[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on...[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a （ ＋ ） with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.展开更多
The synonymous codon usage in the translational initiation and termination regions of genes of severe acute respiratory syndrome (SARS) coronavirus and five other viruses in Coronaviridae was systematically analyzed.T...The synonymous codon usage in the translational initiation and termination regions of genes of severe acute respiratory syndrome (SARS) coronavirus and five other viruses in Coronaviridae was systematically analyzed.The results indicate that most minor codons for these coronaviruses are preferentially used in the initial and terminal region.The minor codons preferentially used in the initial region are thought to have a negative effect on gene expression,which can be explained by the minor codon modulator hypothesis.It also indicates that the minor codons preferentially used in the terminal region may regulate the level of gene expression.The proposed results strongly imply that the minor codon modulator hypothesis can be applied to both some bacteria and some viruses.展开更多
Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ dig...Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.展开更多
Emerging data indicated that HCV subverts the antiviral activity of interferon(IFN);however,whether HCV core protein contributes to the process remains controversial.In the present study,we examined the effect of HCV ...Emerging data indicated that HCV subverts the antiviral activity of interferon(IFN);however,whether HCV core protein contributes to the process remains controversial.In the present study,we examined the effect of HCV core protein on interferon-induced antiviral gene expression and whether the effect is involved in the activation and negative regulation of the Jak/STAT signaling pathway.Our results showed that,following treatment with IFN-α,the transcription of PKR,MxA and 2'-5'OAS were down-regulated in HepG2 cells expressing the core protein.In the presence of HCV core protein,ISRE-dependent luciferase activity also decreased.Further study indicated that the core protein could inhibit the tyrosine phosphorylation of STAT1,whereas the level of STAT1 expression was unchanged.Accordingly,SOCS3,the negative regulator of the Jak/STAT pathway,was induced by HCV core protein.These results suggests that HCV core protein may interfere with the expression of some interferon-induced antiviral genes by inhibiting STAT1 phosphorylation and induction of SOCS3.展开更多
Objectives: In order to investigate the role of mono-cytes and human leukocyte antigen (HLA) class II an-tigen in herpes simplex virus-2 (HSV-2) infection, westudied the effect of HSV-2 infection in vitro on theexpres...Objectives: In order to investigate the role of mono-cytes and human leukocyte antigen (HLA) class II an-tigen in herpes simplex virus-2 (HSV-2) infection, westudied the effect of HSV-2 infection in vitro on theexpression of HLA class II antigen on monocytes.Methods: Monocytes were infected with HSV-2(Strain 333). Culture cells were collected 1, 3, 5 and 7days after infection. The levels of expression of HLAclass II antigen were measured by using alkaline phos-phatase antialkaline phosphatase method (APAAP).Results: The levels of the expression of HLA class IIantigen on monocytes significantly decreased on thefirst day of post-infection, and then gradually returnedto levels found in the controls by the 7th day post-infection.Conclusion: HLA class II antigen expression onmonocytes was inhibited with HSV-2 infection, whichmight be one means of virus escape at an early phase.The expression of HLA class II antigen may play animportant role in herpes simplex viurs-2 pathogenic-ity and immunity.展开更多
A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,elec...A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.展开更多
AIM： To assess the associations of human leukocyte antigen （HI_A） class Ⅱ DQB1＊0301 and/or DRB1＊1101 allele with spontaneous hepatitis C virus （HCV） clearance by meta-analysis of individual dataset from all st...AIM： To assess the associations of human leukocyte antigen （HI_A） class Ⅱ DQB1＊0301 and/or DRB1＊1101 allele with spontaneous hepatitis C virus （HCV） clearance by meta-analysis of individual dataset from all studies published till date. METHODS： To clarify the impact of HLA class Ⅱ polymorphisms on viral clearance, we performed a metaanalysis of the published data from 11 studies comparing the frequencies of DQB1＊0301 and DRB1＊1101 alleles in individuals with spontaneous resolution to