Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy

AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also analyzed.RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.

Upreguiation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved

Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

Hepatitis delta virus(HDV) seems to strongly suppress hepatitis B virus(HBV)replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein(HBx) is essential for HBV replication, determination of HBV X gene(HBX)quasispecies complexity in HBV/HDV infection compared to HBV monoinfection may provide information on the interactions between these two viruses.AIM To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta(CHD) and chronic HBV mono-infected patients.METHODS Twenty-four untreated patients were included: 7/24(29.2%) with HBeAgnegative chronic HBV infection(CI, previously termed inactive carriers), 8/24(33.3%) with HBeAg-negative chronic hepatitis B(CHB) and 9/24(37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels.The HBX 5' region [nucleotides(nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing(MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidencebased indices(number of haplotypes and number of mutations), abundancebased indices(Hill numbers of order 1 and 2), and functional indices(mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity.RESULTS CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL,interquartile range(IQR) 3.5-7.9] than CHD(3.4 logIU/mL, IQR 3-7.6)(P = n.s.)or CI(3.2 logIU/mL, IQR 2.3-3.5)(P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences(CHB2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3 G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype.CONCLUSION The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.

Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes

AIM： To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells.METHODS： HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 μg/mL G418 and named HL-7702/HBV-encoded X protein （HBx） cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively.RESULTS： RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semiquantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than thosein control.CONCLUSION： HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner.

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on human hepatocytes

AIM： To investigate the biological impact of hepatitis B virus X- hepatitis C virus core （HBV X-HCV C） fusion gene on hepatoma cells.METHODS： The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBVX gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colony- forming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion aene on liver cells.RESULTS： MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the AdO and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice.CONCLUSION： Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBVX and HCV C genes on hepatocarcinogenesis in athymic nude mice.

Association between TLR7 copy number variations and hepatitis B virus infection outcome in Chinese

AIM To explore whether copy number variations (CNVs) of toll-like receptor 7 (TLR7) are associated with susceptibility to chronic hepatitis B virus (HBV) infection. METHODS This study included 623 patients (495 males and 128 females) with chronic hepatitis B virus infection (CHB) and 300 patients (135 females and 165 males) with acute hepatitis B virus infection (AHB) as controls. All CHB patients were further categorized according to disease progression after HBV infection (CHB, liver cirrhosis, or hepatocellular carcinoma). Copy numbers of the TLR7 gene were measured using the AccuCopy method chi(2) tests were used to evaluate the association between TLR7 CNVs and infection type. P values, odds ratios, and 95% confidence intervals (CIs) were used to estimate the effects of risk. RESULTS Among male patients, there were significant differences between the AHB group and CHB group in the distribution of TLR7 CNVs. Low copy numberof TLR7 was significantly associated with chronic HBV infection (OR = 0.329, 95% CI: 0.229-0.473, P > 0.001). Difference in TLR7 copy number was also found between AHB and CHB female patients, with low copy number again associated with an increased risk of chronic HBV infection (OR = 0.292, 95% CI: 0.173- 0.492, P < 0.001). However, there were no significant differences in TLR7 copy number among the three types of chronic HBV infection (CHB, liver cirrhosis, or hepatocellular carcinoma). In addition, there was no association between TLR7 copy number and titer of the HBV e antigen. CONCLUSION Low TLR7 copy number is a risk factor for chronic HBV infection but is not associated with later stages of disease progression.

Biological impacts of "hot-spot" mutations of hepatitis B virus X proteins are genotype B and C differentiated

