Association of MDR1 gene polymorphisms with susceptibility to hepatocellular carcinoma in the Chinese population.

OBJECTIVE: The objective of this study was to evaluate the association of MDR1 gene polymorphisms with susceptibility to hepatocellular carcinoma (HCC). METHODS: A total of 689 HCC patients and 680 cancer-free subjects were enrolled. Human MDR1 gene polymorphisms were investigated by created restriction site- polymerase chain reaction (CRS-PCR) and DNA sequencing methods. Multiple logistic regression models were applied to estimate the association between MDR1 gene polymorphisms and susceptibility to HCC. RESULTS: We detected a novel c.4125A>C polymorphism and our findings suggested that this variant was significantly associated with susceptibility to HCC. A significantly increased susceptibility to HCC was noted in the homozygote comparison (CC versus AA: OR=1.621, 95% CI 1.143-2.300, χ2=7.4095, P=0.0065), recessive model (CC versus AC+AA: OR=1.625, 95% CI 1.167-2.264, χ2=8.3544, P=0.0039) and allele contrast (C versus A: OR=1.185, 95% CI 1.011-1.389, χ2=4.4046, P=0.0358). However, no significant increase was observed in the heterozygote comparison (AC versus AA: OR=0.995, 95% CI 0.794-1.248, χ2=0.0017, P=0.9672) and dominant model (CC+AC versus AA: OR=1.106, 95% CI 0.894-1.369, χ2=0.8560, P=0.3549). CONCLUSIONS: These findings suggest that the c.4125A>C polymorphism of the MDR1 gene might contribute to susceptibility to HCC in the Chinese population. Further work will be necessary to clarify the relationship between the c.4125A>C polymorphism and susceptibility to HCC on larger populations of diverse ethnicity.

[The effect of envelope protein mutation on hepatitis B virus assemble].

OBJECTIVE: To investigate the effect of envelope protein mutation on HBV assembly. METHODS: The envelope protein mutated vectors were constructed by the molecular clone in vitro, and then transfected transiently in the cell HepG2. The expression and secretion of S protein was assay by ELISA. HBV DNA was quantitatively evaluated by PCR. After co-transfection with pHBV-mS1S and adwR9 the DNA was quantitatively evaluated by PCR. RESULTS: There was no significant difference in expression and secretion of S protein assayed by ELISA in the cytoplasm and supernatant among pHBV-mS1, pHBV-mS, pHBV-mS1S and the wild HBV adwR9 plasmid. The DNA detected by real-time fluorescence quantitative PCR from those the cytoplasm of mutants was higher than that from the wild HBV adwR9 cytoplasm, especially from the cytoplasm of pHBV-mS1S plasmid. However, the DNA detected by real-time fluorescence quantitative PCR from the supernatant of those mutants was lower than that from the wild HBV adwR9 supernatant, especially from pHBV-mS1S. The DNA detected by real-time fluorescence quantitative PCR from the supernatant co-transfected with pHBV-mS1S and adwR9 was lower than that from the supernatant co-transfected with pcDNA3 and adwR9. CONCLUSION: There was no effect of envelope protein mutation on the expression and secretion of S protein. Envelope protein mutation could interfere the assembly of HBV particle and cause reduction of secretion of HBV.

[Study of mouse marrow cells differentiation into a hepatocyte lineage in vitro].

