Preterminal protein,the achilles heel of adenoviridae:Implications for adenoviral infections

BACKGROUND Adenoviruses pose a serious health risk particularly in the absence of any clinically approved treatment.As adenoviral infections are quite frequent and recent outbreaks manifest more virulent variant strains,the need to develop an effective treatment remains a priority.The adenoviral protein,preterminal protein(pTP),is one of the key common products of the viral lifecycle as it is necessary to initiate viral replication and hence the infection process.This makes pTP a potential chemotherapeutic target in the search for and development of an effective treatment for adenoviral induced infections.Here we report,for the first time,that glycosylation of pTP in situ prevents binding to ssDNA in vitro.AIM To explore whether specific structural tailoring of the adenoviral protein pTP,imparts the potential to scupper the viral replication process.METHODS All chemicals used were of reagent grade.Overexpression of pTP was achieved using the‘BAC to BAC’expression system.The presence and relative concentration of the protein was determined throughout the incubation period by the Bradford assay.The pTP was identified by MALDI-TOFF and sodium dodecyl sulphate polyacrylamide gel electrophoresis.For the removal of the aminosugar,a deglycosylase enzyme kit from PROZYME was used.Purification of cloned pTP(6xHis)was done with a ssDNA cellulose column followed by a Ni-NTA column.His-tags were excised with the Tobacco etch virus protease.Protein fractionation was performed with a fraction collector coupled to a UV detector(280 nm)from Pharmacia.RESULTS The pTP overexpressed in insect cells(Spodoptera frugiperda)(>96 hours),is unable to bind to ssDNA in vitro.Treatment of this unbound protein with a deglycosidase enzyme that is specific for the removal of truncated unsubstituted O-linked Galβ(1-3)GalNAc-α1 disaccharides bound to Thr or Ser in a glycoprotein,restores binding to ssDNA.Data is presented as a linegraph for both the glycosylated and the deglycosylated proteins.Each point represents the mean of triplicate experiments(from different batches).Means and standard deviation were calculated and plotted on a line graph(with error bars).CONCLUSION The finding that glycosylation of cloned pTP in situ prevents binding to ssDNA in vitro could aid in the development of an effective treatment of adenoviral infections and/or as an adjunct to complement other antiadenoviral chemotherapeutic strategies.

Suppression of human colon tumor growth by adenoviral vector-mediated NK4 expression in an athymic mouse model

