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Ai-long Huang

Publications and source records attributed to Ai-long Huang.

At least 19 recordsLinked to original sources
PubMed2006

[Effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer: experiments in vitro and in vivo].

OBJECTIVE: To investigate the effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer. METHODS: Recombinant eukaryotic expression plasmid pshRNA-DNMT1 containing the sequence of the gene of DMNT1 that methylates the specific pyrimidine residue in the DNA promoter region was constructed. Human gastric cells of the line AGS were cultured and transfected with pshRNA-DNMT1. Western blotting was used to detect the protein expression of DNMT1 of the AGS cells, and RT-PCR was used to detect the mRNA expression of DNMT1 of the AGS cells. MTT method was used to dynamically monitor the surviving cells and the cell apoptotic was observed by electron microscopy and TUNEL method. Forty nude the mice were inoculated with suspension of AGS cells. When the tumor reached the size of 5 - 6 mm in diameter the mice were randomly divided into 5 equal groups to be injected intravenously with PBS, liposome, pTZU6 + 1, pshRNA-DNMT1 of medium dose, and pshRNA-DNBMT1 of large dose for 4 times with an interval of 3 days. The tumor size was measured every day. Three days after the last injection the mice were killed and the tumors were taken out to undergo light and electron microscopy and TUNEL method to detect the cell apoptosis. Immunohistochemistry was used to detect the proliferating cell nuclear antigen (PCNA) of the cells. RESULTS: The protein and mRNA expression levels of DNMT1 in the cultured AGS cells 24, 48, and 72 hours after transfection of the pshRNA-DNMT1 group were all lower than those of the control group. The numbers of surviving AGS cells of the pshRNA-DNMT1 group became significantly gradually lower than those of the liposome and pTZU6 + 1 groups since 24 hours after transfection (all P < 0.05). The apoptotic rate of AGS cells in the pshRNA-DNMT1 group was 34.78% +/- 0.52%, significantly higher than those of the liposome and pTZU6 + 1 groups (4.86% +/- 0.17% and 5.12% +/- 0.76% respectively, both P < 0.05). The subcutaneous tumors of the mice of the PGS, liposome, and pTZU6 + 1 groups augmented along with time without significant differences among these 3 groups (all P > 0.05). The tumor of the 2 pshDNMT1 groups began to augment since the 5(th) day and began to be reduced in size since the 10(th) day in comparison with the other 3 groups (all P < 0.05), and the tumor size of the pshRNA-DNMT1 (large dose) group was significantly smaller than that of the pshRNA-DNMT1 (medium dose) group 15 days after the injection (P < 0.05). The rates of cell apoptosis of the pshRNA-DNMT1 (large dose) and pshRNA-DNMT1 (medium dose) groups were both significantly higher than those of the other 3 groups (all P < 0.05) and with a sufficient difference between these 2 pshRNA-DNMT1 groups (P < 0.05). PCNA analysis showed that the proliferation activity of the cells in the pshRNA-DNMT1 groups was significantly suppressed. CONCLUSION: The recombinant plasmid pshRNA-NMT1 effectively and specifically inhibits the expression of the gene DNMT1, thus inhibiting the proliferation and inducing the apoptosis of gastric cancer cells.

Animals↗
PubMed2006

Promoter activity of SARS coronavirus 5' UTR sequence in eukaryotic cells.

