葡聚糖磁性纳米颗粒在基因转染人树突状细胞中的研究

目的评价葡聚糖磁性纳米颗粒作为基因载体在针对人树突状细胞转染中的可行性。方法用紫外分光光度计及琼脂糖凝胶电泳法分析检测葡聚糖磁性纳米颗粒结合DNA的能力,再将葡聚糖磁性纳米颗粒作为基因载体连接绿色荧光蛋白pEGFPC1质粒报告基因在体外转染人树突状细胞,并在转染后72h采用MTT比色法测定这种载体对人树突状细胞增殖和功能的影响以了解其细胞毒性。结果在葡聚糖磁性纳米颗粒表面修饰多聚赖氨酸后,以静电引力作用结合DNA,其DNA结合率可达到75.6%,修饰多聚赖氨酸的葡聚糖磁性纳米颗粒可作为基因载体将报告基因转染至人树突状细胞内并成功表达,在荧光显微镜下可观察到绿色荧光细胞。与相同条件下脂质体为对照,转染后的人树突状细胞的增殖活性及功能略优于对照组,有效的证明了葡聚糖磁性纳米颗粒的细胞毒性稍低于脂质体的细胞毒性。结论葡聚糖磁性纳米颗粒可作为有效转染人树突状细胞的基因载体之一。

Isolation, Expression Characteristics and Chromosomal Locations of Three cDNA Fragments Under Salt Stress in Rice

cDNA libraries were constructed from the leaves of a rice (Oryza sativa L.) salt tolerancevariety Tesan抋i 2 growing in solutions with 150 mmol/L NaCl for 3 h or without salt stress. Three salt-responsive cDNA clones, Ts1, Ts2 and Ts3 were isolated by differential screening. Northern blottinganalysis showed that the transcription levels of Ts1 and Ts2 increased within 3 h salt stress and kept onincreasing within 24 h, while the transcription level of Ts3 reached its peak within 3 h. Sequence analysisindicated that there were no homologies between the three cDNA clones and any known gene. The threecDNA clones were mapped using a doubled haploid (DH) population derived from an indica variety ZYQ8,which was a salt tolerance parent of Tesan抋i 2, with a japonica variety JX17. Ts1, Ts2 and Ts3 werelocated on chromosomes 1, 3 and 7, respectively. It was noted that Ts1, Ts2, and Ts3 were in or near theregions of major or minor salt tolerance quantitative trait loci (QTLs), which were mapped in the same DHpopulation in a parallel study.

Screening of the Metastasis-Associated Genes by Gene Chip in High Metastatic Human Ovarian Cancer Cell Lines

Affymetrix U133A oligonucleotide microarrays were used to study the differences of gene expressions between high （H） metastatic ovarian cancer cell line, HO-8910PM, and normal ovarian tissues （C）. Bioinformatics was used to identify their chromosomal localizations. A total of 1,237 genes were found to have a difference in expression levels more than eight times. Among them 597 were upregulated [Signal Log Ratio （SLR） ≥3], and 640 genes were downregulated （SLR≤-3）. Except one gene, whose location was unknown, all these genes were randomly distributed on all the chromosomes. However, chromosome 1 contained the most differentially expressed genes （115 genes, 9.3%）, followed by chromosome 2 （94 genes, 7.6%）, chromosome 12 （88 genes, 7.1%）, chromosome 11 （76 genes, 6.1%）, chromosomes X （71 genes, 5.7%）, and chromosomes l7 （69 genes, 5.6%）. These genes were localized on short-arm of chromosome （q）, which had 805 （65.1%） genes, and the short arms of No.13, 14, 15, 21, and 22 chromosomes were the only parts of the chromosomes where the differentially expressed genes were localized. Functional classification showed that most of the genes （306 genes, 24.7%） belonged to the enzymes and their regulator groups. The subsequent group was the nucleic acid binding genes （144 genes, 11.6%）. The rest of the top two groups were signal transduction genes （137 genes, 11.1%） and proteins binding genes （116 genes, 9.4%）. These comprised 56.8% of all the differentially expressed genes. There were also 207 genes whose functions were unknown （16.7 %）. Therefore it was concluded that differentially expressed genes in high metastatic ovarian cancer cell were supposed to be randomly distributed across the genome, but the majority were found on chromosomes 1, 2, 12, 11, 17, and X. Abnormality in four groups of genes, including in enzyme and its regulator, nucleic acid binding, signal transduction and protein binding associated genes, might play important roles in ovarian cancer metastasis. Those genes need to be further studied.

