Cell cycle and radiosensitivity of progeny of irradiated primary cultured human hepatocarcinoma cells.

AIM: To evaluate the change of growth characteristics and radiosensitivity of irradiated primary cultured human hepatocarcinoma cells. METHODS: All tumor tissue samples were obtained from 39 hepatocarcinoma patients with a mean age of 49.6 years (range 22-76 years). We divided the samples into irradiated group and non-irradiated group and measured their plating efficiency (PE), population doubling time (PDT), radiosensitivity index SF2 and cell cycle. RESULTS: The PDT of primary culture of hepatocarcinoma cells was 91.0+/-6.6 h, PE was 12.0+/-1.4%, SF2 was 0.41+/-0.05%. The PDT of their irradiated progeny was 124.8+/-5.8 h, PE was 5.0+/-0.7%, SF2 was 0.65+/-0.09%. The primary cultured human hepatocarcinoma cells showed significant S reduction and G(2) arrest in a dose-dependent manner. The progeny of irradiated primary cultured hepatocarcinoma cells grew more slowly and its radiosensitivity increased. CONCLUSION: The progeny of irradiated primary cultured human hepatocarcinoma cells grows more slowly and its radiosensitivity increases.

Cell survival curve for primary hepatic carcinoma cells and relationship between SF(2) of hepatic carcinoma cells and radiosensitivity.

AIM: To establish the cell survival curve for primary hepatic carcinoma cells and to study the relationship between SF(2) of primary hepatic carcinoma cells and radiosensitivity. METHODS: Hepatic carcinoma cells were cultured in vitro using 39 samples of hepatic carcinoma at stages II-IV. Twenty-nine samples were cultured successfully in the fifth generation cells. After these cells were radiated with different dosages, the cell survival ratio and SF(2) were calculated by clonogenic assay and SF(2) model respectively. The relationship between SF(2) and the clinical pathological feature was analyzed. RESULTS: Twenty-nine of thirty-nine samples were successfully cultured. After X-ray radiation of the fifth generation cells with 0, 2, 4, 6, 8 Gy, the cell survival rate was 41%, 36.5%, 31.0%, 26.8%, and 19%, respectively. There was a negative correlation between cell survival and irradiation dosage (r = -0.973, P<0.05). SF(2) ranged 0.28-0.78 and correlated with the clinical stage and pathological grade of hepatic carcinoma (P<0.05). There was a positive correlation between SF(2) and D0.5 (r = 0.773, P<0.05). CONCLUSION: SF(2) correlates with the clinical stage and pathological grade of hepatic carcinoma and is a marker for predicting the radiosensitivity of hepatic carcinomas.

[The inhibitory effects on hepatitis B virus replication by stable expression of DN mutants of hepatitis B virus X gene pRev X-GFP].

OBJECTIVE: Persistent replication of hepatitis B virus (HBV) is one of the major obstacles in HBV infection treatment. Reduction or clearance of HBV propagation would be one of the aims of HBV therapy. The drugs approved in clinical used such as nucleotide analogs or interferon, were limited effects on HBV replication. The newly developing gene therapy method, dominant negative mutants, were be used as new promising HBV therapy strategy, and a dominant negative mutant of HBVX gene pRev X-GFP which we have reported in our previous study has some effects both on HBV replication and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG2 2.2.15 cells without transfection pRev X-GFP in the experiment. To make sure the effects of dominant negative mutant of pRev X-GFP, we established a HBX DN stable express cell clone, and evaluated the effects of HBX dominant negative mutant on HBV replication. METHODS: The X gene mutant, in which a specific point mutation of 3'-end ATG to AAG and fused with human green fluorescence protein (GFP) were cloned into pRev TRE vector, assigned to pRev HBX-GFP dominant mutant (pRev X-GFP). And the plasmid contains the wild type X gene or GFP gene was cloned into the same vector to construct pRev Xwt, pRev GFP constructs. All the constructs then transfected into HepG2 2.2.15 cells by liposome. After 7 days resistance selection of hygromycin (300 microg/ml), and cell clones which stable expression HBX-GFP, HBXwt, GFP were obtained. After reseeding of 106 cells of each clones in 12 wells with a 12 well cell plate and another 12 wells 2.2.15 cell were serve as blank control. The cells and media were harvested after cultured in DMEM with 10% FBS for 3 days. HBV-related DNA was assayed by dot blot and Southern blot. RESULTS: The 100% expression of pRev HBX-GFP, GFP and wild type X constructs were obtained. The stable expressed HBX-GFP can significantly reduce HBV DNA level both in cell media and cells by dot blot and Southern blot analysis, but not for pRev Xwt and pRev GFP. CONCLUSION: The dominant negative mutant pRev HBX-GFP can significantly inhibit the HBV gene expression. It also suggested that X gene might be one of promising target for HBV gene therapy.

[The effects of Chinese national medicine of Huoxueruanjian compound on SMAD signal in hepatic stellate cell and its significance].