OBJECTIVES: To investigate 5'UTR sequence in different SARS-CoV isolates, to identify the secondary structure, and to test the promoter activity of the cDNA sequence corresponding to SARS-CoV 5'UTR in eukaryotic cells. METHODS: 101 SARS-CoV 5'UTR were aligned. One typical sequence containing full 264 nt was then subjected to be predicted its secondary structure. The pGL3-5'UTR and pGL3-a-5'UTR were constructed by substitution of SV40 promoter with SARS-CoV 5'UTR cDNA or its antisense sequence. Then the recombinant plasmids were transfected into HepG2 cells and the luciferase activities were detected. A set of deletion mutant plasmids, of which pGL3-5' UTR-1, pGL3-5' UTR-2, pGL3-5'UTR-3 and pGL3-5'UTR-4 are with 3, 2, 1, and 0 residual stem-loops of 3' termini respectively,were constructed from pGL3-5'UTR and were transfected into HepG2 cells to express reporter gene luc+, with pGL3-5'UTR containing full sequence as control. The luciferase activities expressed by the plasmids were measured. And then the total RNA of the transfected cells was extracted. Subsequently, by 5' Rapid Amplication of cDNA Ends (5'RACE), the PCR product was sequenced. The luciferase expressed by pGL3-5'UTR in various cells, the lung carcinoma cell line A549, hepatoma cell line HepG2, kidney cell Vero E6, cervical cancer cell line HeLa and human umbilical vein endothelial cell line ECV304 were measured and compared with each other. RESULTS: The full sequence of the SARS-CoV 5' UTR is a 264nt, and 18 deletion mutants were found. Totally, 5 site substitutions were found in 101 5'UTR sequences. The SARS-CoV 5'UTR RNA folded to form a stable secondary structure containing four stem-loop domains. The biggest and most complex one is the stem-loop II appearing a pseudoknot. Comparing with pGL3-a-5'UTR, pGL3-5'UTR expressed luciferase obviously. Both pGL3-5'UTR containing full sequence and pGL3-5'UTR-1 containing three stem-loops of 3' termini expressed the luciferase well. However, when lost stem-loop I and II , the pGL3-5'UTR-2, pGL3-5'UTR-3 and pGL3-5'UTR-4 almost didn't express luciferase. The 56th nucleotide of SARS-CoV 5'UTR was found to be the initiation site for transcription. Transfected with expression luciferase plasmid pGL3-5' UTR in which SARS-CoV 5' UTR acts as the promoter, the luciferase could express in five cell lines in different degrees. Ranked by the luciferase activity from the highest to the lowest, the order is A549, HepG2, ECV304, HeLa and Vero E6. CONCLUSIONS: A: The 5'UTR sequences of different SARS-CoV isolates are relatively conserved, and a full sequence would form a secondary structure containing four stem-loop domains. B: The cDNA sequence corresponding to SARS-CoV 5'UTR possessed a promoter activity in eukaryotic cells. C: The promoter domain of the SARS-CoV 5'UTR contains both stem-loop I and II. D: The 56th nucleotide and its down stream TRS of SARS-CoV 5'UTR plays a key role in regulating transcription. E: Cells sourced from various tissues can provide efficient accessory factors for SARS-CoV 5'UTR sequence that acts as a promoter, and the lung-sourced cells may be the most suitable.

5' Untranslated Regions↗
PubMed2006

[Construction and expression of fusion gene encoding Hyper-IL-6 in mammalian cells].

AIM: To construct and express fusion gene encoding Hyper-IL-6 in mammalian cells. METHODS: Using geneSOEing method, human sIL-6R cDNA was fused with IL-6 cDNA by a linker rich in glycine through PCR technique and cloned into pIRES(2)-EGFP. The constructed plasmid was transfected into mammalian cells. The expression of fusion gene encoding Hyper-IL-6 was detected by GFP and RT-PCR and immunocytochemistry. RESULTS: The recombinant plasmid pIRES-HIL-6 was successfully constructed and expressed in mammalian cells. CONCLUSION: The success in construction and expression of human Hyper-IL-6 makes it possible to further study its biological functions.

Animals↗
PubMed2006

[Preparation and characterization of monoclonal antibody against Vibrio cholerae O139].

AIM: To prepare monoclonal antibody (mAb) against Vibrio cholerae O139, which would be used as the gold colloidal reagent strip for rapid detection of O139, and to determine its biological characterization. METHODS: BALB/c mice were immunized by inactivated Vibrio cholerae O139. Anti-O139 mAbs were prepared by using hybridoma technique. The specificity of mAb was determined by indirect ELISA and Western blot. The indirect ELISA was used to identify Ig subgroup, detect its titer in ascites and relative affinity, and analyze antigen-binding epitope of mAbs. RESULTS: Two hybridoma cells (O4D7 and O4D10), secreting anti-O139 mAbs were obtained. The Ig subgroups of O4D7 and O4D10 were IgG2b and IgG3, respectively. The mAb's titer in ascites was 1:10(7). The relative affinity of mAb O4D7 was more than 10(5) and that of O4D10 was more than 10(4). The result of additive ELISA showed that two mAbs could recognize different antigen epitopes. CONCLUSION: The successful preparation of two anti-O139 mAbs provides a powerful tool for the development of a rapid method of detecting Vibrio cholerae O139 infection.

