficient generation of functional hepatocytelike cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells

BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Clinical trial with traditional Chinese medicine intervention ''tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment'' for chronic hepatitis B-associated liver failure

AIM:To study the clinical efficacy of traditional Chinese medicine(TCM)intervention"tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment"("TTK")for treating liver failure due to chronic hepatitis B.METHODS:We designed the study as a randomized controlled clinical trial.Registration number of Chinese Clinical Trial Registry is Chi CTR-TRC-12002961.A total of 144 patients with liver failure due to infection with chronic hepatitis B virus were enrolled in this randomized controlled clinical study.Participants were randomly assigned to the following three groups:(1)a modern medicine control group(MMC group,36patients);(2)a"tonifying qi and detoxification"("TQD")group(72 patients);and(3)a"tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment"("TTK")group(36patients).Patients in the MMC group received general internal medicine treatment;patients in the"TQD"group were given a TCM formula"tonifying qi and detoxification"and general internal medicine treatment;patients in the"TTK"group were given a TCM formula of"TTK"and general internal medicine treatment.All participants were treated for 8 wk and then followed at 48 wk following their final treatment.The primaryefficacy end point was the patient fatality rate in each group.Measurements of various virological and biochemical indicators served as secondary endpoints.The one-way analysis of variance and the t-test were used to compare patient outcomes in the different treatment groups.RESULTS:At the 48-wk post-treatment time point,the patient fatality rates in the MMC,"TQD",and"TTK"groups were 51.61%,35.38%,and 16.67%,respectively,and the differences between groups were statistically significant(P<0.05).However,there were no significant differences in the levels of hepatitis B virus DNA or prothrombin activity among the three groups(P>0.05).Patients in the"TTK"group had significantly higher levels of serum total bilirubin compared to MMC subjects(339.40μmol/L±270.09μmol/L vs 176.13μmol/L±185.70μmol/L,P=0.014).Serum albumin levels were significantly increased in both the"TQD"group and"TTK"group as compared with the MMC group(31.30 g/L±4.77g/L,30.72 g/L±2.89 g/L vs 28.57 g/L±4.56 g/L,P<0.05).There were no significant differences in levels of alanine transaminase among the three groups(P>0.05).Safety data showed that there was one case of stomachache in the"TQD"group and one case of gastrointestinal side effect in the"TTK"group.CONCLUSION:Treatment with"TTK"improved the survival rates of patients with liver failure due to chronic hepatitis B.Additionally,liver tissue was regenerated and liver function was restored.