AIM： To investigate the suppressive effects of adenoviral vector-mediated expression of NK4, an antagonist of hepatocyte growth factor （HGF）, on human colon cancer in an athymic mouse model to explore the possibility of applying NK4 to cancer gene therapy. METHODS： A human colon tumor model was developed by subcutaneous implantation of tumor tissue formed by LS174T cells grown in athymic mice. Fifteen tumorbearing mice were randomized into three groups （n= 5 in each group） at d 3 after tumor implantation and mice were injected intratumorally with phosphate-buffered saline （PBS） or with recombinant adenovirus expressing 13-galactosidase （Ad-LacZ） or NK4 （rvAdCMV/NK4） at a 6-d interval for total 5 injections in each mouse. Tumor sizes were measured during treatment to draw a tumor growth curve. At d 26 after the first treatment, all animals were sacrificed and the tumors were removed to immunohistochemically examine proliferating cell nuclear antigen （PCNA）, microvessel density （represented by CD31）, and apoptotic cells. In a separate experiment, 15 additional athymic mice were employed to develop a tumor metastasis model by intraperitoneal injection （ip） of LS174T cells. These mice were randomized into 3 groups （n = 5 in each group） at d 1 after injection and were treated by ip injection of PBS, or Ad-LacZ, or rvAdCMV/NK4 at a 6-d interval for total two injections in each mouse. All animals were sacrificed at d 14 and the numbers and weights of disseminated tumors within the abdominal cavity were measured. RESULTS： Growth of significantly suppressed human colon tumors were in the athymic mice treatedwith rvAdCMV/NK4 （2537.4± 892.3 mm^3） compared to those treated by either PBS （5175.2 ± 1228.6 mm^3） or Ad-LacZ （5578.8± 1955.7 mm^3） （P 〈 0.05）. The tumor growth inhibition rate was as high as 51%. Immunohistochemical staining revealed a similar PCNA labeling index （75.1% ± 11.2% in PBS group vs 72.8% ± 7.6% in Ad-LacZ group vs 69.3% ± 9.4% in rvAdCMV/ NK4 group） in all groups, but significantly lower microvessel density （10.7 ± 2.4 in rvAdCMV/NK4 group vs 25.6 ± 3.8 in PBS group or 21.3 ± 3.5 in Ad-LacZ group, P 〈 0.05）, and a markedly higher apoptotic index （7.3% ± 2.4% in rvAdCMV/NK4 group vs 2.6 4, 1.1% in PBS group or 2.1% ± 1.5% in Ad-LacZ group, P 〈 0.05） in the rvAdCMV/NK4 group compared to the two control groups. In the tumor metastasis model, the number and weight of disseminated tumors of mice treated with rvAdCMV/NK4 were much lower than those of the control groups （tumor number： 6.2 ± 3.3 in rvAdCMV/ NK4 group vs 22.9 ± 7.6 in PBS group or 19.8 ± 8.5 in Ad-LacZ group, P 〈 0.05; tumor weight： 324 ± 176 mg in rvAdCMV/NK4 group vs 962 ± 382 mg in PBS group or 1116 ± 484 mg in Ad-LacZ group, P 〈 0.05）. CONCLUSION： The recombinant adenovirus, rvAdCMV/ NK4, can attenuate the growth of colon cancer in vivo, probably by suppressing angiogenesis and inducing tumor cell apoptosis, but not by direct suppression of tumor cell proliferation. Moreover, rvAdCMV/NK4 may inhibit peritoneal dissemination of colon cancer cells in a murine tumor metastasis model. These findings indicate that NK4 gene transfer may be an effective tool for the treatment of colon cancer.

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or aftermalignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper wedemonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression inairway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targetinghuman IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β orheat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reducedby shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels ofIκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for thetreatment of inflammatory diseases.

Overexpression of the mTERT gene by adenoviral vectors promotes the proliferation of neuronal stem cells in vitro and stimulates neurogenesis in the hippocampus of mice

We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase（mTERT）,as well as detect its expression and effect on the proliferation of neuronal stem cells.mTERT was am-plified by RT-PCR and then the eukaryotic expression vector of pDC-EGFP-TERT was constructed.After DNA sequence analysis,we detected that there were 293 cells transfected with pDC-EGFP-TERT and helper adenovirus plasmid pBHG lox ΔE1,and three Cre using Lipofectamine 2000 mediation,named Ad-mTERT-GFP,to pack-age adenoviral particles.The Ad-mTERT-GFP was used to infect neuronal stem cells and then the expression and activity of mTERT were detected.In addition,Bromodeoxyuridine labeling test identified the impact of mTERT overexpression on proliferation of neuronal stem cells.The recombinant adenoviral vector confirmed that mTERT was successfully constructed.Overexpression of mTERT stimulated the proliferation of neuronal stem cells both in vitro and in vivo.mTERT overexpression via adenoviral vector carrying mTERT cDNA upregulated the ability of proliferation in neuronal stem cells.

Effects of adenoviral-mediated gene transduction of NK4 on proliferation, movement, and invasion of human colonic LS174T cancer cells in vitro

AIM： To investigate the inhibitory effects of a recombinant adenovirus vector that expresses NK4, a truncated form of human hepatocyte growth factor （HGF）, on human colonic adenocarcinoma cells in vitro to establish a basis for future NK4 gene cancer therapy. METHODS： Cells from the LS174T human colonic adenocarcinoma cell line were infected with recombinant adenovirus rvAdCMV/NK4 and the effects of the manipulation on tumor cell proliferation, scatter, migration, and basement membrane invasion were assessed. Cells infected with a recombinant adenovirus vector （Ad-LacZ） expressing β-galactosidase served as the controls. RESULTS： We found that rvAdCMV/NK4 expression attenuated HGF-induced tumor cell scatter, migration, and basement membrane invasion （P 〈 0.05）, but did not inhibit tumor cell proliferation. CONCLUSION： HGF-induced LS174T tumor cell scatter, migration, and invasion can be antagonized by the recombinant NK4-expressing adenovirus.