Animals↗
PubMed2006

[Inhibition of HCV IRES controlled reporter gene expression by RNA interference].

OBJECTIVE: To develop a RNAi approach that specifically targets the HCV IRES sequence by vector-expressed short hairpin RNA (shRNA) in vitro, and to assess the inhibitory effect of the shRNA on reporter gene expression. METHODS: Eukaryotic expressing plasmids, pIRES-GFP and p5' UTR-Luc containing GFP or luciferase gene controlled by HCV IRES were cotransfected into HepG2 cells with either a RNAi plasmid pshRNA-HCV or a control plasmid pTZU6+1. At 24, 48, 72 hours post transfection, the fluorescence in the transfected cells was studied using fluorescence microscopy. The levels of GFP RNA were determined using RT-PCR and those of protein were determined using Western blot. The activities of luciferase were assayed using a dual luciferase assay system. RESULTS: The introduction of RNAi plasmid efficiently and specifically down-regulated the expression of the reporter gene. RT-PCR showed that the RNAs of GFP gene were distinctly reduced (about 60%) when the pIRES-GFP was cotransfected with pshRNA-HCV, whereas the control vector did not exhibit inhibitory effect on the mRNA level, according to Western blot assay. The luciferase activity also decreased by 60%-70% in comparison to the control plasmid. CONCLUSION: Our results demonstrate that the shRNA targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.

Gene Expression Regulation↗
PubMed2006

[The relationship between histone acetylation modification and IFN-gamma responsive gene regulation].

OBJECTIVES: To study the role of histone modification in the regulation of IFN-gamma-activated gene using chromatin immunoprecipitation technique. METHODS: Real-time PCR was used to measure the mRNA level of interferon-gamma-inducible protein 10 (IP-10) in Hela cells. Chromatin immunoprecipitation combined with Real-time PCR was used to check the histone H4 acetylation level at IFN-stimulated response element (ISRE) locus of IP-10 gene. RESULTS: IP-10 was strongly activated by IFN-gamma. The histone H4 deacetylation happened at the ISRE locus when IP-10 was induced by IFN-gamma. The activation of IP-10 and the deacetylation of histone H4 at the ISRE site induced by IFN-gamma were inhibited or blocked by histone deacetylase (HDAC) inhibitor trichostatin A (TSA). CONCLUSION: The histone H4 deacetylation at the ISRE site is related with the activation of IP-10 by IFN-gamma.

Acetylation↗
PubMed2006

[Screening of proteins interacting with hepatitis C virus NS3 from T7-phage display library].

OBJECTIVE: To screen and identify proteins that interact with hepatitis C virus NS3 by means of T7-phage display system. METHODS: Hepatitis C virus NS3 was expressed by prokaryotic expression and used as a selected molecule to biopan the T7 select human liver cDNA library; the selected positive clones were identified using DNA sequencing and analyzed with BLAST program in GenBank. RESULTS: After BLAST analysis in all the positive clones, the proteins which interacted with the hepatitis C virus NS3 were found to be serpin peptidase inhibitor, clade A, member 1 (SERPINA1) and cyclophilin-LC. CONCLUSION: T7-phage display system is a convenient, rapid and effective method for screening interacting proteins. The proteins thus selected will provide an important means for studying the pathogenesis and carcinogenesis of HCV.

Cell Line↗
PubMed2006

[Inhibition of HBV replication and antigen expression by RNA interference against different targets].