OBJECTIVE: In order to explore the roles of Huoxueruanjian compound on liver fibrogenesis and its molecular mechanism, this paper has investigated the Influence of blood serum with such traditional Chinese medicine compound on the expression of Smad3, Smad7 and procollagen alpha2(I) gene in hepatic stellate cell (HSC). METHODS: HSC-T6 was deal with different Concentration of blood serum medicine with Heluoshugan which was made by routine way. Then expression change of Smad3, Smad7 and procollagen alpha2(I) mRNA among each groups were observed by RT-PCR. Furthermore, the expression change of Smad3 protein were examined by Western blot. RESULTS: Expression of Smad3 and procollagen alpha2(I) mRNA as well as Smad3 protein had been downregulated after treating with blood serum medicine of Heluoshugan (P<0.01, P<0.05, respectively). The expression of procollagen alpha2(I) mRNA changed at the same tendency as those of Smad3. The role of blood serum medicine was significant difference between different concentration, P<0.05. And the expression of procollagen alpha2(I) mRNA changed in concentration-dependent manner. Blood serum medicine has no effects on the Smad7 mRNA. CONCLUSION: The anti-fibrosis roles of HuoXueruanjian Compound maybe influence the function of TGF-beta and Smad by nonspecific action, thereby inhibit the transcription of procollegan alpha2(I) mRNA and decrease the production of ECM. As regards Smad3, it may be facilitating the development of liver fibrosis when its expression increases. Otherwise, it manifest with anti-fibrosis role.

[The association between polymorphism of endothelial nitric oxide synthase gene and cirrhotic portal hypertension].

OBJECTIVE: To study whether the liver cirrhosis and portal hypertension are associated with a -786T-->C mutation at promoter and VNTR polymorphism in intron 4 and a 894 G-->T mutation at exon 7 of the eNOS. METHODS: A case control study of 106 patients with liver cirrhosis due to HBV was performed in comparison with 108 controls with the help of PCR-SSCP or RFLP. RESULTS: There was no difference in the gene frequency of allele G of promoter between LC(+) group and other groups. The frequencies of the T and TG genotype at exon7 and the a allele and ab genotype in intron 4 were significantly higher in portal hypertension group (LC(+)) than in liver cirrhosis group alone and control group (P < 0.05). Patients of the liver cirrhosis with coexistence of the T and a alleles had a higher incidence of portal hypertension (P < 0.05) than those with only one of the two alleles or without any of the two alleles. Multivariate logistic regression analysis revealed that VNTR polymorphism in intron 4 and 894 G-->T mutation at exon 7 of the eNOS gene are independent risk factors for the occurrence of portal hypertension in patients with liver cirrhosis. CONCLUSION: The T allele at exon 7 and a allele in intron 4 are associated with the occurrence of portal hypertension in patients with liver cirrhosis. The ocurrence of portal hypertension with liver cirrhosis is higher in patients who have both T and a allele than patients who have either T or a allele alone, which is an independent risk in occurrence of portal hypertension, respectively. TGab may be susceptibility genotype of portal hypertension.

Inhibition of hepatitis B virus by a novel L-nucleoside, beta-L-D4A and related analogues.

AIM: To explore the inhibition of beta-L-D4A on hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome. METHODS: 2.2.15 cells were plated at a density of 5X104 per well in 12-well tissue culture plates, and treated with various concentrations of beta-L-D4A for 6 days. In the end, 5 microl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethylene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with HindIII. Both DNAs were subjected to Southern blot, hybridized with a (32)P-labeled HBV probe and autoradiographed. Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED(50) was calculated. Then Hybond-N membrane was washed and rehybridized with a (32)P-labeled mtDNA-specific probe, and effect of beta-L-D4A on mitochondrial DNA was studied. 2.2.15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. ID(50) was calculated. Structure-activity relationships between D2A and D4A were also studied as above. RESULTS: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. ED(50) was 0.2 microM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID(50) at 200 microM. CONCLUSION: beta-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxicity and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.

[Correlation between polymorphism of TaqI of ET-1 gene and cirrhotic portal hypertension].

OBJECTIVE: To investigate the correlation between polymorphism of TaqI of endothelin (ET)-1 gene intron 4 and cirrhotic portal hypertension and to search new risk factor of portal hypertension. METHODS: Peripheral venous blood was extracted from 106 patients with cirrhosis after hepatitis B (PH+ group) and 108 healthy blood donors (PH- group). PCR-RFLP was used to analyze the polymorphism of TaqI of ET-1 gene. The plasma ET-1 concentration was detected with immunoassay. Multivariate logistic regression analysis was made to analyze the risk factors. RESULTS: The C allele frequency in the PH+ group was 25.4%, significantly higher than that of the controls (16.7%, P < 0.05). The frequency of CC + TC genotype in PH+ group was 46.2%, significantly lower than that in the controls (29.6%, P < 0.05). In the PH+ group, the thickness of spleen was greater, hemorrhage rate was higher, and III degrees ascites was more in C allele carrier than in T allele carriers (P < 0.05). The plasma ET-1 concentration was higher in PH+ group than in PH- group. In the PH+ group, the plasma ET-1 concentrations in those with CC genotype and those with TC genotype were significantly higher than in those with TT genotype (P < 0.05). Correlation analysis showed that ET-1 gene polymorphism was positively correlated with plasma ET-1 concentration (R = 0.808 2). Multivariate logistic regression analysis showed that gradation of liver function, diameter of portal vein, and ET-1 gene polymorphism were independent risk factors of portal hypertension. CONCLUSION: Polymorphism of TaqI of ET-1 gene is correlated with the pathogenesis of cirrhotic portal hypertension. It may be one of the risk factors of portal hypertension.

