Design,delivery and efficacy testing of therapeutic nucleic acids used to inhibit hepatitis C virus gene expression in vitro and in vivo

Despite major achievements in the treatment ofchronic hepatitis C with the combination ofinterferons and the nucleoside analog ribavirin themajority of patients with chronic hepatitis C virus(HCV) infection cannot be treated effectively.Toimprove this response rate we used antisensetechnologies to inhibit HCV translation as possibleadditional option for experimental treatment.Antisense oligodeoxynucleotides(ODN) are

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.

Vascular damage and anti-angiogenic effects of tumor vessel-targeted adenovirus-mediated herpes simplex virus thymidine kinase gene

AIM： To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase （HSV-tk） targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS： Recombinant adenovirus containing kinase domain insert with receptor （KDR） or cytomegalovirus （CMV） promoter-controlled HSV-tk gene （AdKDR-tk and AdCMV-tk） was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells （HUVEC） and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir （GCV）, the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups： ganciclovir group （Ⅰ）, Ad group （Ⅱ）, AdCMV-tk group （Ⅲ） and AdKDR-tk group （Ⅳ）. Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir （GCV） was given at a dose of 100 mg·kg^-1·d^-1 （ip） started on the following day and lasted 10 d. Microvessel density （MVD） of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and alter the last GCV dose.RESULTS： Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk- transfected HUVEC and HepG2 decreased from 100% to （28.94 ± 5.67）% and （75.45 ± 2.91）% at proper order, respectively （P 〈 0.01）, while the survival rate of AdCMV- tk-transfected HUVEC and HepG2 declined from 100% to （17.56 ± 2.48）% and （23.15± 5.72）%, respectively （P 〉 0.05）. Compared with group I, there was a decrease of tumor weight by 14.7% in group Ⅲ and by 23.6% in group Ⅳ. And there was a distinct difference between group M and Ⅳ （P 〈 0.05）. The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 （microvessels/mm^2） in group Ⅰ, Ⅱ, M and IV, respectively. And there was a marked difference between group M and Ⅱ （P 〈 0.05）, Ⅳ and Ⅱ （P 〈 0.01）, and Ⅳ and M （P 〈 0.01）. CONCLUSION： KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.

Effect of hepatitis C virus infection on expression of several cancer-associated gene products in hepatocellular carcinoma

AIM To study hepatocarcinogenesis of hepatitis C virus (HCV). METHODS Expression of HCV antigens (CP10, NS3 and NS5) and several cancer associated gene products (ras p21, c myc, c erbB 2, mutated p53 and p16 protein) in the tissues of hepatocellular carcinoma (HCC, n =46) and its surrounding liver tissue were studied by the ABC (avidin biotin complex) immunohistochemical method. The effect of HCV infection on expression of those gene products in HCC was analyzed by comparing HCV antigen positive group with HCV antigen negative group. RESULTS Positive immunostaining with one, two or three HCV antigens was found in 20 (43 5%) cases, with either of two or three HCV antigens in 16 (34 8%) cases, and with three HCV antigens in 9 (19 6%) cases. Deletion rate of p16 protein expression in HCC with positive HCV antigen (80%, 16/20) was significantly higher than that in HCC with negative HCV antigen. Whereas no significant difference of the other gene product expression was observed between the two groups. CONCLUSION HCV appears related to about one third of cases of HCC in Chongqing, the southwest of China, and it may be involved in hepatocarcinogenesis by inhibiting the function of p16 gene, which acts as a negative regulator of cell cycle.

Cooperative Therapeutic Effects of Herpes Simplex Virus Thymidine Kinase Gene/Ganciclovir System and Chemotherapeutic Agents on Prostate Cancer in vitro

The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir （HSV-tk/GCV） approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer （HRPC） cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction （RT-PCR） and strept avidin-biotin complex （SABC） im- munohistochemical method. The killing effect of GCV, cisplatin （CDDP）, etoposide （VP-16）, vincristine （VCR）, methotrexate （MTX）, 5-fluorouracil （5-Fu）, and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry （FCM）. The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38 % and 35.51%, and the proportion of necrosis being 33.05 % and 28.87 %, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment. Our results highlight the potential for such new combination therapies for future treatments of HRPC.