Chinese and western medicine research progress in child adenoviral pneumonia

Adenoviral pneumonia is one of the more serious types of community-acquired pneumonia in children.It easily develops into severe pneumonia with many extrapulmonary complications and a high mortality rate.However,there is still no effective treatment for adenovirus.Therapeutic drugs are conducive to clinical treatment.Traditional Chinese medicine often achieves satisfactory results through staged syndrome differentiation.Currently,heat,sputum,stasis,and poison are considered to be the main pathological products.Fever,phlegm,and stasis are present in its development.Analyze the progress of traditional Chinese and western medicine in children's adenoviral pneumonia,and have a clearer understanding of the law of syndrome differentiation and its prescription medication,providing ideas for clinical treatment of adenoviral pneumonia.

Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

BACKGROUND： We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter- 1 （GLUT 1） in rats, OBJECTIVE： This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-X^TM system. DESIGN： An in vitro cell-based experiment. SETTING： This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS： Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5 a and human embryonic kidney 293 cells （HEK293 cells） used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody （Chemicon, U.S.A.） and primers （Shanghai Boya Bioengineering Co., Ltd） were also used. METHODS： E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES： Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively. RESULTS： Results demonstrated that recombinant adenoviral vectors successfully expressed GLUT1 protein, with a relative molecular mass of 55000 in HEK293 cells. These results suggest that recombinant adenoviral vectors obtained by homologous bacterial recombination feature high efficiency, rapidness, and simplicity. CONCLUSION： We successfully amplified the rat GLUT1 gene and constructed replication-defective adenoviral vectors expressing GLUT1. The replication-defective adenoviral vectors proved to successfully express the target gene in HEK293 cells.

Adenoviral Vectors in Veterinary Vaccine Development: Potential for Further Development

Vaccines are an integral part of veterinary disease prevention. However there are still a significant number of veterinary diseases for which vaccines do not currently exist or where currently available vaccines do not provide adequate immunity. Adenoviruses have transitioned from tools for gene replacement therapy to bona fide vaccine delivery vehicles because of their ability to elicit potent cell-mediated and humoral responses making them ideal for use against viruses and other intracellular pathogens. Adenoviral vector based vaccines are likely to play a significant role in overcoming these problems in the future. However, this vector is under utilized in veterinary vaccine development at this time. This review focuses on adenoviral vector based vaccines developed to date and explores the potential for veterinary vaccine development based upon this platform: advantages and potential disadvantages of this technology are discussed as well as the potential for developing efficacious commercial veterinary adenoviral vector based vaccines.

5-Fluorouracil-related enhancement of adenoviral infection is Coxsackievirus-adenovirus receptor independent and associated with morphological changes in lipid membranes

AIM： To evaluate the mechanism underlying the effects of 5-Fluorouracil （5-FU） on adenoviral infection. METHODS： Low and high Coxsackievirus-Adenovirus Receptor （CAR） expressing human colon carcinoma cell lines were treated with 5-FU and two El-deleted adenoviral constructs, one transferring GFP （Ad/CMV- GFP） the other bax （Ad/CEA-bax）. The number of infected cells were monitored by GFP expression. To evaluate the effects of 5-FU in a receptor free system, Ad/GFP were encapsulated in liposomes and treated with 5-FU. Ad/GFP release was estimated with PCR and infection of 293 cells with the supernatant. Electron microscopy of the Ad5-GFP-liposome complex was made to investigate morphological changes of the liposomes after 5-FU. RESULTS： Infection rates of all cell lines increased from 50% to 98% with emerging 5-FU concentrations. The enhanced viral uptake was independent of the CAR expression. Additionally, 5-FU treated liposomes released 2-2.5 times more adenoviruses. Furthermore, 5-FU- treated liposomes appeared irregular and porous-like. CONCLUSION： adenoviral uptake is enhanced in the presence of 5-FU irrespective of CAR and is associated with morphological changes in membranes making the combination of both a promising option in gene therapy.

