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Objective: To investigate the effect of Liuwei Dihuangwan on connexin 32 (Cx32) in hepatoma cell line CBRH7919 and its gap junction intercellular communication (GJIC), and furthermore study its mechanism of enhancing the bystander killing effect of suicide gene therapy. Method: Liuwei Dihuangwan (32 g·kg·d-1) and the same volume of normal saline were given to the rats by intragastrical administration. Blood was taken to prepare the medicated serum of Liuwei Dihuangwan and blank control serum, respectively. The hepatoma cell line CBRH7919 were treated by control serum and medicated serum of Liuwei Dihuangwan in different concentrations. There were four groups in experiment:the blank control group (volume fraction of 10%), medicated serum high dose group of Liuwei Dihuangwan (the volume fraction of 10%), medicated serum middle dose group of Liuwei Dihuangwan (the volume fraction of 5%), and medicated serum low dose group of Liuwei Dihuangwan (the volume fraction of 2.5%). The expression levels of Cx32 protein and mRNA in hepatoma cell line CBRH7919 were detected by indirect immunofluorescence assay (ⅡA) and Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) assay. The fluorescence redistribution after photobleaching (FRAP) method was used to detect the function of GJIC of hepatoma cell line CBRH7919. Result: ① The indirect immunofluorescence assay (ⅡA) analysis indicated that as compared with the blank control group, the cx32 expression of CBRH7919 cells was up-regulated in a concentration-dependent manner in each dose group of the serum containing Liuwei Dihuangwan (PPPPConclusion: The mechanism of medicated serum of Liuwei Dihuangwan in enhancing the bystander killing effect of suicide geneis related to gap junction. Liuwei Dihuangwan may enhance the function of GJIC by increasing the localization of cx32 on the cell membrane of CBRH7919 cells and increasing the expression of cx32 mRNA and protein to achieve the synergistic action.
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OBJECTIVE@#To construct a decellularized matrix of human fatty liver as the scaffold for three-dimensional (3D) culture of hepatocarcinoma cells.@*METHODS@#Human fatty liver decellularized matrix (hFLM) was prepared by repeated freezingthawing, perfusion with gradient SDS and 1% Triton X-100 through the portal vein and hepatic artery, and repeated agitation with Triton X-100. HepG2 cells were cultured in the prepared hFLM, and the cell survival, morphology, proliferation and cellular expressions of the adhesion molecules were detected.@*RESULTS@#The decellularization procedure shortened the time for scaffold preparation and preserved the 3D ultrastructure and the composition of the extracellular matrix. HepG2 cells cultured in hFLM scaffold maintained proliferation for up to 15 days and showed a growth pattern with a long lag phase and a slow growth rate, which was similar to the growth pattern . The cultured HepG2 exhibited a low expression of E-cadherin and a high expression of vimentin, which was consistent with the xenograft but opposite to 2D cultured cells. However, the lack of adequate nutrient transport in this hepatocarcinoma cell model led to a slowdown of cell proliferation in the later stage. The PCNA index of HepG2 cells cultured in hFLM was lowered by 29.3% on day 12 as compared with that on day 6.@*CONCLUSIONS@#We established a new protocol for preparing hFLM and confirmed the feasibility of constructing hepatocarcinoma cell models using the hFLM scaffold.
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Humans , Carcinoma, Hepatocellular , Extracellular Matrix , Fatty Liver , Liver Neoplasms , Tissue Engineering , Tissue ScaffoldsABSTRACT
Objective To investigate the role of interleukin-22 (IL-22)-regulated autophagy in hydrogen peroxide (H2 O2 )-induced hepatocarcinoma cell damage. Methods HepG2 cells were transfected with pEGFP-LC3 and then cultured in RPMI 1640 medium free of fetal bovine serum (FBS) or containing 1％ or 10％ FBS. These cells were pretreated with rapamycin or an autophagy inhibitor (3-MA) and then stimulated with recombinat human IL-22 (rhIL-22). GFP-LC3 puncta formation and autophagy signaling ac-tivation were measured. MTT assay was performed to detect cell viability. Results rhIL-22 significantly promoted GFP-LC3 puncta formation and LC3-Ⅱ expression in HepG2 cells treated with different stimulation protocols. The autophagy pathway inhibitor, 3-MA, dramatically suppressed the rhIL-22-activated autophagy signals. rhIL-22 attenuated H2 O2-mediated HepG2 cell death and that could be inhibited by 3-MA. Conclu-sion IL-22 promoted the activation of autophagy signaling pathways and alleviated H2 O2-mediated HepG2 cell damage.
