To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates.AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC).On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all<0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all<0.01).Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.
Albumins ; biosynthesis ; metabolism ; Alginates ; Ammonia ; metabolism ; Animals ; Cell Aggregation ; physiology ; Cell Culture Techniques ; methods ; Cell Line, Transformed ; physiology ; Chitosan ; Diazepam ; metabolism ; Glucuronic Acid ; Hep G2 Cells ; cytology ; physiology ; Hepatocytes ; cytology ; physiology ; Hexuronic Acids ; Humans ; Liver, Artificial ; Rotation

Based on their ability to differentiate into multiple cell types including hepatocytes, the transplantation of mesenchymal stem cells (MSCs) has been suggested as an effective therapy for chronic liver diseases. The aim of this study was to evaluate the safety, efficacy and therapeutic effects of MSCs in patients with chronic liver disease through a literature-based examination. We performed a systematic review (SR) and meta-analysis (MA) of the literature using the Ovid-MEDLINE, EMBASE and Cochrane Library databases (up to November 2014) to identify clinical studies in which patients with liver diseases were treated with MSC therapy. Of the 568 studies identified by the initial literature search, we analyzed 14 studies and 448 patients based on our selection criteria. None of the studies reported the occurrence of statistically significant adverse events, side effects or complications. The majority of the analyzed studies showed improvements in liver function, ascites and encephalopathy. In particular, an MA showed that MSC therapy improved the total bilirubin level, the serum albumin level and the Model for End-stage Liver Disease (MELD) score after MSC treatment. Based on these results, MSC transplantation is considered to be safe for the treatment of chronic liver disease. However, although MSCs are potential therapeutic agents that may improve liver function, in order to obtain meaningful insights into their clinical efficacy, further robust clinical studies must be conducted to evaluate the clinical outcomes, such as histological improvement, increased survival and reduced liver-related complications, in patients with chronic liver disease.
Cell Differentiation/physiology ; Cell- and Tissue-Based Therapy/adverse effects/*methods ; Hepatocytes/cytology ; Humans ; Liver/physiopathology/surgery ; Liver Diseases/*therapy ; Liver Function Tests ; Mesenchymal Stem Cell Transplantation/adverse effects/*methods ; Mesenchymal Stromal Cells/*cytology

OBJECTIVETo investigate whether RNA interference (RNAi) of LXRα gene in donor rats with fatty liver improves liver graft function after transplantation.

METHODSFifty donor SD rats were fed a high-fat diet and 56% alcohol to induce macrovesicular steatosis exceeding 60% in the liver. The donor rats were injected via the portal veins with 7 × 10⁷ TU LXRα-RNAi-LV mixture (n=25) or negative control-LV (NC-LV) vector (n=25) 72 h before orthotopic liver transplantation. At 2, 24, and 72 h after the transplantation, the recipient rats were sacrificed to examine liver transaminases, liver graft histology, immunostaining (TUNEL), and protein and mRNA levels of LXRα.

RESULTSLentivirus-LXRα RNAi inhibited LXRα gene expression at both the mRNA and protein levels in the liver graft and reduced the expressions of SREBP-1c and CD36 as compared with the controls, resulting also in reduced fatty acid accumulation in the hepatocytes. The recipient rats receiving RNAi-treated grafts showed more obvious reduction in serum ALT, AST, IL-1β and TNF-α levels, and exhibited milder hepatic pathologies than the control rats after the transplantation. TUNEL assay demonstrated a significant reduction in cell apoptosis in LXRα-RNAi-LV-treated liver grafts, and the rats receiving treated liver grafts had a prolonged mean overall survival time.

CONCLUSIONLXRα-RNAi-LV treatment of the donor rats with fatty liver can significantly down-regulate LXRα gene expression in the liver graft and improve the graft function and recipient rat survival after liver transplantation.

Animals ; Fatty Liver ; genetics ; surgery ; Gene Expression Regulation ; Hepatocytes ; cytology ; Lentivirus ; Liver ; physiology ; Liver Transplantation ; Liver X Receptors ; Orphan Nuclear Receptors ; genetics ; RNA Interference ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley

OBJECTIVEIn the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo.

METHODSHuman cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time.

RESULTSWe observed that treatment with pristimerin, at as low as 1 μmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes.