GENE ENGINEERING EB VIRUS MEMBRANE ANTIGEN IN DETECTION OF MA-IgA ANTIBODY(COMPARISON WITH VCA-IgA AND EA-IgA ANTIBODIES)

With gene engineering EB virus membrane antigen as the diagnostic antigen, indirect immunofluo-rescence (IF) assay was used to detect IgA antibody against EB virus membrane antigen (MA-IgA) in sera from 202 nasopharyngeal carcinoma (NPC) patients and 315 controls (normal and patients with other tumors). MA-IgA antibody was positive in 96.8% of the pretreatment NPC patients with a GMT of 1:36.3. MA-IgA detection by this method was more sensitive than EA-IgA detection by IE. In contrast, patients with tumors other than NPC were negative for MA-IgA antibody. 9.1% of VCA-IgA positive persons were MA-IgA positive with a GMT of less than 1:5. No MA-IgA positive was found in VCA-IgA negatives. The results indicated that this method was relatively specific. In the treatment group, the positive rate and GMT of MA-IgA antibody declined with increase in survival time and the decline was faster than VCA-IgA. When recurrence or distant metastasis developed, similar to VCA-IgA and EA-IgA antibodies, the positive rate and GMT of MA-IgA antibody increased to its pretreatment level. Therefore, MA-IgA detection might be valuable in the early diagnosis and monitor of NPC.

Transcriptomic analyses reveal new genes and networks response to H5N1 influenza viruses in duck(Anas platyrhynchos)

H5N1 influenza represents one of the great challenges to public health.Some H5N1 viruses(i.e.,A/goose/Hubei/65/05,GS/65) are weakly pathogenic,while the others(i.e.,A/duck/Hubei/49/05,DK/49) are highly pathogenic to their natural hosts.Here,we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 virus.We demonstrated that,compared to the GS/65 virus,the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways(i.e.,RIG-I and MDA5) in ducks.We found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes,while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 virus.We also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis(WGCNA).Moreover,we first demonstrated that the duck BCL2 L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication efficiently.These analyses,in combination with our comprehensive transcriptomic data,provided global view of the molecular architecture for the interaction between host and H5N1 viruses.

Expression of Porcine Interleukin-2 and Porcine Interleukin-6 and Their Adjuvant Effects on Gene Deleted Vaccine of Pseudorabies Virus(TK^-/gG^-/LacZ^+)

Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene deleted vaccine of pseudorabies virus Ea strain(TK-/gG-/LacZ+)was mixed with the two different purified recombinant proteins each, or both, with the doses of 2, 5 or 10 μg ml-1. Ten groups of pseudorabies negative antibody swines were immuned twice with tested vaccines with different doses, or control vaccine, respectively. The antibody liters of the test groups were detected by neutralization test, and the daily weight gains of swines were calculated and analyzed statistically. In the study, all the neutralizing antibody ti-ters in test groups were higher than the control group, and the recombinant proteins appeared a dose dependent adjuvant effect. The tested vaccines with 2 μg ml-1 pIL-2 and with 10 μg ml-1 pIL-2/pIL-6 got significant and extremely significant differences, compared with the vaccines without pILs. The difference of the daily weight gain indicated the potential positive influence of pIL-2 and pIL-6 on immune protection.

CONSTRUCTION AND EXPRESSION OF ADENO-ASSOCIATED VIRUS-BASED PLASMID EXPRESSING VECTORS CONTAINING hIL-2 GENE OR mIFN-γ GENE

Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.

Variation and evolution of NP genes of human avian H_5N_1 virus strains

In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of strains were retrieved. The sequences were analyzed by DNAStar 5.0, and the evolutionary speed was studied with reference to the epidemiological data. It was found that NP genes of 45 strains during 1997-2006 were homologically classified into three groups： strains in 1997-1998, strains in 2004-2005 and strains from 2003 to 2006. There were 35 substitutions in NPs in all strains accounting for a ratio of 7.03% （35/498）. An additional glycoprotein domain （NGT430-432） was found in NP genes in the strains of 2003-2006, the mutation of N370S in GD-01-06 resulted in occurrence of one more glycoprotein domain （NES368-370）. In the synonymous variation, Ks values in NP were 2.03 × 10^-5-2.55 × 10^-5 Nt/d and K. values in NP were 1.58 × 10^-6-3.10 × 10^-6 Nt/d. There didn＇t exist obviously selective pressure. An additional glycoprotein domain in every strain of 2003-2006 and one more in strain GD-01-06 might change the antigenicity of human avian H5 N1 influenza virus. The variation on human avian H5 N1 influenza strains occurred frequently in the natural world, which would result in high probability of human-human transmission along with the natural evolution of the virus.