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ObjectiveTo a nalyze the morphologic features of SMMC-7721 cannibalistic cells that induced by triazole Schiff base derivative(LH-37) in vitro. Methods The SMMC-7721 cells (1×10~4/ml)were cultured in the medium containing of 1×10~(-5) mol/L LH-37 for 24h,48h.The character of cells was detected by Papanicolaou and Wright′s Staining. Immunohistochemical method was used to observe the cleaved Caspase-3 positive cells. The ultrastructure of cannibalism cells was observed by JEM 100CX-II transmission electronic microscope. Results Microscopic analysis demonstrated the complete internalization of one cell within another. We noted that some cannibalistic cells in small aggregates appeared to be inside of large vacuoles, suggesting that they were internalized within a neighboring cell. The proportion of cannibalistic cells were increased after SMMC-7721 cells were cultured in the presence of LH-37 for 48 hours. The proportion of the cannibalistic cells in control and LH-37 group was 0.47% and 5.23% respectively . Many internalized cells were positive for cleaved caspase-3 staining . Ultrastructural analysis of engulfed cells from 24 hours exhibited evidence of live-cell internalization consistent with cannibalism, The most common fate for internalized cells was death after treatment with LH-37 for 48 hours, as evidenced by nuclear degradation and the eventual disappearance of some cells within the enveloping cell . Conclusion The data presented indicate that LH-37 can lead to an increase of cannibalism in human hepatocarcinoma cell in vitro.
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In order to investigate the inhibitory effects of all trans-retinoitc acid (ATRA) on differentiation and apoptosis of Walker-256 hepatocellular carcinoma cells and the therapeutic effects of ATRA combined with transarterial chemoembolization (TACE) on rat Walker-256 transplanted hepa-tocarcinoma, Walker-256 hepatocarcinoma cell lines were treated with ATRA at different concentrations. After culture for 48 h, the inhibitory rate of cell proliferation was determined by MTT assay; the changes of Fas and Bcl-2 mRNA expression were determined by RT-PCR, and the expression levels of Caspase3 and Caspase8 proteins were detected by Western blot. Twenty-seven Wistar rat models of hepatocarcinoma were set up successfully by implanting Walker-256 cell lines. The tumor volume at the 11th day after implantation (Vpreoperatioi) was measured by magnetic resonance imaging (MRI). The 27 rats were randomly and equally divided into three groups, and the therapy scheme was performed as follows: group A (ATRA 0.1 mg+mitomycin 0.05 mL+lipiodol 0.05 mL+gelfoam powder 0.025 mg); group B (mitomycin 0.05 mg+lipiodol 0.05 ml+gelfoam 0.025 mg; group C (0.9% NaCl 0.2 mL). After another 11 days, MRI was performed once again to measure the tumor volume (Vpostoperation)- The expression of factor VIII and Ki-67 in the tumor tissues was detected by immuno-histochemistry. The results showed that ATRA could suppress proliferation of Walker-256 cell lines. After treatment of Walker-256 cell lines with ATRA, the expression of Fas mRNA was significantly up-regulated and the Bcl-2 mRNA was significantly down-regulated by ATRA at the concentration of 10 umol/L as compared with the control group (P<0.05). After treatment with 10 umol/L ATRA for 48 h, the Caspase3 and Caspase8 were significantly activated as compared with the control group (P<0.05). Significant difference existed in growth rate among the three groups (P<0.01) and between either two groups (P<0.05). The expression rate of factor VIII and Ki-67 was gradually increased from group A, group B to group C. The study suggests that ATRA could inhibit the proliferation of Walker-256 cells and the effectiveness of the combined therapy (ATRA+TACE) for treating transplanted hepatoma of rats is superior to that of TACE alone.