AIM: To investigate the biological impacts of 'hot-spot'mutations on genotype B and C HBV X proteins (HBx).METHODS: Five types of'hot-spot' mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M+V131I,I127T+K130M+V131I, or K130M+V131I+F132Y, respectively,were generated by means of site-directed mutagenesis.To evaluate the anti-proliferative effects, HBx or related mutants' expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drug-resistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored (increase of the colonies indicated the reduction of the anti-proliferation activity,and vice versa). Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVβ to Chang cells, which were lysed 48 h post-transfection and the intra-cellular β-galactosidase activities were monitored (increase of the β-galactosidase activities indicated the reduction of the transactivation activity, and vice versa). All data obtained were calculated by paired-samples t-test.RESULTS: As compared to standard genotype B HBx,mutants of I127T and I127T+K130M+V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M+V131I, and K130M+V131I+F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity,compared to standard genotype C HBx, F132Y and K130M+V131I mutants showed lower activities, and K130M+V131I +F132Y mutant, on the other hand, showed higher activity,while the mutants of I127T and I127T+K130M+V131I showed no differences.CONCLUSION: 'Hot-spot' mutations affect the antiproliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most 'hot-spot' mutations on HBx are genotype B and C differentiated.

Relationship between hepatitis B virus associated primary hepatocellular carcinoma and alteration of tumor suppressor gene p53

Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.

Hepatitis B virus infection and the risk of hepatocellular carcinoma

Epidemiological studies have provided overwhelming evidence for a causal role of chronic hepatitis B virus(HBV) infection in the development of hepatocellular carcinoma(HCC).However,the pathogenesis of HBV infection and carcinogenesis of HBV-associated HCC are still elusive.This review will summarize the current knowledge on the mechanisms involved in HBV-related liver carcinogenesis.The role of HBV in tumor formation appears to be complex,and may involve both direct and indirect mechanisms.Integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion,and it has been shown to enhance the host chromosomal instability,leading to large inverted duplications,deletions and chromosomal translocations.It has been shown that the rate of chromosomal alterations is increased significantly in HBV-related tumors.Prolonged expression of the viral regulatory HBV x protein may contribute to regulating cellular transcription,protein degradation,proliferation,and apoptotic signaling pathways,and it plays a critical role in the development of hepatocellular carcinoma.

Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA: Different quasispecies?

BACKGROUND Different forms of pregenomic and other hepatitis B virus(HBV)RNA have been detected in patients’sera.These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity,and predicting hepatitis B eantigen seroconversion or viral rebound after nucleos(t)ide analog cessation.Data on serum HBV-RNA quasispecies,however,is scarce.It is therefore important to develop methodologies to thoroughly analyze this quasispecies,ensuring the elimination of any residual HBV-DNA.Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection.AIM To establish a next-generation sequencing(NGS)methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies.METHODS Thirteen untreated chronic hepatitis B patients,showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA>5 Log10 IU/mL and HBV-RNA>4 Log10 copies/mL was taken from each patient.HBV-RNA was treated with DNAse I to remove any residual DNA,and the region between nucleotides(nt)1255-1611 was amplified using a 3-nested polymerase chain reaction protocol,and analyzed with NGS.Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies.RESULTS No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies.HBV quasispecies complexity showed heterogeneous behavior among patients.The Rare Haplotype Load at 1%was greater in DNA than in RNA quasispecies,with no statistically significant differences(P=0.1641).Regarding conservation,information content was equal in RNA and DNA quasispecies in most nt positions[218/357(61.06%)].In 102 of the remaining 139(73.38%),HBV-RNA showed slightly higher variability.Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies,3 of them coincided between the 2 quasispecies:nts 1258-1286,1545-1573 and 1575-1604.The 2 hyper-variable sequence fragments also coincided:nts 1311-1344 and 1461-1485.Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA,respectively.CONCLUSION Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA.Thanks to this,we have been able to compare both quasispecies in the present study.

COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma

AIM:To investigate the hepatitis B virus (HBV) x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations. METHODS: HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods. RESULTS: The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non- cancerous liver tissues (11/19 vs 18/19, P = 0.019). In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19, P < 0.001). The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20/45) - 56.8% (25/45) tumors and 40.9% (18/45) - 52.3% (23/45) non-tumor tissues. CONCLUSION: HBx deletion, especially the COOH- terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.