The adenoviral E1A protein relieves gene repression by receptors in v/vo displaces corepressors and unliganded thyroid hormone

The human adenovirus type 5 early region 1A （E1A） is one of two oncogenes present in the adenovirus genome and functions by interfering with the activities of cellular regulatory proteins. The E1A gene is alternatively spliced to yield five products. Earlier studies have revealed that E1A can regulate the function of thyroid hormone （T3） receptors （TRs）. However, analysis in yeast compared with transfection studies in mammalian cell cultures yields surprisingly different effects. Here, we have examined the effect of E1A on TR function by using the frog oocyte in vivo system, where the effects of E1A can be studied in the context of chromatin. We demonstrate that different isoforms of E1A have distinct effects on TR function. The two longest forms inhibit both the repression by unliganded TR and activation by T3-bound TR. We further show that E1A binds to unliganded TR to displace the endogenous corepressor nuclear receptor corepressor, thus relieving the repression by unliganded TR. On the other hand, in the presence of T3, E1A inhibits gene activation by T3-bound TR indirectly, through a mechanism that requires its binding domain for the general coactivator p300. Taken together, our results thus indicate that E1A affects TR function through distinct mechanisms that are dependent upon the presence or absence of T3.

A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells

We report here a method for the use of poly-L-lysine （PLL） to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells （BMSCs） in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein （GFP）-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research.

Inhibition of KIT RNAi mediated with adenovirus in gastrointestinal stromal tumor xenograft

AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transplanted GISTs were established in nude mice. AdEGFP-U6-KIT was intratumorally injected. The volume, inhibition ratio of tumor and CD117 expression of GIST graft tumor in nude mice were compared between test and control groups. RESULTS: The length of KIT shRNA was determined to be about 50bp by agarose electrophoresis. Gene se-quencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFPU6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 10 11 viral particles/mL and 5.67 × 10 7 plaque forming units/mL, respectively. The mean volume of the grafted tumor was significantly smaller in test mice than in control mice (75.3 ± 22.9 mm 3 vs 988.6 ± 30.5 mm 3 , t = -18.132, P < 0.05). The inhibition ratio of the tumors was 59.6% in the test group. CD117 positive expression was evident in two cases (20%) in the test group and 10 cases (100%) in the control group (χ 2 = 10.2083, P < 0.005). CONCLUSION: AdEGFP-U6-KIT is successfully constructed, and KIT RNAi mediated with Admax vector system can effectively inhibit the expression of the KIT gene and the growth of GIST in nude mice.

Analysis of the expression of coxsackievirus and adenovirus receptor in five colon cancer cell lines

AIM： To investigate the expression of coxsackievirus and adenovirus receptor （CAR） and adenovirus-mediated reporter gene transfer in five human colon cancer cell lines.METHODS： Expression of CAR-specific mRNA and protein was analyzed by reverse transcriptase polymerase chain reaction and Western blotting, respectively. Adenovirusbased gene delivery was evaluated by infection of cells with adenoviral vector carrying the green fluorescent protein （GFP） gene.RESULTS： All the colon cancer cell lines examined （HT29, LS180, SW480, SW948 and SWlll6） expressed CAR full-length mRNA and an alternatively-spliced variant that lacks the transmembrane coding exon. All cell lines were detected as CAR-positive by Western blot analysis. Further, all cells we examined were efficiently infected with adenoviral vector-GFP.CONCLUSION： The data indicated that the five colon cancer cell lines tested expressed adenovirus primary receptor and could be efficiently infected by adenoviral vectors. Therefore, these cell lines will be useful for adenovirus-based gene transfer and research.