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Objective:To investigate the expression of livin and survivin induced by 5-Fu agent,and determine the role of livin and survivin in anti-chemotherapy of hepatocellular carcinoma(HCC). Methods:Low concentration of 5-Fu was added in cultured HepG2 cells.The expression of livin and survivin was detected by RT-PCR and western-blot on 24h and 48h after 5-Fu treatment.Results:Livin and survivin mRNA 1eve1 was increased by 0.6 ,1.1 and 1.5,2.2 times,respectively.and thant of the protein expression was increased by 1.3 ,2.7 and 0.9,1.9 times,respectively.Conclusion:livin and survivin was expression in HepG2.A high expression level of livin and survivin was induced in HepG2 by 5-Fu,which reduced to apoptosis by 5-Fu.It's valuable to study the curative effect of 5-Fu to hepatocelluar carcinoma further.
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Objective To investigate the mechanism of Depression proliferation in human hepatocarcinoma cells by Abscisic acid.Methods To detect protein expression o of P53,Ki-67 and Cyclin D1 by immunocyte chemistry; detect mRNA expression of P53 and telomerase by RT-PCR. Results The protein expression level of mtP53, Cyclin D1, Ki-67 and the mRNA expression level of mtP53 and hTERT all decreased in cells treated by ABA,HMBA and ABA+ HMBA(P
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Objective To investingate the effect of 5-Aza2'-deoxycytidine(5-Aza-CdR)on cell growth and to explore the possibility of re-expression of the hypermethylated and silenced RUNX3 gene in hepatocarcinoma cell line HepG2.Methods The change in expression of the tumor suppressor gene RUNX3 mRNA in cultured HepG2 cells was observed by RT-PCR before and after 5-Aza-CdR treatment.Activity of cell growth was observed by MTT assay and colony-forming test.The cell cycle was analyzed by flow cytometry.Apoptotic morphology was observed by transmitting electron microscopy.Results The gene was reactivated by two different doses of 5-Aza-CdR treatment in HepG2 cell without expressing RUNX3.The hepatocarcinoma cell line treated with 5-Aza-CdR displayed a slowed growth rate in contrast to the control group.The colony formation rate of HepG2 cell treated with 5-Aza-CdR decreased dramatically(P
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Objective: To investigate the suppressive effects of nedaplatin on human liver cancer cell line SMMC7721 and the interactions between nedaplatin and adriamycin or mitomycin or fluorouracil. Methods: The cytotoxic index of nedaplatin alone or combined with other chemotherapy agents on SMMC cells were detected by MTT method. Results: SMMC was sensitive to nedaplatin with a positive correlation between cytotoxic index and nedaplatin concentration. There were significant synergism in the cytotoxic effects of nedaplatin combined with adriamycin or Mitomycin or Fluorouracil on SMMC cells. Conclusion: Nedapltin is a promising agent in the treatment of human liver cancer.
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Objective:To find out the role of JNK/SAPK signaling-transduction pathway in the effect of elemene against hepatocarcinoma, offering the clue to ilustrate the molecular mechanisms of antitumor effects of elemene. Methods: The detection of the distribution of elemene in Hca-F cells was detected by gas chomatography and apoptotic changes in elemene treated. SMMC7721 cells were examined by TEM. After elemene treatment, the activation of JNK/SAPK in HepG2 cellls and the DAXX gene expression in SMMC7721 cells were detected by Western blotting and RT-PCR respectively. Results: Gas chomatography showed that elemene was detected at 8. 42 minute. The SMMMC7721 cells treated by elemene for 3 hours began to show typical apoptotic changes . The JNK/SAPK activity in HepG2 cell treated with heat shock was the highest of all groups and the group treated with elemene was the next and the control group is the lowest one. There was no DAXX gene expression in SMMC7721 cells treated with elemene. Conclusion: Elemene can diffuse into cells. Tumor cell apoptosis treated with elemene may be induced by JNK/SAPK activating and DAXX signal pathway may not play key role in JNK/SAPK activation induced by elemene.
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Objective To study the mechanism of killing and apoptosis-inducing effects of Capparis spinosa alkaloid (CSA) on human hepatocarcinoma cell line HepG2. Methods The killing effect of CSA on human hepatocarcinoma cell line HepG2 was studied by MTT method. Morphological observation of HepG2 cells was carried out by fluorescence microscope. Results The CSA had obvious cytotoxicity on the HepG2 in a dose-dependent manner and its IC50 value was 142.82 ?g/mL. The HepG2 cells showed the characteristic morphologic changes of apoptosis by the function of CSA and the apoptosis percentage is higher than that of the natural one. The progress of cells cycle from S phase to G2 phase had been blocked by CSA. The intracellular Ca2+ level had been increased by the function of CSA, which was positively related with drug concentration. Conclusion CSA has obviously killing and apoptosis-inducing effects on human hepatocarcinoma cell line HepG2 and calcium overload might also be invovled in these events.