HBV-HCC中HBx与免疫微环境的交互作用

乙型肝炎病毒X蛋白(HBx)是乙型肝炎病毒X基因(HBX)将自身DNA整合至人基因组,进而合成的多功能蛋白。HBX基因的表达受肝细胞免疫、微环境和机体免疫的监视和调控,其表达的蛋白也可通过激活肝星状细胞、参与机体免疫调节、调控炎性细胞因子和诱导细胞外基质重塑等参与肝细胞癌(HCC)抑制性免疫微环境的形成。HBx与免疫微环境的相互作用是影响乙肝病毒相关肝细胞癌(HBV-HCC)发生、发展的主要因素之一。深入研究HBx与免疫微环境相互作用机制,探索促进HBV-HCC抑制性免疫微环境形成的机制,有助于开发新型抗HCC药物,改善患者预后。本文就HBx与HBV-HCC免疫微环境的研究进展进行综述。

The function of HBx in HBV-induced hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the second cause cancer death in the world. HCC is frequently diagnosed at advanced stages with intrahepatic metstasis or vascular invasion and has a poor prognosis with a high mortality rate. In the world, hepatitis B virus(HBV) caused over 50% HCC, making it the most common carcinogen after tobacco, and the encoded protein of the HBV X gene(HBx) plays a critical role in the tumorigenesis of HBV-related HCC. There is a lot of literature about the function of HBx. In this article we summary the main function of HBx in HCC.

肝细胞癌患者B和C基因型乙型肝炎病毒X蛋白的变异特点

背景与目的X蛋白是乙型肝炎病毒(hepatitisBvirus,HBV)最重要的致病因子之一,它在HBV相关性肝细胞癌(hepatocellularcarcinoma,HCC)的发生发展中起着重要作用。目前已知B、C基因型HBV相关性HCC的临床及病理表现不尽相同,但目前尚不清楚这种差别是否与B、C基因型HBVX基因之间的差别有关。因此本实验拟研究B、C基因型间HBVX蛋白氨基酸差异及其在HCC患者中的变异及特点,初步探讨其与HCC发生、发展的关系。方法PCR扩增22份乙型肝炎病毒表面抗原(HBsAg)阳性HCC患者血清HBVX基因,克隆、测序并以VectorNTI6.0软件分析其基因型。以DNAMAN软件对标准HBV及HCC来源的HBVX蛋白进行氨基酸同源分析。结果检测的22个HBVX基因片段均属于B或C基因型。B、C基因型HBVX蛋白之间存在14个氨基酸的差异,HCC患者来源的B、C型HBVX蛋白存在4个氨基酸的共有变异,C型HBVX蛋白尚有6个型特异性变异。这些差异或变异氨基酸均位于X蛋白B、T细胞表位或反式激活区及调节区内。结论B、C基因型HBVX蛋白之间存在氨基酸差异,且在HCC中发生基因型特异性变异,这些差异或变异氨基酸可能导致X蛋白免疫学功能及反式激活功能的不同。

靶向整合于MHCC97-H细胞的HBx基因shRNA表达载体的构建和鉴定

目的构建靶向整合于人肝癌MHCC97-H细胞乙型肝炎病毒x基因(HBxgene)基因的shRNA真核载体。方法依据从人肝癌细胞株MHCC97-H中克隆的HBx基因序列,设计合成X169、X220及MOCK(无关对照)3对寡核酸,经退火成互补双链并克隆至pGenesil-Ⅰ载体;经酶切、PCR和测序鉴定;将鉴定的shRNA质粒脂质体转染MHCC97-H细胞,流式细胞分析转染效率。结果酶切、PCR及测序证实pshRNA-X169/X220/MOCK质粒构建符合设计,转染48h后MHCC97-H细胞可激发绿色荧光,pshRNA-X220转染效率最低。结论成功构建靶向整合于人肝癌细胞HBVx基因特异性shRNA表达载体,并成功转染人肝癌细胞株MHCC97-H细胞,为沉默HBx基因实验性肝癌基因治疗奠定基础。