those with persistent infection. As we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. RESULTS： Meta-analyses yielded summary estimatesodds ratio （OR） of 2.36 [95%CI （1.62, 3.43）, P〈0.00001] and 2.02 [95%CI （1.56, 2.62）, P〈0.00001] for the effects of DQB1＊0301 and DRB1＊1101 alleles on spontaneous clearance of HCV, respectively. CONCLUSION： These results support the hypothesis that specific HLA class Ⅱ alleles might influence the susceptibility or resistance to persistent HCV infection. Both DQB1＊0301 and DRB1＊1101 are protective alleles and present HCV epitopes more effectively to CD4^＋T lymphocytes than others, and subjects with these two alleles are at a lower risk of developing chronic HCV infection. Large, multi-ethnic confirmatory and welldesigned studies are needed to determine the host genetic determinants of HCV infection.展开更多
AIM： To elucidate the role of the peroxisome proliferator-activated receptor α （PPARα） and its target gene camitine palmitoyl acyl-CoA transferase 2A （CPT1A） in the pathogenesis of hepatitis C virus （HCV） inf...AIM： To elucidate the role of the peroxisome proliferator-activated receptor α （PPARα） and its target gene camitine palmitoyl acyl-CoA transferase 2A （CPT1A） in the pathogenesis of hepatitis C virus （HCV） infection.METHODS： Liver samples were collected from the patients with chronic HCV infection and controls. HepG2 cells were transfected with vector pEF352neo carrying. Two independent clones （clone N3 and N4） stably expressing HCV core protein were analyzed. Total RNA was extracted from cells and liver tissues. PPARα and CPT1A mRNAs were quantified by real-time polymerase chain reaction （PCR） using SYBR Green Master. Total extracted proteins were separated by polyacrylamide gel electrophoresis, and electroblotted. Membranes were incubated with the anti-PPARα antibody, then with a swine anti-rabbit IgG conjugated to horseradish peroxidase for PPARα. Protein bands were revealed by an enhanced chemiluminescence reaction for PPARα. For immunohistochemical staining of PPARα, sections were incubated with the primary goat polyclonal antibody directed against PPARα at room temperature.RESULTS： Real-time PCR indicated that the PPARα level and expression level of CPT1A gene in hepatitis C patients lowered significantly as compared with the controls （1.8±2.8 vs 13±3.4, P = 0.0002; 1.1±1.5 vs 7.4±1, P = 0.004）. Western blot results showed that the level of PPARα protein in the livers of hepatitis C patients was lower than that in controls （2.3±0.3 vs 3.6±0.2, P = 0.009）. The immunohistochemical staining results in chronic hepatitis C patients indicated a decrease in PPARα staining in hepatocytes compared with those in the control livers. The in vitro studies found that in the N3 and N4 colon stably expressing HCV core protein, the PPARα mRNA levels were significantly lower than that in the controls.CONCLUSION： The impaired intrahepatic PPARα expression is associated with the pathogenic mechanism in hepatic injury during chronic HCV infection. HCV infection reduced the expression of PPARα and CPT1A at the level of not only mRNAs but also proteins. PPARα plays an important role in the pathogenesis of chronic HCV infection, but the impaired function of this nuclear receptor in HCV infection needs further studies.展开更多
AIM: To evaluate the possible value of FasL in gastric cancer gene therapy by investigating the effects of FasL expression on human gastric cancer cell line. METHODS: An adenoviral vector encoding the full-length huma...AIM: To evaluate the possible value of FasL in gastric cancer gene therapy by investigating the effects of FasL expression on human gastric cancer cell line. METHODS: An adenoviral vector encoding the full-length human FasL cDNA was constructed and used to infect a human gastric cancer (SGC-7901) cell line. FasL expression was confirmed by X-gal staining, flow cytometric analysis and RT-PCR. The effect of FasL on cell proliferation was determined by clonogenic assay, cytotoxicity was detected by MTT assay, and cell viability was measured by trypan blue exclusion. The therapeutic efficiency of Ad-FasL in vivo was investigated with a xenograft tumor model in nude mice. RESULTS: SGC-7901 cells infected with Ad-FasL showed increased expression of FasL, resulting in significantly decreased cell growth and colony-forming activity when compared with control adenovirus-infected cells. The cytotoxicity of anti-Fas antibody (CH-11) in gastric cancer cells was stronger than that of ActD (91±8 vs60±5,P<0.01), and the cytotoxicity of Ad-FasL was stronger than that of CH-11 (60±5 vs50±2, P<0.05). In addition, G1-phase arrest (67.75±0.39 vs 58.03±2.16, P<0.05) and apoptosis were observed in Ad-FasL-infected SGC-7901 cells, and the growth of SGC-7901 xenografts in nude mice was retarded after intra-tumoral injection with Ad-FasL (54% vs 0%,P<0.0001). CONCLUSION: Infection of human gastric carcinoma cells with Ad-FasL induces apoptosis, indicating that this target gene might be of potential value in gene therapy for gastric cancer.展开更多