The Effect of Helicobacter Pylori Infection on Expression of hMSH2, hMLH1 and p53 in Gastric Carcinogenesis

Objective： To detect the expression of hMSH2, hMLH1 and p53 in gastric epithelial cells with and without Helicobactcr pylori （H. pylori） infection and investigate the relationship between H. pylori infection and these genes in gastric carcinogenesis. Methods： H. pylori infection was detected by rapid urease tests. Expression of hMSH2, hMLHland p53 in gastric cancer （GC） tissue, its adjacent mucosa, gastritic mucosa and normal mucosa was assessed by immunohistochemistry SP method. Results： Positive expression rate of hMSH2 in GC tissue （62.7%） was higher than those in adjacent mucosa （29.4%）, gastritic mucosa （32.4%） and normal mucosa （30.0%） （P〈0.001）. Positive rate of hMSH2 in poorly differentiated adenocarcinoma （76.4%） was higher than those in other carcinomas （54.3%, 53.1%） （P〈0.05）. Positive expression rate of hMLH1 in GC tissue （64.3%） mucosa （82.4%） and normal mucosa （80.0%） was lower than those in adjacent mucosa （84.4%）, gastritic （P〈0.01）. Positive rate of hMLH1 in mucoid carcinoma （43.7%） was lower than those in other carcinomas （78.2%, 64.7%） （P〈0.01）. Positive expression rate of p53 in GC tissue （51.9%） was higher than those in adjacent mucosa （3.1%）, gastritic mucosa （0.0%） and normal mucosa （0.0%） （P〈0.001）. Positive rate of p53 in well differentiated adenocarcinoma （32.6%） was lower than those in other carcinomas （58.8%, 68.7%） （P〈0.01）. Positive rates of hMSH2 and hMLH1 in GC with H. pylori infection were lower than those without the infection, respectively （P〈0.05）. Positive rate of p53 in GC with H. pylori infection （61.4%） was higher than that without the infection （40.6%） （P〈0.05）. Conclusion： Gastric carcinogenesis may be associated with abnormal expression of hMSH2, hMLH1 and p53; H. pylori infection affecting expression of these genes may be one of its carcinogenic mechanisms.

Agroinoculation as a Simple Way to Deliver a Tobacco Mosaic Virus- Based Expression Vector

烟草花叶病毒(TMV)表达载体30B是一个目前广泛应用的植物病毒表达载体,但用其生产外源蛋白时,必须先将它体外转录成RNA,才能被用来接种宿主植物。由于RNA体外转录费用昂贵、操作复杂,因此限制了30B表达载体的进一步应用。针对这一不足,我们用农杆菌接种法(agroinoculation)接种该病毒载体,即将30B cDNA置于花椰菜花叶病毒(CaMV)的35S启动子和终止子之间,再将整个表达框架插入到农杆菌T-DNA的左边界和右边界之内,构建成质粒p35S-30B,将转入该质粒的农杆菌注射到植物的叶片中,30B cDNA随T-DNA进入植物细胞后,被转录成可自我复制的RNA形式,进而发生系统侵染。为了检测此接种方式的可行性,绿色荧光蛋白(GFP)报告基因被克隆到p35S-30B中,构建成p35S-30B∶∶GFP,用含有该质粒的农杆菌进行注射操作。证实该病毒载体可通过简便的农杆菌接种法侵染Nicotiana benthamiana,在被接种植物的系统叶中,GFP的表达量可占植物总可溶蛋白的5.2％。

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

Relationship between abnormality of FHIT gene and EBV infection in gastric cancer