OBJECTIVE: To explore whether bone marrow stem cells (MSCs) from adult mice can be induced to differentiate into hepatocytes by hepatocyte growth factor (HGF) alone and the time phase characteristics in the differentiation progress. METHODS: Adult mouse MSCs were treated with or without 100 ng/ml HGF, on days 0, 7, 14, 21, and 28. The morphologic characteristics of the cells were examined; the albumin (ALB), AFP mRNA was analyzed sub-quantively using reverse transcription polymerase chain reaction (RT-PCR) and immumohistochemistry techniques. The expression of ALB, AFP and CK19 were detected by using anti-ALB, AFP and CK19 antibodies. RESULTS: Freshly isolated adult mouse MSCs expressed ALB and AFP mRNA weakly; in the group without HGF, no ALB mRNA was detected on day 7. The expression of AFP mRNA was reduced significantly on day 7, and could not be detected anymore after day 14. In the HGF treated group, ALB mRNA was not detected on day 7, but the positive lane appeared again on day 14, and the expression of ALB mRNA was increased on day 21 but reduced in the following days. The AFP mRNA was positive at all times, however it tended to decrease after day 14 in the HGF treated groups. The result of immumohistochemistry was consistent with that of RT-PCR, and CK19 was always negative. CONCLUSION: Adult mouse MSCs can be induced into hepatocyte differentiation in vitro. The optimal time for the induction was 2 to 3 weeks.

[Development of hygromycin-resistant packaging cell line for hepatitis B virus-derived vectors].

OBJECTIVE: To cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles. METHODS: Free of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: Hygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium. CONCLUSION: After the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.

[Replication and encapsidation of HBV mutants with the truncated C gene].

OBJECTIVE: To evaluate the replication and encapsidation of HBV mutants with the truncated C gene. METHODS: The HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome. RESULTS: The C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay. CONCLUSION: The C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.

[The anti-HBV effect and mechanism of C gene truncated mutant in vitro].

OBJECTIVE: To explore the effect and mechanism on HBV replication in C gene truncated mutant. METHODS: Protein expression of C gene truncated vector and wild C gene vector were assay by SDS-PAGE Western blot. Constructed C gene truncated expression vector was cotransfected with wild HBV genome; virus load was detected by PCR in the culture medium and the cell. The formation of core particle was assay by Native western blot. RESULTS: The recombinant vectors can efficiently express. Virus load of the cotransfected group by pcDNA3-deltaC and adwR9 was lower than that of control group in the culture medium and the cell. Protein band of the co-expressed group by pcDNA3-deltaC and pcDNA3-C showed slightly weaker than that of the co-expressed group by pcDNA3 and pcDNA3-C. CONCLUSION: C gene truncated mutant could interfere with the formation of core particle and reduce of HBV replication

[Therapeutic effect of levamisole plus HBV vaccine and dipyridamole on patients chronically infected by HBV with precore mutation].

OBJECTIVE: To evaluate the incidence of precore mutation in HBeAg negative HBV infected patients and the therapeutic effect of the immune therapy (levamisole + HBV vaccine + dipyridamole) on patients chronically infected by HBV with precore mutation. METHODS: The precore region of HBV from the HBeAg (-) chronic hepatitis patients was sequenced and the patients suffered from HBV with precore mutation were treated with immune therapy. RESULTS: The precore mutation rate was 10/12. The therapeutic effect of the immune therapy on the precore mutation patients (5/7) was better than that on the HBsAg(+), HBeAg(+) patients (2/11), P less than 0.05. CONCLUSION: The precore mutation rate was quite high in the HBsAg(+), HBeAg(-) patients we studied. The immune-therapy has some therapeutic effects on the patients with precore mutation. But the number of cases was too small, further study is needed.

[Approach to transforming hepatitis B virus as a gene therapeutic vector].

OBJECTIVE: To evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy. METHODS: A fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot. RESULTS: The HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon. CONCLUSION: It is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.

[Anti-HBV effects of genetically engineered replication-defective HBV with combined expression of antisense RNA and dominant negative mutants of core protein and construction of first-generation packaging cell line for HBV vector].

OBJECTIVE: To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein. METHODS: Full length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: The mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, P<0.01), 66.54% 4.45% (t=42.3, P<0.01), and 73.68% 5.07% (t=51.9, P<0.01) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean inhibitory rates of HBeAg were 4.46% 4.25%, 52.86% 1.32% (t=36.2, P<0.01), 26.36% 1.69% (t=22.3, P<0.01), and 59.28% 2.10% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium. CONCLUSIONS: It has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.