Anatomical distributional defects in mutant genes associated with dominant intermediate Charcot-Marie-Tooth disease type C in an adenovirus-mediated mouse model

Dominant intermediate Charcot-Marie-Tooth disease type C（DI-CMTC） is a dominantly inherited neuropathy that has been classified primarily based on motor conduction velocity tests but is now known to involve axonal and demyelination features.DI-CMTC is linked to tyrosyl-t RNA synthetase（YARS）-associated neuropathies,which are caused by E196 K and G41 R missense mutations and a single de novo deletion（153-156 del VKQV）.It is well-established that these YARS mutations induce neuronal dysfunction,morphological symptoms involving axonal degeneration,and impaired motor performance.The present study is the first to describe a novel mouse model of YARS-mutation-induced neuropathy involving a neuron-specific promoter with a deleted mitochondrial targeting sequence that inhibits the expression of YARS protein in the mitochondria.An adenovirus vector system and in vivo techniques were utilized to express YARS fusion proteins with a Flag-tag in the spinal cord,peripheral axons,and dorsal root ganglia.Following transfection of YARS-expressing viruses,the distributions of wild-type（WT） YARS and E196 K mutant proteins were compared in all expressed regions; G41 R was not expressed.The proportion of Flag/green fluorescent protein（GFP） double-positive signaling in the E196 K mutant-type mice did not significantly differ from that of WT mice in dorsal root ganglion neurons.All adenovirus genes,and even the empty vector without the YARS gene,exhibited GFP-positive signaling in the ventral horn of the spinal cord because GFP in an adenovirus vector is driven by a cytomegalovirus promoter.The present study demonstrated that anatomical differences in tissue can lead to dissimilar expressions of YARS genes.Thus,use of this novel animal model will provide data regarding distributional defects between mutant and WT genes in neurons,the DICMTC phenotype,and potential treatment approaches for this disease.

Adenovirus-expressed preS2 antibody inhibits hepatitis B virus infection and hepatic carcinogenesis

AIM:To investigate the inhibitory effect of hepatitis B virus (HBV) preS2 antibody (preS2Ab) against HBV in-fection and HBV-associated hepatic carcinogenesis.METHODS:An adenoviral vector carrying the full-length light and heavy chains of the HBV preS2Ab gene,Ad315-preS2Ab,was constructed.Enzyme linked immunosorbent assay (ELISA) and Western blotting analyses were used to determine the preS2Ab expres-sion levels in vitro.Immunofluorescent techniques were used to examine the binding affinity between the expressed HBV preS2Ab and HBV-positive liver cells.ELISAs were also used to determine hepatitis B surface antigen (HBsAg) levels to assess the inhibitory effect of the preS2Ab against HBV infection in L02 cells.The inhibitory effect of preS2Ab against hepatic carcinogen-esis was studied with diethylnitrosamine (DEN)-induced hepatocellular carcinomas (HCCs) in HBV transgenic mice.RESULTS:The expression of HBV preS2Ab increased with increases in the multiplicity of infection (MOI) of Ad315-preS2Ab in L02 cells,with 350.87 ± 17.37 μg/L of preS2Ab when the MOI was 100 plaque forming units (pfu)/cell.The expressed preS2Abs could recog-nize liver cells from HBV transgenic mice.ELISA results showed that L02 cells expressing preS2Ab produced less HBsAg after treatment with the serum of HBV pa-tients than parental L02 cells expressing no preS2Ab.HBV transgenic mice treated with Ad315-preS2Ab had fewer and smaller cancerous nodes after induction with DEN than mice treated with a blank Ad315 vec-tor or untreated mice.Additionally,the administration of Ad315-preS2Ab could alleviate hepatic cirrhosis and decrease the serum levels of alanine transaminase and aspartate transaminase.CONCLUSION:Adenovirus-mediated HBV preS2Ab expression could inhibit HBV infection in L02 cells,and then inhibit DEN-induced hepatocellular carcinogenesis and protect hepatic function in HBV transgenic mice.

Construction of recombinant human nerve growth factor beta adenovirus and evaluation of its function An in vitro and in vivo study