The stability of E protein gene of the Japanese encephalitis live attenuated vaccine virus SA_(14)-14-2

The purpose of this study was to understand the stability and possibility of back mutation of Japanese encephalitis （JE） attenuated vaccine virus strain SA14-14-2 HKs on molecular level. The E genes of the SA14-14-2 HKs vaccine virus and its PHK cells passaged virus （SA14-14-2 HK17 ）, its mouse brain passaged virus （SA14-14-2 SM1 ） were sequenced and compared with the E gene of parental SAI4 virus. The total RNA was extracted from infected Vero cells and amplified by RT-PCR. The RT-PCR products were purified and cloned into T-vector. Positive clones were screened, identified and sequenced. There were twelve nucleotides and eight amino acids substitutions between SA14 parent virus and SA14-34-2 PHKs vaccine virus. The SA14-14-2 PHK17 virus showed two additional mutations （E-331 and E-398） which were not back mutations. Although five additional mutations were found in SA14-34-2 SMt virus, only one （E-307） was back mutation. Genetic characteristics of the attenuated vaccine virus SA14- 34-2 were stable when it was passaged 37 times on PHK cells or one time in mouse brains.

THE INTRACELLULAR FORM OF EPSTEIN-BARR VIRUS GENOME IN NASOPHARYNGEAL CARCINOMA

This work was supported by the National Natural Science Foundation of China (No. 39370766). Object: To study the existent form of EBV genome in nasopharyngeal carcinoma (NPC) biopsies, in a trans planted NPC tumor SUNT 1 and its corresponding epithelial cell line SUNE 1. Methods: By using polymerase chain reaction (PCR) amplification of Epstein Barr virus (EBV) BamHI W fragment, EBV DNA was detected in 20/20 biopsy specimens of poorly differentiated, as well as in a nude mouse xenografted NPC tumor (SUNT 1, from passage 1 to 34) and in the corresponding epithelial cell line (SUNE 1, from passage 1 to 62). The intracellular form of EBV genome was studied by analyzing the terminal structure using a LMP2A probe and an “ in situ lysing gel” technique. Results: A single EBV fused terminal DNA fragment was detected in 19 biopsy specimens, two hybridized bands were seen in one specimen. These results indicate that an episomal form of EBV genome is predominantly present in most NPC biopsy specimens, but insertion of the genome into the host chromosome could not be excluded. Conclusion: The finding suggests that EBV infection precedes clonal amplification of transformed cells, or in a rare case, that a single EBV infected clone is predominant in the development of NPC. Linear form of EBV DNA was detected in the 20th passage of SUNE 1; this may imply the in vitro activation of the productive cycle of EBV.

Ambisense polarity of genome RNA of orthomyxoviruses and coronaviruses

Influenza viruses and coronaviruses have linear single-stranded RNA genomes with negative and positive sense polarities and genes encoded in viral genomes are expressed in these viruses as positive and negative genes,respectively.Here we consider a novel gene identified in viral genomes in opposite direction,as positive in influenza and negative in coronaviruses,suggesting an ambisense genome strategy for both virus families.Noteworthy,the identified novel genes colocolized in the same RNA regions of viral genomes,where the previously known opposite genes are encoded,a so-called ambisense stacking architecture of genes in virus genome.It seems likely,that ambisense gene stacking in influenza and coronavirus families significantly increases genetic potential and virus diversity to extend virus-host adaptation pathways in nature.These data imply that ambisense viruses may have a multivirion mechanism,like"a dark side of the Moon",allowing production of the heterogeneous population of virions expressed through positive and negative sense genome strategies.

The association between the genetic polymorphisms of LMP2/LMP7 and the outcomes of HCV infection among drug users