AIM： To examine the role of E-cadherin and betacatenin in carcinogenesis and to assess their prognostic implication in Epstein-Barr virus-associated gastric carcinomas （EBV-GCs）. METHODS： We compared the frequency...AIM： To examine the role of E-cadherin and betacatenin in carcinogenesis and to assess their prognostic implication in Epstein-Barr virus-associated gastric carcinomas （EBV-GCs）. METHODS： We compared the frequency of E-cadherin and beta-catenin expression in 59 EBV-GCs and 120 non-EBV-GCs, and examined the association between patients＇ prognosis and the expressions of these proteins. RESULTS： Neither the cellular-membranous nor the cytoplasmic E-cadherin expression showed any difference between EBV-GCs and non-EBV-GCs. On the other hand, loss of membranous expression of beta- catenin occurred more frequently in non-EBV-GCs than EBV-GCs [odds ratio = 0.41; 950 confidence interval （CI）, 0.19-0.90]. Furthermore, the nuclear and/or cytoplosmic expression of beta-catenin was seen more frequently in EBV-GCs than non-EBV-GCs （odds ratio = 2.23; 95% CI, 0.97-5.09）, and was observed in a larger proportion of carcinoma cells of EBV-GCs than non-EBV-GCs （P = 0.024）. Survival analysis for non-EBV-GC revealed that lymph node metastasis was significantly associated with poor prognosis （P 〈 0.001）. Among EBV- GCs, the depth of invasion （P = 0.005）, lymph node metastasis （P = 0.004） and an intestinal type by Lauren classification （hazard ratio = 9.47; 95% CI, 2.67-33.6） were significantly associated with poor prognosis. On the other hand, nuclear and/or cytoplasmic expression of beta-catenin was associated with a better prognosis in patients with EBV-GC （hazard ratio = 0.32; 95% CI, 0.11-0.93）. CONCLUSION： We observed more frequent preservation of beta-catenin in cell membrane and accumulation in nuclei and/or cytoplasm in EBV-GCs than in non-EBV- GCs. Factors involved in the prognosis of EBV-GCs and non-EBV-GCs are different in the two conditions.展开更多
基金Supported by the Natural Science Foundation Program of Shandong Province(2007ZRA16001)~~
文摘[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus（PPV）. [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a （ ＋ ） with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.
文摘The synonymous codon usage in the translational initiation and termination regions of genes of severe acute respiratory syndrome (SARS) coronavirus and five other viruses in Coronaviridae was systematically analyzed.The results indicate that most minor codons for these coronaviruses are preferentially used in the initial and terminal region.The minor codons preferentially used in the initial region are thought to have a negative effect on gene expression,which can be explained by the minor codon modulator hypothesis.It also indicates that the minor codons preferentially used in the terminal region may regulate the level of gene expression.The proposed results strongly imply that the minor codon modulator hypothesis can be applied to both some bacteria and some viruses.
基金National Natural Science Foundation of China(30100160,30271179)
文摘Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.
文摘Emerging data indicated that HCV subverts the antiviral activity of interferon(IFN);however,whether HCV core protein contributes to the process remains controversial.In the present study,we examined the effect of HCV core protein on interferon-induced antiviral gene expression and whether the effect is involved in the activation and negative regulation of the Jak/STAT signaling pathway.Our results showed that,following treatment with IFN-α,the transcription of PKR,MxA and 2'-5'OAS were down-regulated in HepG2 cells expressing the core protein.In the presence of HCV core protein,ISRE-dependent luciferase activity also decreased.Further study indicated that the core protein could inhibit the tyrosine phosphorylation of STAT1,whereas the level of STAT1 expression was unchanged.Accordingly,SOCS3,the negative regulator of the Jak/STAT pathway,was induced by HCV core protein.These results suggests that HCV core protein may interfere with the expression of some interferon-induced antiviral genes by inhibiting STAT1 phosphorylation and induction of SOCS3.