OBJECTIVES: To observe the inhibition of HBV replication and antigen expression by RNA interference aimed at different parts of the HBV genome. METHODS: Following the rules of shRNA expression vector design and construction, we constructed seven kinds of sequence specific vectors and two kinds of mutant shRNA expression ones. We then cotransfected those shRNA and HBV expression vectors into HepG2 cells using lipofectamine2000. The level of HBV replication was investigated using Southern blot and the antigen expression using ELISA. RESULTS: The replication of HBV DNA was inhibited by many shRNAs, especially the ones against P1, S2, C2, S1 and X. The inhibition rate against P1 was as high as 95%. Results obtained with ELISA showed that the shRNAs targeting C2, C1 and S2 had high rates of inhibition to HBsAg. CONCLUSION: The replication and antigen expression of HBV could be inhibited by shRNAs aimed at four different open read frames, and higher inhibition rates of HBV replication and surface antigen expression could be obtained by P1 and C2, respectively.

Gene Expression↗
PubMed2005

Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice.

BACKGROUND: RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs (siRNAs) have sufficiently inhibited hepatitis B virus (HBV) replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects. METHODS: A mutant RNAi vector (pSI-C mut) with two base pairs different from the original target gene sequence at the RNAi vector (pSI-C) was constructed according to the method described in this study. A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload. pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein (GFP) gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay (ELISA) assay was used to measure the concentration of HBV surface antigen (HBsAg) in mouse serum, immunohistochemical straining method was used to visualize the expression of HBV core protein (HBcAg) in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detected by reverse transcriptase PCR (RT-PCR) analysis. RESULTS: Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection (p.i.), the OD values were shown to be 5.07 +/- 1.07 in infecting group and 0.62 +/- 0.59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group (P < 0.01). Liver intracellular synthesis of viral core protein was sharply reduced to 0.9% +/- 0.1%, compared with 5.4% +/- 1.2% of positive hepatocytes in infecting group (P < 0.01), and the transcriptional level of HBV C mRNA was greatly reduced by 84.7%. However, the irrelevant RNAi control plasmid (pSIGFP), and the pSI-C mut did not show the same robust inhibitory effects as pSI-C. CONCLUSION: pSI-C exert efficient and specific inhibitory effects on HBV replication and expression in mice models.

Animals↗
PubMed2005

Potent and specific inhibition of SARS-CoV antigen expression by RNA interference.

BACKGROUND: Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression. METHODS: The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot. RESULTS: The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription. CONCLUSIONS: Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.

Antigens, Viral↗
PubMed2005

[RNA interference inhibits replication and expression of hepatitis B virus in mice].

OBJECTIVE: To develop a mouse model of acute hepatitis B virus infection and to observe the RNA interference-mediated inhibition of HBV replication and expression in the mouse model. METHODS: Thirty Balb/c mice were randomly divided into 5 equal groups: group A to be injected with pHBV1.3, naked plasmid containing 1.3 time HBV ayw type whole genome eukaryotic expression vector, via the caudal vein as infection group; group B to be injected with pHBV1.3 and pSI-C, plasmid expressing HBV-C specific short hairpin RNA, as interference group; group C to be injected with pHBV1.3 and pSI-C mut, a mutant RNAi vector, as mutant interference group; group D to be injected with pHBV1.3 and pGFP, siRNA transcription vector targeting green fluorescence protein (GFP); and group E to be injected with PBS as blank controls. Six days after blood was collected and the mice were killed and their livers were taken out. ELISA was used to measure the concentration of HBsAg in the serum. Immunohistochemistry and RT-PCR were used to detect the expression of HBcAg and HBV C mRNA in the liver. RESULTS: Three days after injection the HBsAg expression in the sera of the infection group was strongly positive. Six days after injection expression of HBsAg was negative in the interference group and blank control group, and was positive in the infection group, mutant interference group, and irrelevant group, however, with significantly lower OD values in the latter 2 groups compared with in the infection group (both P < 0.05). Six days after injection immunohistochemistry showed that HBcAg expression in liver was positive in the infection group, weakly positive in the mutant interference group and irrelevant interference group, and was negative in the blank control group and interference group. RT-PCR showed clear expression of HBV C mRNA in the infection group, mutant interference group, and irrelevant interference. CONCLUSION: RNAi technique specifically and effectively inhibits the replication and expression of HBV. siRNA has significant potential to become a new type antiviral drug. The establishment of an animal model of acute HBV infection in mice by hydrodynamic injection of naked plasmid has solved, to a certain degree, the problem of lack of appropriate animal model of HBV infection.