Objective：To investigate a possible association of LMP2/LMP7 genes with chronic hepatitis C virus（HCV） infection,and to assess whether LMP2/LMP7 genes could influence the outcomes of HCV infection among drug users.Methods：Genomic DNAs of 362 anti-HCV sero-positive drug users and 225 control drug users were extracted from the peripheral blood leukocytes.The sero-positive patients were divided into those who had persistent infection and those who had spontaneously cleared the infection.Polymorphisms of LMP genes were determined by PCR combined with restriction fragment length polymorphism（RFLP）.Results：The distribution of LMP2 genotypes among the control,persistent infection and spontaneous clearance groups were not different.However,the LMP7 codon 145 Gln/Lys,Lys/Lys,and Gln/Lys＋Lys/Lys genotypes were found significantly more frequent in the persistent infection group than in control group（OR=1.75,95%CI=1.06～2.90;OR=3.16,95%CI=1.23-8.12;OR=1.94,95%CI=1.21-3.12,respectively）.Similarly,the frequencies of the codon 145 Gln/Lys,Lys/Lys,and Gln/Lys＋Lys/Lys genotypes were found significantly more frequent in the persistent infection group than in the spontaneous clearance group（OR=1.64,95%CI=1.04-2.57;OR=2.40,95%CI=1.09-5.28;OR=1.76,95%CI=1.152.69,respectively）.Stratified analysis indicated that combined genotype Gln/Lys＋Lys/Lys of the LMP7 gene was related to an increasing susceptibility to HCV infection（OR=1.91,95%CI=1.02-3.55;OR=2.19,95%CI=1.243.89;OR=1.91,95%CI=1.05-3.48,OR=2.86,95%CI=1.41-5.78,respectively）and the risk of persistent HCV infection（OR=1.94,95%CI=1.12-3.34;OR=2.02,95%CI=1.21-3.38;OR=1.78,95%CI=1.12-2.85,OR=2.23,95%CI=1.09-4.58,respectively）among30-year-old,males,the injection drug user（IDU）subjects and/or the shorter duration drug users（≤5 y）.Conclusion：These results suggest that polymorphism of the LMP7 gene may have an influence on the outcomes of HCV infection,and is one of the factors accounting for the genetic susceptibility to HCV infection among drug users.

A TOM1 homologue is required for multiplication of Tobacco mosaic virus in Nicotiana benthamiana

The AtTOM1 gene of Arabidopsis thaliana had been shown to be essential for the efficient multiplication of Tobacco mosaic virus(TMV) in A.thaliana.In this study,we cloned an AtTOM1-like gene from Nicotiana benthamiana named as NbTOM1.Sequence alignment showed that NbTOM1 is closely related to AtTOM1 homologues of N.tabacum and Lycopersicon esculentum with 97.2% and 92.6% nucleotide sequence identities,respectively.Silencing of NbTOM1 by a modified viral satellite DNA-based vector resulted in complete inhibition of the multiplication of TMV in N.benthamiana.The result suggests that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants.

Entry of hepatitis C virus into the cell: A therapeutic target

Several receptors have been identified as implicated on viral entry into the hepatocyte; and, this interaction between the virus and potential receptors could modulate infection, spontaneous viral clearance, persistence of the infection and the widespread of the virus as outbreak. Nevertheless, the playing role of each of them remains controversial. The NiemannPick type C1 like 1 gene (NPC1L1) receptor has been recently implicated on hepatitis C virus (HCV) entry into the cell and ezetimibe, an anti-cholesterol drug seems to block that, emerging the idea to control hepatitis C outbreak modulating lipid-related receptors. Hepatitis C infection seems to modulate lipid metabolism according to host genetic background. Indeed, it circulates like a lipoviroparticle. The main aim of this field of vision would be to discuss the role of hepatocyte receptors implicated on virus entry, especially NPC1L1 and the therapeutic options derived from the better knowledge about HCV-lipidsreceptors interaction.

Study on PRRSV Receptor Genes Differential Expression in Lung Tissues in Different Breeds of Pigs after Infecting with HP-PRRSV