CONCLUSIONThese studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

Animals ; Cell Line ; Dependovirus ; genetics ; physiology ; Gene Expression ; drug effects ; Genetic Therapy ; Genetic Vectors ; genetics ; physiology ; Hepatocytes ; metabolism ; virology ; Humans ; Liver ; cytology ; metabolism ; virology ; Mice ; Transgenes ; drug effects ; Triterpenes ; pharmacology

Vitamin D3 up-regulated protein 1 (VDUP1) is a potent growth suppressor that inhibits tumor cell proliferation and cell cycle progression when overexpressed. In a previous study, we showed that VDUP1 knockout (KO) mice exhibited accelerated liver regeneration because such animals could effectively control the expression of cell cycle regulators that drive the G1-to-S phase progression. In the present study, we further investigated the role played by VDUP1 in initial priming of liver regeneration. To accomplish this, VDUP1 KO and wild-type (WT) mice were subjected to 70% partial hepatectomy (PH) and sacrificed at different times after surgery. The hepatic levels of TNF-alpha and IL-6 increased after PH, but there were no significant differences between VDUP1 KO and WT mice. Nuclear factor-kappaB (NF-kappaB), c-Jun-N-terminal kinase (JNK), and signal transducer and activator of transcription 3 (STAT-3) were activated much earlier and to a greater extent in VDUP1 KO mice after PH. A single injection of TNF-alpha or IL-6 caused rapid activation of JNK and STAT-3 expression in both mice, but the responses were stronger and more sustained in VDUP1 KO mice. In conclusion, our findings provide evidence that VDUP1 plays a role in initiation of liver regeneration.
Animals ; Blotting, Western ; Carrier Proteins/*genetics/metabolism ; Cell Proliferation ; *Gene Expression Regulation ; Hepatectomy ; Hepatocytes/*cytology/physiology ; JNK Mitogen-Activated Protein Kinases/genetics/metabolism ; Liver/*physiology ; Male ; Mice, Knockout ; NF-kappa B/genetics/metabolism ; Polymerase Chain Reaction ; *Regeneration ; STAT3 Transcription Factor/genetics/metabolism ; Thioredoxins/*genetics/metabolism

OBJECTIVETo investigate the characteristics of phase II metabolic enzymes in mouse embryonic stem (ES) cell-derived liver tissue.

METHODSMature hepatocytes were differentiated from embryonic stem cells in cultured mouse embryoid bodies (EB) at d18. Western blot was used to detect the expression of uridine 5'-diphosphate glucronosyl transferase (UGT1a1,UGT1a6) and microsomal glutathione S-transferases 1(mGST1) during the differentiation course.The derived liver tissue was incubated with UDPGA and 7-HFC,the formation of 7-HFC glucuronide was detected by HPLC to examine the total activities of UGT1a1 and UGT1a6. Furthermore, the microsomes were incubated with CDNB and GSH,and the mGST1 activity was measured by spectrometry.

RESULTSAn increase tendency of UGT1a1 expression was noticed during the differentiation course. UGT1a6 and mGST1 were not detected in the earlier stage until d18 of differentiation. The metabolic activity of mGST1 in the derived hepatocytes was 7.65 nmol/min/mg on d18.

CONCLUSIONThe ES cell-derived liver tissue possesses partial metabolic function of phase II enzymes on d18 of differentiation,which might be used as a model for in vitro research on hepatic pathophysiology and phase II drug metabolism.

Animals ; Cell Differentiation ; Embryoid Bodies ; cytology ; Embryonic Stem Cells ; cytology ; Glucuronosyltransferase ; physiology ; Glutathione Transferase ; physiology ; Hepatocytes ; cytology ; enzymology ; Mice

BACKGROUNDHuman embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.

METHODSDonated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propagate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells.

RESULTSWe generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.

CONCLUSIONSThis newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo development research, along with clinical treatments for diabetes and some hepatic diseases.

Cell Differentiation ; physiology ; Cell Line ; Embryonic Stem Cells ; cytology ; Hepatocytes ; cytology ; metabolism ; Humans ; Karyotyping ; Pancrelipase ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVEThe aim of this study was to transfect TPT1 into cell lines SMMC-7721 and L-02, seperately, and to observe the changes of biological behaviors of the cell lines.