文摘Objectives: In order to investigate the role of mono-cytes and human leukocyte antigen (HLA) class II an-tigen in herpes simplex virus-2 (HSV-2) infection, westudied the effect of HSV-2 infection in vitro on theexpression of HLA class II antigen on monocytes.Methods: Monocytes were infected with HSV-2(Strain 333). Culture cells were collected 1, 3, 5 and 7days after infection. The levels of expression of HLAclass II antigen were measured by using alkaline phos-phatase antialkaline phosphatase method (APAAP).Results: The levels of the expression of HLA class IIantigen on monocytes significantly decreased on thefirst day of post-infection, and then gradually returnedto levels found in the controls by the 7th day post-infection.Conclusion: HLA class II antigen expression onmonocytes was inhibited with HSV-2 infection, whichmight be one means of virus escape at an early phase.The expression of HLA class II antigen may play animportant role in herpes simplex viurs-2 pathogenic-ity and immunity.
文摘A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.
基金Supported by the National Natural Science Foundation of China,No. 30200232
文摘AIM： To assess the associations of human leukocyte antigen （HI_A） class Ⅱ DQB1＊0301 and/or DRB1＊1101 allele with spontaneous hepatitis C virus （HCV） clearance by meta-analysis of individual dataset from all studies published till date. METHODS： To clarify the impact of HLA class Ⅱ polymorphisms on viral clearance, we performed a metaanalysis of the published data from 11 studies comparing the frequencies of DQB1＊0301 and DRB1＊1101 alleles in individuals with spontaneous resolution to those with persistent infection. As we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. RESULTS： Meta-analyses yielded summary estimatesodds ratio （OR） of 2.36 [95%CI （1.62, 3.43）, P〈0.00001] and 2.02 [95%CI （1.56, 2.62）, P〈0.00001] for the effects of DQB1＊0301 and DRB1＊1101 alleles on spontaneous clearance of HCV, respectively. CONCLUSION： These results support the hypothesis that specific HLA class Ⅱ alleles might influence the susceptibility or resistance to persistent HCV infection. Both DQB1＊0301 and DRB1＊1101 are protective alleles and present HCV epitopes more effectively to CD4^＋T lymphocytes than others, and subjects with these two alleles are at a lower risk of developing chronic HCV infection. Large, multi-ethnic confirmatory and welldesigned studies are needed to determine the host genetic determinants of HCV infection.
基金Supported by the National Natural Science Foundation of China,No.30300458
文摘AIM： To elucidate the role of the peroxisome proliferator-activated receptor α （PPARα） and its target gene camitine palmitoyl acyl-CoA transferase 2A （CPT1A） in the pathogenesis of hepatitis C virus （HCV） infection.METHODS： Liver samples were collected from the patients with chronic HCV infection and controls. HepG2 cells were transfected with vector pEF352neo carrying. Two independent clones （clone N3 and N4） stably expressing HCV core protein were analyzed. Total RNA was extracted from cells and liver tissues. PPARα and CPT1A mRNAs were quantified by real-time polymerase chain reaction （PCR） using SYBR Green Master. Total extracted proteins were separated by polyacrylamide gel electrophoresis, and electroblotted. Membranes were incubated with the anti-PPARα antibody, then with a swine anti-rabbit IgG conjugated to horseradish peroxidase for PPARα. Protein bands were revealed by an enhanced chemiluminescence reaction for PPARα. For immunohistochemical staining of PPARα, sections were incubated with the primary goat polyclonal antibody directed against PPARα at room temperature.RESULTS： Real-time PCR indicated that the PPARα level and expression level of CPT1A gene in hepatitis C patients lowered significantly as compared with the controls （1.8±2.8 vs 13±3.4, P = 0.0002; 1.1±1.5 vs 7.4±1, P = 0.004）. Western blot results showed that the level of PPARα protein in the livers of hepatitis C patients was lower than that in controls （2.3±0.3 vs 3.6±0.2, P = 0.009）. The immunohistochemical staining results in chronic hepatitis C patients indicated a decrease in PPARα staining in hepatocytes compared with those in the control livers. The in vitro studies found that in the N3 and N4 colon stably expressing HCV core protein, the PPARα mRNA levels were significantly lower than that in the controls.CONCLUSION： The impaired intrahepatic PPARα expression is associated with the pathogenic mechanism in hepatic injury during chronic HCV infection. HCV infection reduced the expression of PPARα and CPT1A at the level of not only mRNAs but also proteins. PPARα plays an important role in the pathogenesis of chronic HCV infection, but the impaired function of this nuclear receptor in HCV infection needs further studies.