HBV X蛋白反式调节基因XTP11的克隆化研究

目的:应用寡核苷酸基因芯片技术及生物信息学分析,筛选并克隆乙肝病毒X蛋白(HBxAg)反式激活新型靶基因。方法:以HBxAg表达质粒pcDNA 3 1(-) X转染HepG2细胞,以空载体pcDNA 3. 1(-)为平行对照,提取mRNA并进行寡核苷酸基因芯片分析。对于所获基因片段序列分析表明,其中之一为新型基因片段,与GenBank中注册的已知功能基因序列没有同源性。通过序列同源性搜索和比对和电子拼接,根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷酸信号序列,确定新型基因序列。从转染了pcDNA 3 . 1(-) X的HepG2细胞提取总RNA ,以逆转录聚合酶链反应(RT- PCR)技术,扩增获得该新基因的全长序列,并测序证实。结果:该新基因命名为XTP11,在GenBank中注册,注册号为AY740 5 2 0。该基因的编码序列全长为13 44个核苷酸(nt) ,编码产物由44 8个氨基酸残基(aa)组成。结论:HBxAg反式激活新型靶基因XTP11的筛选与克隆,为进一步研究HBxAg反式激活作用的分子生物学机制奠定基础。

舒林酸对HBV X基因转染的肝癌细胞Wnt信号通路的影响

目的探讨非甾体类抗炎药舒林酸(Sulindac)对HBV X基因转染的人肝癌SMMC7721细胞系Wnt信号通路的影响。方法用脂质体转染方法将HBx真核表达载体pcDNA3-X瞬时转染SMMC7721细胞(SMMC7721/HBx),以转染空载体pcDNA3的SMMC7721细胞组(SMMC7721/pcDNA3)及未转染质粒的SMMC7721细胞组(SMMC7721)为对照,于转染后72 h用RT-PCR和Western blot检测HBx的表达;并以各种浓度的舒林酸于瞬时转染后24 h干预SMMC7721/HBx组和SMMC7721/pcDNA3组至72 h,Western blot和免疫细胞化学检测β-catenin的表达,Western blot检测cyclinD1的表达。结果RT-PCR和Western blot显示,SMMC7721/HBx组在RNA及蛋白水平均有HBx的表达,而SMMC7721/pcDNA3组及SMMC7721组均无表达。与SMMC7721/pcD-NA3组及SMMC7721组相比,Western blot示SMMC7721/HBx组β-catenin、cyclinD1的表达水平较高,免疫细胞化学显示SMMC7721/HBx组β-catenin的表达水平较高,核染色较深,而SMMC7721/pcDNA3组及SMMC7721组之间则无明显差异。Westernblot和免疫细胞化学示一定浓度的舒林酸干预SMMC7721/HBx组和SMMC7721/pcDNA3组细胞48 h后可下调β-catenin和cy-clinD1的表达,而对SMMC7721/HBx组的下调较SMMC7721/pcDNA3组明显。结论HBx基因成功瞬时转染入SMMC7721细胞,激活Wnt信号通路,并同时上调cyclinD1的表达。舒林酸可通过下调β-catenin抑制肝癌细胞的Wnt信号通路,下调cyclinD1的表达,且HBx阳性的肝癌细胞对舒林酸较敏感。