Animals↗
PubMed2005

[Detection and comparison of transcriptional activities of tumor-specific survivin and alpha-fetoprotein promoters in human hepatocellular carcinoma cells].

OBJECTIVE: To detect and compare the transcriptional activities of tumor-specific survivin and AFP promoters in various hepatocellular carcinoma cell lines and to lay some groundwork for targeting gene therapy in human hepatocellular carcinoma. METHODS: The fragment of survivin and AFP promoters were acquired by PCR amplification and were cloned into the reporter plasmid pGL3-Basic, which contained a luciferase gene. The constructed eukaryotic expression plasmid pGL3-SUR and pGL3-AFP, in which the expression of the luciferase was derived by survivin or the AFP promoter, were transfected into three HCC cell lines. At 24 hours post transfection (p.t.), the activity of the luciferase was determined with Dual-Luciferase Reporter Assay System. A pGL3-CMV, containing the CMV promoter controlled luciferase gene, was used as a positive control. RESULTS: Both survivin and AFP promoters had transcriptional activities in all three HCC cell lines and the transcriptional activity of the survivin promoter was much higher than the AFP promoter (52-98 times) and reached a level of 16% approximately 21% of the transcriptional activity of the CMV promoter. CONCLUSION: Our data reveals that the survivin promoter possesses a high transcriptional activity in all three established HCC cell lines and may serve as a useful tool for transcriptional targeting gene therapy of HCCs.

Carcinoma, Hepatocellular↗
PubMed2005

[A method of HPRE synthesis via transcription by T7 RNA polymerase in vitro].

OBJECTIVE: To synthesize highly pure HBV post-transcriptional regulatory element (HPRE) via transcription in vitro by T7 RNA polymerase. METHODS: HPRE gene was amplified by PCR from a template containing HBV complete genomic DNA and cloned into plasmid pGEM-11zf. The cloned DNA sequence was transcribed by T7 RNA polymerase. RESULTS: The construction of HPRE gene recombinant plasmid and production of HPRE via transcription in vitro was successful. CONCLUSION: In vitro transcription by T7 RNA polymerase can be used to synthesize highly pure HPRE.

DNA-Directed DNA Polymerase↗
PubMed2005

[An experimental study on antiviral effects of IFN alpha].

OBJECTIVE: To investigate the effects of different subtypes IFN alpha (IFN alpha2b, IFN alpha2a, and IFN alpha1b) transduction molecular STAT1, STAT2, IFNAR, PKR, and RNase L, and to study the differences of their antiviral effects and to evaluate the key signaling transduction molecules. METHODS: (1) After HepG2 cells were treated with IFN alpha2b, IFN alpha2a, or IFN alpha1b, the mRNA levels of STAT1, STAT2, IFNAR, PKR, and RNase L were detected by RT-PCR. (2) After HepG2 cells were treated with 1000 U/ml IFN alpha2b, IFN alpha2a, or IFN alpha1b, the protein expression levels of STAT1 and IFNAR were examined by Western blot. RESULTS: RT-PCR results: (1) IFNAR, STAT1, and STAT2 mRNA expression levels were slightly higher in the IFN alpha1b group than those in the IFN alpha2b group (P > 0.05). The mRNA expression levels in IFN alpha1b or IFN alpha2b groups were significantly higher than in the IFN alpha2a group (P < 0.05). (2) The PKR mRNA expression showed no significant differences among IFN alpha1b, IFN alpha2b, and IFN alpha2a groups. (3) The RNase L mRNA expression was very weak. We could not compare the differences of the RNase L mRNA levels in different groups by RT-PCR. Western blot results: (1) The IFNAR, and STAT1 protein expressions were greatly up-regulated after IFN alpha induction compared with the untreated group (P < 0.05). (2) The IFNAR, and STAT1 protein expression levels in IFN alpha1b group were slightly higher than the IFN alpha2b group. IFNAR, and STAT1 protein levels of IFN alpha1b or IFN alpha2b group were significantly higher than IFN alpha2a group (P < 0.05). CONCLUSION: STAT1, STAT2, IFNAR mRNA and protein expressions could all be markedly up-regulated after IFN alpha treatment. Effects of IFN alpha1b or IFN alpha2b were greatly stronger than IFN alpha2a. The PKR mRNA expression also was greatly up-regulated after IFN alpha treatment. Expression levels of PKR in IFN alpha1b, IFN alpha2b, and IFN alpha2a groups were all similar. The mRNA level results were consistent with the protein level results. Our results showed that the antiviral activity of IFN alpha1b or IFN alpha2b were stronger than that of IFN alpha2a. The signal transduction molecules STAT1, STAT2, and IFNAR could be regarded as a key index to evaluate antiviral activity of IFN alpha. Further confirmation is still needed to see whether PKR could be regarded as a key index.