[ Objective] In order to study the susceptibility molecular mechanism of highly pathogenic porcine reproductive and respiratory syndrome virus （ HP- PRRSV） JXA1 isolate on Tibetan pig, Zangmei pig and Yorkshire pig. [ Method ] In the study, real-time quantitative RT-PCR method was established to compare and analyze the differential expression of five porcine reproductive and respiratory syndrome virus （PRRSV） receptor genes （HSPG2, SIGLEC1, CD163, VIM and NMMHC-H A） in lung tissues in Tibetan pig, Zangmei pig and Yorkshire pig before the challenge and at the 4th ,7th and 14th days after the challenge with JXAI isolate. [ Results ] HSPG2 expression in Tibetan pig lung tissues increased significantly at the 4th and 14th days after the challenge with JXAI （ P 〈 0.05 ）, while decreased significantly at the 7th day after the challenge （P 〈 0.05 ）, HSPG2 expression in Zangmei pig lung tissues increased significantly at the 14th day after the challenge （P〈0.05）. SIGLECl expression in Tibetan pig lung tissues increased significantly at the 4th and 14th days after the infection（P 〈 0.05 ）, while SIGLEC 1 expression in Yorkshire pig decreased significantly at the 4th, 7th and 14th days after the challenge （P 〈0. 05 ）. CD163 expression in lung tissues of Tibetan pig and Zangmei pig both increased significantly at the 14th day after the challenge （P 〈 0.05 ）, while CD163 expression in lung tissues of Yorkshire pig decreased significantly at the 7th and 14th days after the challenge （ P 〈 0. 05 ）. VIM expression in lung tissues of Tibetan pig increased significantly at the 7th day after the challenge （ P 〈 0. 05 ）, while which of Yorkshire pig at the 7th day after the challenge decreased significantly （ P 〈 0. 05 ）. NMMHC-II A expression in lung tissues of Zangmei pig increased significantly at the 4th day after the challenge （ P 〈 0. 05 ）, and which of Yorkshire pig increased significantly at the 4th and 14th days after the challenge （P 〈 0. 05 ）. [ Conclusion] SIGLEC1 and VIM genes might be the potential key genes affecting the susceptibility of JXA1 isolate on Tibetan pig, Zangrnei pig and Yorkshire pig. Key words JXA1 isolate; Tibetan pig; Zangmei pig; Yorkshire pig; Porcine reproductive and respiratory syndrome virus receptor genes; Differential expression

Effects of herpes simplex virus thymidine kinase/acyclovir system on growth of human pulmonary adenocarcinoma A549 cell line in vitro and in vivo

Objective: To observe the effect of anciclovir (ACV) treatment on tumors induced by inoculation of TK gene-transfected human pulmonary adenocarcinoma A549 cells in nude mice. Methods: A recombinant plasmid containing TK gene was constructed and transfected into A549 cells by electroporation. The sensitivity of the transgenic cells (A549-TK) to ACV was examined by MTT assay in vitro and for in vivo observation, inoculation of A549-TK and A-549 cells into nude mice was separately performed to induce tumor growth, the response of which to ACV treatment was observed, and the tumor tissues were pathologically examined. Results: A recombinant plasmid containing TK gene was successfully constructed and transfected into A549 cells. The sensitivity of A549-TK cells to ACV was 43 times higher than that of A549 cells. The tumors induced by A549-TK cells showed no significant increase in size after ACV treatment (P>0. 05) , and light microscopy revealed local tissue necrosis, karyoklasis, and nuclei disappearance. Conclusion: A549-TK cells acquires sensitivity to ACV both in vitro and in vivo, and ACV can inhibit the growth of tumors induced by A549-TK cell inoculation in nude mice.

Adeno-associated virus mediated apoA-I and apoA-IMilano expression in skeletal muscular cells

Objective： To construct recombinant adeno-associated virus type 2（rAAV2） vectors encoding human apoA-I/apoA-lMilano protein, and explore an effective and safe method to prevent and treat the atherosclerotic diseases. Methods： Human apoA-I cDNA with a His-tag in the upward stream of cDNA sequence were obtained with RT-PCR and PCR, and human apoA-IMilano cDNA was prepared by QuikChange Site-Directed Mutagenesis Kit. After extracted rAAV vectors with a most economic and convenient method, the particle numbers of rAAV vectors were measured by Dot-blot, and the purity was assayed by SOS-Page. The expression efficiency of the apoA-I or apoA-IMilano in C2C12 infected by rAAV vectors were detected by ELISA method. Results： ApoA-I cDNA was gained by RT-PCR and a His-tag was added in the upward stream of apoA-I cDNA successfully. ApoA-I cDNA was mutanted to apoA-IMilano cDNA successfully by QuikChange Site-Directed Mutagenesis Kit. The both titres of the rAAV vectors of apoA-I and apoA-IMilano were about 2×10^14/L, and the result of SOS-Page showed that the purity of the rAAV vectors was satisfied. The expression level of apoA-I was （0.39±0.04） μg/ml and the apoA-IMilano was （0.31±0.03） μ/ml in the DMEM culture medium at the first 24h after transfection. Conclusion： The success of the rAAV vectors construction, purification and the expression of apoA-I and apoA-IMilano in C2C12 cells mediated by these vectors, makes possible to inject rAAVA and rAAVAM vectors into mice muscle, and rises a new hope on finding a new way to prevent and treat atherosclerotic diseases and cardiovascular disease.