AIM: To examine the aberrant expression of fragile histidine triad (FHIT) gene and protein in gastric cancer, and to evaluate the role of FHIT gene and the relationship between FHIT gene and EBV infection in gastric carcinogenesis. METHODS: FHIT transcripts were detected by nested RT-PCR in 30 cases of gastric cancer and their products were sequenced.FHIT protein was detected by Western blot. EBV infection was detected by PCR method in 50 cases of gastric cancer. RESULTS: The wild type transcripts were detected in all 30 matched normal tissues of gastric cancer.Aberrant transcripts were found in 11/30 (36.7%) gastric cancerous tissues. Sequencing analysis of the aberrant fragments found an RT-PCR product missing exons 5-7 in one case of gastric cancer, and another product missing exons 4-7. Four of ten (40.0%) cases of primary gastric cancer showed absent or decreased expression of FHIT protein as compared with their matched normal tissues.EBV was detected in 5/50 (10%) gastric cancers,among which 4/5 (80%) had aberrant transcripts of FHIT gene. CONCLUSION: Loss of FHIT gene or FHIT protein plays an important role in carcinogenesis,development and progression of gastric cancer.EBV infection might influence carcinogenesis of gastric cancer by inducing the abnormality of FHIT gene.

Down-regulation of PTEN expression due to loss of promoter activity in human hepatocellular carcinoma cell lines

AIM: To investigate the regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene expression in human hepatocellular carcinoma (HCC) cell lines.METHODS: The mRNA and protein levels of PTEN were detected by Northern blot and Western blot in HCC cell lines, respectively. Plasmids containing different fragments of PTEN promoter with Luciferase reporter were constructed and transiently transfected into HCC cell lines to study the promoter activity. DNA analysis and RT-PCR were performed to detect the mutation of PTEN promoter and PTEN cDNA.RESULTS: Either protein or mRNA levels of PTEN in L02 cells (as a control) were significantly higher than that in HCC cell lines. The profile of PTEN promoter activity in 8 cell lines was closely correlated with levels of PTEN mRNA and PTEN protein. Furthermore, the sequence analysis of 8 cells lines showed no mutation in the region of PTEN promoter and PTENcDNA.CONCLUSION: PTEN expression is down-regulated in HCC cell lines probably due to loss of activity of PTEN promoter.

Establishment and primary application of a mouse model with hepatitis B virus replication

AIM： To establish a rapid and convenient animal model with hepatitis B virus （HBV） replication.METHODS： A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen （HBcAg） and hepatitis B surface antigen （HBsAg） in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen （HBeAg） was detected by enzyme- linked immunosorbent assay （ELISA）. Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid （polyIC） or phosphate-buffered saline （PBS）.RESULTS： After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS.CONCLUSION： A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.

Cloning of α-β fusion gene from Clostridium perfringens and its expression

AIM： To study the cloning of α-β fusion gene from Closindium perfringens and the immunogenidty of 0-6 fusion expression. METHODS： Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of α-toxin and β-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of α-β fusion gene binding. In order to verify the exact location of the α-β fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed α-β fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. RESULTS： The protective co-toxin gene （cpa906） and the β-toxin gene （cpb930） were obtained. The recombinant plasmid pZCPAB carrying α-β fusion gene was constructed and transformed into BL21（DE3）. The recombinant strain BL21（DE3）（pZCPAB） was obtained. After the recombinant strain BL21（DE3）（pZCPAB） was induced by 42℃, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with α-βfusion protein could neutralize the toxicity of α-toxin and β-toxin. CONCLUSION： The obtained α-toxin and β-toxin genes are correct. The recombinant strain BL21（DE3）（pZCPAB） could produce α-β fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with α-β fusion protein could neutralize the toxicity of α-toxin and β-toxin.

Toll-like receptor 4 plays an anti-HBV role in a murine model of acute hepatitis B virus expression

AIM： Toll-like receptor 4 （TLR4） has been shown to be important for bacterial infection, especially to lipopolysaccharide signaling. Its possible role in HBV infection is studied in the present study. MATERIALS AND METHODS： pHBV3.6 plasmid, containing full-length HBV genome was used in the murine model of acute HBV expression by hydrodynamics in vivo transfection. TLR4 normal or mutant mouse strain was compared to investigate the possible role of TLR4 in acute HBV expression. RESULTS： After pHBV3.6 injection, the infiltrating leukocytes expressed TLR4 were observed nearby the HBsAg-expressing hepatocytes. The HBV antigenemia as well as the replication and transcription were higher in TLR4-mutant C3H/HeJ mice than in normal C3H/ HeN mice. The HBV-specific immune responses were impaired in the liver or spleen of the C3H/HeJ mice. Their inducible nitric oxide synthase （iNOS） expression on the hepatic infiltrating cells was also impaired. When adoptively transferring splenocytes from C3H/HeN mice to C3H/HeJ mice, the HBV replication was inhibited to the level as that of C3H/HeN. CONCLUSION： These results suggest that TLR4 plays an anti-HBV role in vivo through the induction of iNOSexpression and HBV-specific immune responses after HBV expression.