叶下珠水提物对裸鼠人肝癌移植瘤HBx和VEGFR3表达的影响

目的探讨乙型肝炎病毒x基因(hepatitis B virux x gene,HBx)和血管内皮生长因子受体3(vascular endothelial growth factor receptor 3,VEGFR3)基因与乙型肝炎相关肝癌的相关性,以及叶下株水提物(aqueous extract of phyllanthus urinaria L,AEP)对HBx和VEGFR3蛋白表达的干预作用。方法将60只Balb/c-nu裸鼠随机分为10组。分别复制转染HBx、氯霉素乙酞转移酶(chloramphenicol acetyltransferase,CAT)和VEGFR3基因的HepG2肝癌细胞的裸鼠皮下移植瘤模型,用AEP进行干预,以生理盐水、环磷酰胺(cyclophosphamide,CTX)作为对照,共治疗6周。观察模型复制后不同时间各组裸鼠的移植瘤生长状况,采用酶联免疫吸附试验检测各组裸鼠血清甲胎蛋白(alpha fetal protein,AFP)水平,采用Western blot方法检测移植瘤组织中HBx和VEGFR3蛋白表达水平。结果肝癌移植瘤模型复制成功。各组裸鼠体质量均随着时间显著增长。实验结束时,接种HepG2-HBx细胞的各组中,AEP处理组肿瘤质量显著低于CTX处理组(P<0.05),其AFP水平显著高于NS处理组(P<0.05),其移植瘤组织中HBx表达水平显著低于NS组(P<0.05)。接种相同肝癌细胞的各组中,AEP处理组VEGFR3表达水平最低,但与CTX组VEGFR3表达水平比较,差异无统计学意义(P>0.05);AEP组和CTX组VEGFR3表达水平显著低于NS组(P<0.05)。对于相同的处理因素,接种HepG-HBx细胞和HepG-CAT细胞的裸鼠移植瘤组织中VEGFR3表达水平均显著低于接种HepG-VEGFR3细胞的裸鼠(P<0.01)。结论 AEP通过直接抑制HBx蛋白和VEGFR3蛋白表达,及通过抑制HBx蛋白表达而间接抑制VEGFR3蛋白表达,达到对肝癌移植瘤生长的抑制作用。

慢病毒介导HBV X基因稳定表达HepG2细胞系的建立

目的:构建乙型肝炎病毒X基因(HBV X)重组慢病毒表达载体,建立稳定表达HBVX蛋白(HBx)的HepG2细胞系.方法:应用PCR法从质粒pIERES2-EGFP-HBV中扩增X基因片段,克隆至慢病毒载体pZac2.1,应用PCR、酶切和测序鉴定正确后,经病毒包装,感染HepG2细胞,经嘌呤霉素筛选稳定表达细胞株,RT-PCR、免疫组织化学、Western blot鉴定HBx的表达.结果:酶切鉴定和基因序列测定证实长度为489bp的HBx基因成功克隆至慢病毒表达载体pZac2.1-HBx;重组慢病毒经包装、纯化后获得滴度为1×108TU/mL,通过感染HepG2细胞株和嘌呤霉素筛选,8-10d形成生长形态良好的单克隆细胞株HepG2-HBx;RT-PCR鉴定显示细胞株HepG2-HBx在3d,14d,30d和2mo后均可见稳定表达的HBx mRNA;利用免疫组织化学和蛋白免疫印迹法鉴定,细胞株HepG2-HBx可稳定表达HBx蛋白.结论:成功构建了HBV X重组慢病毒载体,获得稳定表达HBx的HepG2细胞系,为进一步研究HBx的生物学功能及致病机制提供细胞模型.

HBV X基因转染对HepG2肝癌细胞凋亡的影响及其机制

目的 :研究HBVX基因转染对HepG2肝癌细胞凋亡及凋亡相关因子表达的影响。 方法 :用脂质体转染法将HBx真核表达载体pcDNA3 /HBx瞬时转入HepG2细胞 ,以未转染的HepG2细胞及转染空载体 pcDNA3 的细胞为对照。RT PCR法检测HBx基因的表达 ;MTT法检测各组细胞的增殖活性 ;TUNEL法检测各组的凋亡情况。β actin为内参 ,半定量RT PCR法检测凋亡相关基因Bax、Bcl xL、c myc的表达量变化。结果 :pcDNA3 X转染HepG2细胞后 ,RT PCR扩增出HBVX片段 ,空质粒组及正常对照组均未扩增出相应片段。HepG2细胞转染HBx基因后 ,相对于对照组细胞增殖能力明显下降 ,凋亡增多 ,差异有显著性意义 (P <0. 0 1) ;转染HBx的细胞Bax、Bcl xL、c mycmRNA相对表达量较转染空质粒组和未转染质粒组明显增高 ,差异有显著性意义 (P <0 . 0 5 )。结论 :成功将HBx基因转染入HepG2细胞 ,并在细胞中表达 ,转染HBx基因可同时上调Bax、Bcl xL、c mycmRNA表达 ,促进HepG2细胞凋亡 ,抑制增殖。