基金Supported by the Medical Scientific Foundation of Jiangsu Province, No. H200147
文摘AIM: To evaluate the possible value of FasL in gastric cancer gene therapy by investigating the effects of FasL expression on human gastric cancer cell line. METHODS: An adenoviral vector encoding the full-length human FasL cDNA was constructed and used to infect a human gastric cancer (SGC-7901) cell line. FasL expression was confirmed by X-gal staining, flow cytometric analysis and RT-PCR. The effect of FasL on cell proliferation was determined by clonogenic assay, cytotoxicity was detected by MTT assay, and cell viability was measured by trypan blue exclusion. The therapeutic efficiency of Ad-FasL in vivo was investigated with a xenograft tumor model in nude mice. RESULTS: SGC-7901 cells infected with Ad-FasL showed increased expression of FasL, resulting in significantly decreased cell growth and colony-forming activity when compared with control adenovirus-infected cells. The cytotoxicity of anti-Fas antibody (CH-11) in gastric cancer cells was stronger than that of ActD (91±8 vs60±5,P<0.01), and the cytotoxicity of Ad-FasL was stronger than that of CH-11 (60±5 vs50±2, P<0.05). In addition, G1-phase arrest (67.75±0.39 vs 58.03±2.16, P<0.05) and apoptosis were observed in Ad-FasL-infected SGC-7901 cells, and the growth of SGC-7901 xenografts in nude mice was retarded after intra-tumoral injection with Ad-FasL (54% vs 0%,P<0.0001). CONCLUSION: Infection of human gastric carcinoma cells with Ad-FasL induces apoptosis, indicating that this target gene might be of potential value in gene therapy for gastric cancer.
基金Supported by Grants-in-Aid for Scientific Research on Priority Areas (12218231, 17015037, and 18014024) of the Ministry of Education, Culture, Sports, Science and Technology, Japan
文摘AIM： To examine the role of E-cadherin and betacatenin in carcinogenesis and to assess their prognostic implication in Epstein-Barr virus-associated gastric carcinomas （EBV-GCs）. METHODS： We compared the frequency of E-cadherin and beta-catenin expression in 59 EBV-GCs and 120 non-EBV-GCs, and examined the association between patients＇ prognosis and the expressions of these proteins. RESULTS： Neither the cellular-membranous nor the cytoplasmic E-cadherin expression showed any difference between EBV-GCs and non-EBV-GCs. On the other hand, loss of membranous expression of beta- catenin occurred more frequently in non-EBV-GCs than EBV-GCs [odds ratio = 0.41; 950 confidence interval （CI）, 0.19-0.90]. Furthermore, the nuclear and/or cytoplosmic expression of beta-catenin was seen more frequently in EBV-GCs than non-EBV-GCs （odds ratio = 2.23; 95% CI, 0.97-5.09）, and was observed in a larger proportion of carcinoma cells of EBV-GCs than non-EBV-GCs （P = 0.024）. Survival analysis for non-EBV-GC revealed that lymph node metastasis was significantly associated with poor prognosis （P 〈 0.001）. Among EBV- GCs, the depth of invasion （P = 0.005）, lymph node metastasis （P = 0.004） and an intestinal type by Lauren classification （hazard ratio = 9.47; 95% CI, 2.67-33.6） were significantly associated with poor prognosis. On the other hand, nuclear and/or cytoplasmic expression of beta-catenin was associated with a better prognosis in patients with EBV-GC （hazard ratio = 0.32; 95% CI, 0.11-0.93）. CONCLUSION： We observed more frequent preservation of beta-catenin in cell membrane and accumulation in nuclei and/or cytoplasm in EBV-GCs than in non-EBV- GCs. Factors involved in the prognosis of EBV-GCs and non-EBV-GCs are different in the two conditions.