Helicobacter pylori upregulates prion protein expression in gastric mucosa: A possible link to prion disease

AIM： Pathological prion protein （PrP^sc） is responsible for the development of transmissible spongiform encephalopathies （TSE）. While PrPc enters the organism via the oral route, less data is available to know about its uptake and the role of gastrointestinal inflammation on the expression of priori precursor PrPc, which is constitutively expressed in the gastric mucosa.METHODS： We studied PrPc expression in the gastric mucosa of 10 Helicobacter pylori-positive patients before and after successful H pylori eradication compared to non-infected controls using RT-PCR and Western blotting. The effect of central mediators of gastric inflammation, i.e., gastrin, prostaglandin E2 （PGE2）, tumor necrosis factor alpha （TNF-α） and interleukin 1 beta （IL-1β） on PrPc expression was analyzed in gastric cell lines.RESULTS： PrPc expression was increased in H pyloriinfection compared with non-infected controls and decreased to normal after successful eradication. Gastrin, PGE2, and IL-1β dose-dependently upregulated PrPc in gastric cells, while TNF-α had no effect.CONCLUSION： H pylori infection leads to the upregulation of gastric PrPc expression. This can be linked to H pylori induced hypergastrinemia and increased mucosal PGE2 and IL-1β synthesis. H pylori creates a milieu for enhanced propagation of prions in the gastrointestinal tract.

Chronic hepatitis C virus infection:Prevalence of extrahepatic manifestations and association with cryoglobulinemia in Bulgarian patients

AIM: To assess the prevalence of extrahepatic manifestations in Bulgarian patients with chronic hepatitis C virus (HCV) infection and identify the clinical and biological manifestations associated with cryoglobulinemia. METHODS: The medical records of 136 chronically infected HCV patients were reviewed to assess the prevalence of extrahepatic manifestations. Association between cryoglobulin-positivity and other manifestations were identified using χ2 and Fisher’s exact test. Risk factors for the presence of extrahepatic manifestations were assessed by logistic regression analysis. RESULTS: Seventy six percent (104/136) of the patients had at least one extrahepatic manifestation. Clinical manifestations included fatigue (59.6%), kidney impairment (25.0%), type 2 diabetes (22.8%), paresthesia (19.9%), arthralgia (18.4%), palpable purpura (17.6%), lymphadenopathy (16.2%), pulmonary fi brosis (15.4%), thyroid dysfunction (14.7%), Raynaud’s phenomenon (11.8%), B-cell lymphoma (8.8%), sicca syndrome (6.6%), and lichen planus (5.9%). The biological manifestations included cryoglobulinproduction (37.5%), thrombocytopenia (31.6%), and autoantibodies: anti-nuclear (18.4%), anti-smooth muscle (16.9%), anti-neutrophil cytoplasm (13.2%) and anti-cardiolipin (8.8%). All extrahepatic manifestations showed an association with cryoglobulin-positivity, with the exception of thyroid dysfunction, sicca syndrome, and lichen planus. Risks factors for the presence of extrahepatic manifestations (univariate analysis) were: age ≥ 60 years, female gender, virus transmission by blood transfusions, longstanding infection (≥ 20 years), and extensive liver fi brosis. The most signifi cant risks factors (multivariate analysis) were longstanding infection and extensive liver fi brosis. CONCLUSION: We observed a high prevalence of extrahepatic manifestations in patients with chronic HCV infection. Most of these manifestations were associated with impaired lymphoproliferation and cryoglobulin production. Longstanding infection and extensive liver fi brosis were signifi cant risk factors for the presence of extrahepatic manifestations in HCV patients.