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Objective:To identify the human hepatocarcinoma cell line with the highest reactivity to hALR and to construct the phage-displayed library with its cDNA as the basis of the selection for special protein binding hALR from this library.Methods:The reactivity to hALR of HepG2,7701 and QGY was compared by 3?H-TdR methods in vitro.The mRNA of QGY with the highest reactivity to hALR was isolated with PloyATtract System 1000 kit.The phage-displayed library with cDNA fragments of QGY was constructed withT7Select10-30rient Express cDNA Cloning System and Random Primer kit.The quality of this library was evaluated by phage titer assay and PCR.Results:hALR could stimulate the proliferation of QGY,HepG2,and 7701 in dose-dependent way in vitro,but QGY showed the highest reactivity to hALR.The phage-displayed library with cDNA of QGY was successfully constructed and contained approximate 2?107 primary recombinants.It was indicated by PCR that the size of the cDNA inserts was from 0.2 to 2kb,the average 0.6kb.Thorough information about mRNA of QGY cells could be soundly represented by this library.Conclusion:The results show that QGY had the highest reactivity to the hALR among three strains detected.The high quality phage-displayed library is successfully constructed with cDNA of QGY.
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Objective: To further investigate the molecular mechanism of photodynamic therapy. Methods: We used cDNA microarray technique to explore the gene expression profiles of HEPG2 cells after photodynamic therapy with hematoperphyrin monomethyl ether(HMME) in HEPG2 cells. After treated with HMME for 60 min, the HEPG2 cells were irradiated with laser, and observed by microscope with H E staining. To prepare the probes, mRNA from both control and treated cells were isolated and purified, then reversely transcribed to cDNA with the incorporation of fluorecent labeled dUTP. The probes were hybridized with a cDNA microarray representing the 1 538 genes originated from human hepatocarcinoma cells. The fluorencent signals of Cy3 and Cy5 were scanned and analyzed. Results: After laser irradiation, the HEPG2 cells showed the typical feature of apoptosis. The gene expression profiles were also changed greatly. Among the 1 538 target genes, 389(2.47%) different expression genes were detected. Most of the changed genes (nearly 80%) were down regulated. They were functionally related to cell proliferation cycle, replication, metabolism and so on. Several apoptosis associated genes were detected among those up regulated genes, encoding the key proteins involved in apoptosis signal transduction, such as CCP32,AIF,Mch2. Conclusion: The HMME photodynamic therapy can initiate the apoptosis process of HEPG2 cells, which may be regulated by mitochondial pathway.[
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Objective To investigate the method and optimum conditions of electric transfection,and the major influential factors of electransfection efficiency and the survival rate of dendritic cells. Methods RNA was extracted from human hepatocarcinoma cell line(Bel 7402).Purified monocytes as precursor DC-s(pDC-s) were separated from human peripheral blood cells(PBMCs) by density gradient centrifugalization with lymphocyte gradation fluid and adherence method,pDCs were incubated in RPMI-1640 medium containing rhGM-CSF(8?10~5IU/L) and rIL-4(5?10~5IU/L) for 7 days and made them fully differentiate into immature DCs(imDCs).The total RNA human hepatocarcinoma cell and green fluorescent protein(GFP) were electransfected respectively into imDCs by electroporation apparatus with different electric voltages,times of impulse,cell concentrations,temperatures and electroporation buffers.Numbers of green fluorescence positive cells and the total cell number were counted respectively under fluorescent microscope,and visible light microscope.One day after the electric transfection,the cells were stained with 0.4% trypan blue,and electransfection efficiency and the cell survival rate were counted. Results Electransfection efficiency was increased to the highest value,up to about 49.7% when imDCs with the concentration of 5?10~6 cells/ml were mixed with 40?g-total RNA of human hepatocarcinoma cell,the electric voltage of electroporation apparatus was set at 300V,and the time of impulse was 500Us.Conclusion Electric transfection provides a technical possibility to make human hepatocarcinoma RNA into imDCs.The major influential factors of the electransfection efficiency were electric voltage and impulsing time.As receptor cells,the imDCs growing condition was also an important influential factor.