[Establishment and identification of highly expressing and replicating hepatitis B virus genome transgenic mouse models].

OBJECTIVE: To establish a highly expressing and replicating hepatitis B virus (HBV) genome transgenic mouse models for screening anti-HBV drugs and investigating the pathogenesis of hepatitis B. METHODS: Elongated HBV genome as the investigated gene was transducted into the pronuclei of the fertilized eggs of mice by the technique of microinjection, then the eggs were transplanted into the oviducts of the pseudopregnant mice. All the newborn mice were screened and identified by PCR and Southern blot detecting genomic DNA in tail tissue, then the positive mice were examined plasma HBsAg, HBeAg by ELISA and plasma HBV DNA by Southern blot. RESULTS: Among the 61 offsprings, 18 were positive for tail tissue HBV DNA examination, 7 of which were positive for replication and expression detection. CONCLUSION: Transgenic mice with elongated HBV genome possess high efficiency of replication and expression, which can be used for further investigation.

[Clinical study of oligonucleotide microarray on monitoring the lamivudine-resistance mutations in hepatitis B virus].

OBJECTIVE: To evaluate the mutations of lamivudine-resistance using oligonucleotide microarray in hepatitis B virus (HBV) infected patients. METHODS: A randomized clinical trial was conducted on 20 lamivudine-treated patients for 18 months and 10 patients as controls. The serum HBV DNA was amplified by PCR and the lamivudine-resistance mutations in YMDD region were assayed by 4 sites microarray developed before. RESULTS: This microarray could clearly differentiate the wide-type from mutated-type HBV with lamivudine-resistance mutations. The rate of mutations in YMDD region increased with the time of lamivudine treatment (chi2=6.69, P<0.01). The most common mutated type was M539V+L515M and next M539I. Continuous administration of lamivudine was no benefit for inhibiting the replication of HBV with YMDD mutation but helpful for wide-type HBV. CONCLUSION: The routine serum HBV DNA assay by PCR may introduce prejudice in monitoring HBV inhibitory effect by lamivudine, while the microarray technique can avoid this and is one of the best ways to monitor the lamivudine-resistance mutations in HBV. There is no effect of lamivudine on HBV with YMDD mutation in clinical practice.

[Effect and mechanism of beta-L-D4A (a novel nucleoside analog) against hepatitis B virus].

OBJECTIVE: To explore the effect and the molecular targets of anti-hepatitis B virus (HBV) by beta-L-D4A in vitro. METHODS: 2.2.15 cells were cultured and treated with various concentrations of beta-L-D4A for 6 hours, then the effect of anti-HBV was examined by Southern blot and the replicating core particles from the cells were isolated. The endogenous polymerase reaction and activity gel experiment were performed to monitor the activities of the DNA polymerase and reverse transcriptase. RESULTS: The replication of HBV DNA was inhibited in a dose-dependent manner. The endogenous polymerase reaction showed both the two enzymatic activities were irreversibly inactivated in a concentration -dependent manner, with IC50 at 0.51 micromol/L and 0.55 micromol/L, respectively. But the activities of DNA polymerase and reverse transcriptase were found to remain active by activity gel with exogenous templates. CONCLUSIONS: The mechanism of inhibiting HBV replication by beta-L-D4A may be in that either the DNA replication priming is blocked or the elongation of DNA chain is terminated irreversibly.

Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro.

AIM: To study the preparation and cleavage activity of HpRz directed against the transcript of HBV core gene in vitro. METHODS: HpRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32p-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme in vitro. 32p-labeled pKC transcript containing HBV core region as target-RNA was transcribed using T7 RNA polymerase and purified by denaturing PAGE. Cold HpRz transcript was incubated with 32p-labeled target-RNAs under different conditions and radio autographed after denaturing polyacrylamide gel electrophoresis. RESULTS: HpRz has the specific ability of cleavage of target RNA at 37 degrees and 12 mM MgCl2. Km=26.31 nmol/L, Kcat=0.18/min. These results revealed that the design of HpRz was correct. CONCLUSION: HpRz prepared in this study possesses specific catalytic activity from the identification of cleavage activity. These results indicate that hairpin ribozyme may intracellularly inhibit the replication of HBV, therefore it may become a novel potent weapon for the treatment of hepatitis B.