Effects of Exogenous CC10 Transfection on CyclinDl Protein and mRNA Expression in A549 Lung Cancer Cells

Objective： To investigate the effects of exogenous CC10 gene transfection on cell cycle and the expression of cyclinD1 protein and mRNA in A549 cells. Methods： A549 cells in all test groups （group A to E） and control group （group F） were transfected with exogenous CC10 gene by liposome for 8, 16, 24, 36, 48 and 0 h respectively. CC10 protein expression was detected in A549 cells by Western blot. The growth inhibitory rate was detected by MTT method. Flow cyometry analysis （FCS） and AnnexinV-PI staining were used to determine the changes of cell cycle progression and apoptosis rate in all groups. CyclinD1 protein and mRNA expression in A549 cells was detected by the methods of immunocytochemistry and RT-PCR. Results： Exogenous CC10 gene could inhibit the growth of A549 cells, and the growth inhibitory rates in all test groups （from group A to E） were 24.7%, 33.1%, 44.3%, 61.7% and 74.2% respectively, and that in group F was 6.24%. CC10 blocked the cell cycle progression at G0/G1 and induced apoptosis gradually. In A549 cells of test groups, the expression of cyclinD1 protein and mRNA was significantly decreased. Conclusion： The inhibitory effects of the transfection of exogenous CC10 gene on G0/G1 cycle of lung cancer cells might be related with the down-regulation of cyclinD1 gene.

Immunogenicity and immunoprotection of recombinant PEB1 in Campylobacter-jejuni-infected mice

AIM： To construct a prokaryotic expression vector carrying Campylobacterjejuni peblA gene and express it in Escherichia coli. Immunoreactivity and antigenicity of rPEB1 were evaluated. The ability of rPEB1 to induce antibody responses and protective efficacy was identified. METHODS： peblA gene was amplified by PCR, target gene and prokaryotic expression ptasmid pET28a （＋） was digested with BamHI and XhoI, respectively. DNA was ligated with T4 DNA ligase to construct recombinant plasmid pET28a（＋）-peblA. The rPEB1 was expressed in E. coli BL21 （DE3） and identified by SDS-PAGE. BALB/c mice were immunized with rPEBI. ELISA was used to detect the specific antibody titer and MTT method was used to measure the stimulation index of spleen lymphocyte transformation. RESULTS： The recombinant plasmid pET28a （＋）-peblA was correctly constructed. The expression output of PEB1 protein in pET28a （＋）-peblA system was approximately 33% of total proteins in E. coil The specific IgG antibody was detected in serum of BALB/c mice immunized with rPEB1 protein. Effective immunological protection with a lower sickness incidence and mortality was seen in the mice suffering from massive C. jejuni infection. CONCLUSION： rPEB1 protein is a valuable candidate for C. jejuni subunit vaccine.

Characterization of flgK gene and FlgK protein required for H pylori Colonization-from cloning to clinical relevance

AIM： To characterize the role of flgK and its protein product in Hpylori colonization. METHODS： The PCR cloning method identified the flgK gene. An isogenic flgK mutant was constructed by gene replacement and confirmed by Southern blot analysis and PCR analysis. The recombinant FlgK protein （r-FlgK） was purified. Electron microscopy （EM） was applied to demonstrate the flagella of H pylori. An in vitro motility test was assessed in semisolid medium. The densities of H pylori colonization with either the wild-type strain or its flgK mutant were compared among BALB/c mice with or without pre-immunization with r-FlgK. The serological responses to r-FlgK were analyzed for 70 clinical patients with different densities of H pylori colonization. RESULTS： From a duodenal ulcer strain, the flgK gene was cloned and it contained 1821 bp, with a 95.7% identity to the published sequences. No flagella were observed under EM for the mutant strain, which had a loss of motility. Hpylori density was lower in the BALB/c mice inoculated by the mutant or with pre-immunization with r-FlgK compared to unimmunized mice or mice inoculated by the wild-type strain （P 〈 0.05）. In the H pylori-infected patients, the serological responses to r-FlgK were uniformly low in titer.CONCLUSION： FlgK encoded by flgK is important for flagella formation and H pylori motility. Deficiency in FlgK or an enhanced serological response to r-FlgK can interfere with Hpylori colonization. FlgK of Hpylori could be a novel target for vaccination.

Character of HBV (hepatitis B virus) polymerase gene rtM204V/I and rtL180M mutation in patients with lamivudine resistance

Objectives: To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance. Methods: One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment. Results: One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL 180M/M204V mutation (41.28%), 28patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations. Conclusions: HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.