METHODSThrough lipofectamine, the eukaryotic report expression vector containing TPT1 ORF (open reading frame), pEGFP-N3TPT1, were transducted into hepatocarcinoma cell line SMMC-7721 cells and normal liver cell line L-02 cells, seperately. The transduction was repeated three times in 24 hrs. The differences of biological behaviors between the pEGFP-N3TPT1 and pEGFP-N3 groups were studied by RT-PCR, MTT assay, soft agar colony formation assay and cell cycle analysis.

RESULTSThe pEGFP-N3TPT1 transfected cells had a high mRNA level in the two cell lines (P < 0.05) compared with the pEGFP-N3 controls. The ability of proliferation and the soft agar colony formation were enhanced in the SMMC-7721 transducted cells with pEGFP-N3TPT1 compared with that transducted with pEGFP-N3 (P < 0.05), and the cell cycle analysis showed that the cells in the phase G(2)+S/M increased after pEGFP-N3TPT1 transduction. In the L-02 cell line, we obtained similar results, pEGFP-N3TPT1 enhanced the colony formation in plate (P < 0.05), but not make it form colony in soft agar.

CONCLUSIONSTPT1 can enhance malignant phenotype of SMMC-7721 cells and promote the growth of L-02 cells, but not transform L-02 into malignant phenotype.

Biomarkers, Tumor ; biosynthesis ; genetics ; physiology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Cell Transformation, Neoplastic ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatocytes ; cytology ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Open Reading Frames ; RNA, Messenger ; metabolism ; Transfection

OBJECTIVE: To determine the regulation effect of bone morphogenetic protein-4 (BMP-4) on the proliferation and differentiation of rat hepatic precursor cells. METHODS: We used Noggin (200 ng/mL) as the function blocking control of BMP-4, and the hepatic precursor cells of WB-F344 were treated with recombinant BMP-4 at 50 ng/mL at different time points. The proliferation of WB-F344 cells were tested by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The ultrastructural characters of differentiated WB-F344 cells regulated by BMP-4 were observed under a transmission electron microscope. RT-PCR was used to examine mRNA expression of specific molecular markers for different cellular phenotypes potentially differentiated from the WB-F344 cells. RESULTS: At different time points, the absorbance values in the BMP-4 treatment groups were higher than those in the control groups of Noggin and blank treatment (P<0.01). The WB-F344 cells treated with BMP-4 exhibited typical ultrastructural characters of well-differentiated epithelial cells. The hepatocyte mRNA markers were more significantly promoted in the differentiated WB-F344 cells in the BMP-4 treatment group than those in the other 2 control groups. CONCLUSION BMP-4 can promote the proliferation and directional differentiation towards hepatocytes of rat hepatic precursor cells of WB-F344.
Animals ; Bone Morphogenetic Protein 4 ; antagonists & inhibitors ; genetics ; physiology ; Carrier Proteins ; pharmacology ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Hepatocytes ; cytology ; Rats ; Recombinant Proteins ; Stem Cells ; cytology

OBJECTIVETo compare macroporous microcarrier Cytopore 2 and Cultispher S for their effects in microgravity culture of CL-1 cells.

METHODSCL-1 cells were cultured on Cytopore 2 and Cultispher S respectively in a rotational cell culture system (RCCS) in a volume of 50 ml. Dynamic morphological observation and cell counting and functional test were carried out during the cell culture.

RESULTSThe cells were capable of adhering to and proliferating on both of the microcarriers, reaching the growth peak on day 9 of cell culture with the maximum cell density of 4x10(6)/ml on cultispher S. Albumin was significantly higher in the supernatant of Cytopore 2 than in that of Cultispher S on days 10, 11, and 12 (P<0.05), and the urea level in the supernatant of cytopore 2 was also significantly higher on days 10 and 11 (P<0.05).

CONCLUSIONThe cells cultured on Cytopore 2, though with a smaller cell number, display better functions than those cultured on Cultispher S under RCCS conditions.

Cell Culture Techniques ; instrumentation ; methods ; Cell Division ; physiology ; Cell Line ; Cellulose ; Gelatin ; Hepatocytes ; cytology ; Humans ; Liver, Artificial ; Porosity ; Tissue Engineering ; methods ; Tissue Scaffolds ; Weightlessness