Antiviral Agents↗
PubMed2005

[The promoter activity of the DNA sequence corresponding to HCV 5'UTR in HepG2].

OBJECTIVE: To study the promoter activity in HepG2 cells of the DNA sequence corresponding to the HCV 5'UTR. METHODS: Plasmids, 5'UTR-Luc(+) and 5'UTR-Luc(-) carrying the forward and reverse DNA sequences corresponding to the HCV 5'UTR respectively were constructed, and subsequently transfected into HepaG2 cells. The luciferase activity and the mRNA of the luciferase gene were then detected. The 5'UTR sequence was cloned into a GFP vector to make 5'UTR-EGFP, and then the GFP expression was confirmed by fluorescence microscopy. RESULTS: 5'UTR-Luc(+) had an obvious luciferase activity whereas 5'UTR-Luc(-) had nearly no luciferase activity. The former had a high level of luciferase mRNA while the latter could not be detected. An intense green fluorescence expression was observed in the cells transfected with the plasmid of 5'UTR-EGFP. CONCLUSION: The forward DNA sequence corresponding to HCV 5'-UTR had an obvious promoter activity in hepG2 cells. It may play an important role in the replication of HCV.

5' Untranslated Regions↗
PubMed2004

[Study on the replication of Hepatitis B Virus in primary hepatocytes from heterogenous species].

OBJECTIVES: By studying the possibility and degree of the replication and expression of human hepatitis B virus (HBV) genes in normal liver cells from heterogenous species, such as primary duck hepatocytes (PDH) and primary rat hepatocytes (PRH), to investigate the species-specificity of HBV infection and replication. METHODS: PDH and PRH isolated by in situ perfusion with low concentration collagenase were transfected with complete HBV genome by electroporation (transfection group, about 1.19 10(12) copies of linear HBV DNA/1 10(7) PRH/PDH). From 1 day to 15 days after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH/PDH were measured with IMX System, HBcAg was assayed with western blotting, Immunol dot blotting and Immunocytochemistry. Moreover, HBV S-mRNA and X-mRNA were tested with RT-PCR. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PRH/PDH electroporated only was used as control group. RESULTS: HBsAg in the lysates of transfected PDH group were 15.24 (1day), 14.55 (3 days) and 5.13 ( 5 days; P/N values, positive>or=2.1), HBeAg all was negative (<2.1), and both were negative in the supernatants of transfected group. Viral antigen production in transfected rat hepatocytes: HBsAg in the lysates of transfected hepatocytes was positive, P/N values ranging from 2.17 to 93.41, The average P/N values was 14.74+/-31.82, and could be maintained for 15 days after transfection. Whereas, HBsAg in the supernatants of transfected group was only found positive on 1 day after transfection, which P/N value was 6.66. HBeAg and HBcAg in the lysates of PRH of transfection group were positive during the first 3 days following transfection, P/N values was around 2.8. The total amount of HBV DNA in the transfected PDH and PRH groups was strongly positive by dot blotting, whereas that of the control group was negative. Southern blot analysis of intracellular total HBV DNA indicated that there were relaxed circular (RC), covalently closed circular (ccc) and single-stranded (SS) HBV DNA replicative intermediates in the transfected PDH and PRH groups, there was no integrated HBV DNA in the cellular genome. Control groups were negative at all. CONCLUSION: Expression of HBV genes and production can occur in hepatocytes from nonmammalian species or mammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.

Animals↗