PREDOMINANT EXPRESSION OF HUMAN Aγ-IN CONTRAST WITH β-GLOBIN GENE IN MEL CELLS TRANSFECTED WITH THE CONSTRUCT μLCRAγψβδ

A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and adult globins.Similar patterns of human globin gene expression were displayed in the two MEL cell lines transfected with the construct.Inducible expression of the Aγ and β gene was observed during induced cell differentiation.However,the expression level of the Aγ globin gene is much higher than that of the β globin gene in either uninduced or induced MEL transformants.No γ to β switching happened in the stable MEL transformants following a continuous culture.The much more effective enhance of the μLCR on the Aγ globin gene than that on the β globin gene is resulted probably from the fact that the distance between the LCR and the β globin gene is much longer than that between the LCR and the Aγ globin gene in the construct,in comparison with other constructs containing HS2 or μLCR linked to both of γ and β globin genes in different order.Two suggestions can be derived from these results:1) A competition between the γ and β globin gene for interaction with the LCR may indeed present,but only an enough long distance difference between the LCR to the γ and to the β gene can effectively influence the competition;2) Unlike transgenic mice,MEL cells are incapable of reconstructing the regulatory information involved in developmental control when it is provided by a fragment of the β globin gene cluster with limited length.

Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma

AIM： To investigate the expression and methylation status of the secreted frizzled-related protein 2 （SFRP2） in esophageal squamous cell carcinoma （ESCC） and ex- plore its role in ESCC carcinogenesis.METHODS： Seven ESCC cell lines （KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1） and one immortalized human esophageal epithelial cell line （Het- 1A）, 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction （RT-PCR） was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expres- sion and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2＇-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we es-tablished stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro.RESULTS： SFRP2 mRNA was expressed in the im- mortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2＇-deoxycytidine could restore the expres- sion of SFRP2 mRNA in the three ESCC cell lines lack- ing SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue （0.939 ± 0.398 vs 1.51 ± 0.399, P 〈 0.01）. SFRP2 methylation was higher in tumor tissue than in paired normal tissue （95% vs 65%, P 〈 0.05）. The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by in- troducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro （47.17% 4± 15.61% vs 17% ：1： 3.6%, P = 0.031） and tumor growth in nude mice （917.86：1：249.35 mm3 vs 337.23 ± 124.43 mm3, P 〈 0.05）. Using flow cytom- etry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells （P = 0.025）. The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts. CONCLUSION： Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.

Screening Helicobacter pylori genes induced during infection of mouse stomachs

AIM:To investigate the effect of in vivo environment on gene expression in Helicobacter pylori(H.pylori) as it relates to its survival in the host.METHODS:In vivo expression technology(IVET) systems are used to identify microbial virulence genes.We modified the IVET-transcriptional fusion vector,pIVET8,which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors,pIVET11 and pIVET12.Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H.pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase.Additionally,each vector contains a kanamycin resistance gene.We used a mouse macrophage cell line,RAW 264.7 and mice,as selective media to identify specific genes that H.pylori expresses in vivo.Gene expression studies were conducted by infecting RAW 264.7 cells with H.pylori.This was followed by real time polymerase chain reaction(PCR) analysis to determine the relative expression levels of in vivo induced genes.RESULTS:In this study,we have identified 31 in vivo induced(ivi) genes in the initial screens.These 31 genes belong to several functional gene families,including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs.Virulence factors,vacA and cagA,were found in this screen and are known to play important roles in H.pylori infection,colonization and pathogenesis.Their detection validates the efficacy of these screening systems.Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H.pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae.Transcription profiles of allivi genes were confirmed by real time PCR analysis of H.pylori RNA isolated from H.pylori infected RAW 264.7 macrophages.We compared the expression profile of H.pylori and RAW 264.7 coculture with that of H.pylori only.Some genes such as cag A,vac A,lpx C,mur I,tlp C,trx B,sod B,tnp B,pgi,rbf A and inf B showed a 2-20 fold upregulation.Statistically significant upregulation was obtained for all the above mentioned genes(P < 0.05).tlp C,cag A,vac A,sod B,rbf A,inf B,tnp B,lpx C and mur I were also significantly upregulated(P < 0.01).These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal.CONCLUSION:The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H.pylori gene expression in the host environment.