Exogenous delivery of nerve growth factor （NGF） promotes neural regeneration. However, the short half-life limits delivery efficacy. Therefore, a long-term, efficient, local delivery tool or scheme is needed. The purpose of this study was to construct a functioning, recombinant, adenoviral vector carrying human NGF-β （hNGF-β） DNA, and to measure expression of the constructed vector in vitro and in vivo. rhNGF-β adenoviral vector containing full-length hNGF-β cDNA was generated by homologous recombination in Escherichia CoIL The rhNGF-β adenovirus was packaged and amplified in human embryonic kidney HEK293 cells. Transformation efficiency, expression and function of rhNGF-β adenovirus for primary Schwann cells, Schwann cell lines, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts were evaluated. Subsequently, expression of rhNGF-β adenovirus at the peripheral nerve of rat was also assessed. Recombinant adenoviral vector carrying hNGF-β was successfully constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. Green fluorescent protein expression was observed in 90% of rhNGF-β adenovirus-infected cells （primary Schwann cells, Schwann cell line, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts） compared with non-infected cells. Total mRNA isolated from rhNGF-β adenovirus-infected cells exhibited strong expression. Maximum NGF release was induced by primary cultured Schwann cells at 4 days after infection, which steadily continued for 14 days. PC-12 cells exposed to media conditioned with rhNGF-β adenovirus-infected Schwann cells exhibited increased neurite extension. In vivo experiment revealed that the injected rhNGF-β adenovirus was transfected into the cells at the injected site and promoted expression of NGF, p75NTR and brain derived neurotrophic factor at the sciatic nerve and dorsal root ganglia.

EFFECT ON BIOLOGICAL BEHAVIOR OF CHEMOTHERAPY-RESISTANT TUMOR CELLS BY HUMAN WILD-TYPE p53,GM-CSF AND B7-1 GENES VIA RECOMBINANTADENOVIRUS

Objective: To explore the effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes mediated via recombinant adenovirus. Methods: p53-abnormal KB-v200 (VCR resistant) and KB-s (VCR sensitive) cell lines were used as model tumor cells, which are resistant and sensitive to chemotherapeutic drugs respectively. After infected with recombinant adenovirus carrying human wild-type p53, GM-CSF and B7-1 genes, changes in biological behavior (including drug sensitivity) of these two kinds of gene-transduced cancer cells were observed. Results: Both of the cell lines were susceptible to adenovirus, all of three exogenous genes (p53, GM-CSF and B7-1) could be effectively expressed in these cell lines, their growth was suppressed, and apoptosis was induced. The drug-pumping-out function of Pgp glycoprotein on the cytomembrane of drug-resistant KB-v200 cells was markedly affected 48h after transfection of the recombinant adenovirus, revealed by increase of the amount of rhodamine 123 accumulation in the cells. The MTT assay also indicated the reversal of their sensitivity to VCR drugs.In vivo experiment in nude mice it was demonstrated reduction of tumorigenicity of the KB-v200 cells or KB-s cells infected with the recombinant adenovirus, and increase of their sensitivity to VCR. Conclusion: The clinical application of this recombinant adenovirus carrying agents might be more effective in treatment of tumors with multidrug resistance(MDR).

核苷酸结合寡聚化结构域样受体蛋白6重组腺病毒过表达载体的构建与鉴定

目的构建及鉴定携带小鼠核苷酸结合寡聚化结构域样受体蛋白6(NLRP6)基因(NM_133946.2)的过表达重组腺病毒载体。方法合成小鼠NLRP6基因的编码序列,经酶切后插入腺病毒载体(GL2003),转化大肠杆菌感受态细胞DH-5α进行培养,挑取转化子,进行聚合酶链反应(PCR)鉴定后送测序进行比对。采用蛋白质印迹法(Western blot)验证重组质粒中NLRP6蛋白的表达。利用Admax包装系统获得重组腺病毒载体Ad-NLRP6,导入HEK293细胞,经扩增纯化后测定病毒滴度。结果双酶切及DNA测序证明NLRP6成功构建入表达质粒中,同时检测其携带的FLAG标签蛋白,表明NLRP6表达水平显著升高。包装并生产携带NLRP6的重组腺病毒,进一步将病毒感染HEK293细胞,Ad-NLRP6细胞表达绿色荧光蛋白提示感染成功,收集病毒,经扩增纯化后测定病毒滴度为4×10^(10)PFU/mL。结论成功构建携带小鼠NLRP6基因的过表达重组腺病毒载体,这一结果为进一步研究NLRP6分子学功能提供了有效工具。

重组腺病毒介导IFN-γ基因转染的巨噬细胞的体外免疫效应功能分析

巨噬细胞既是免疫效应细胞又是抗原提呈细胞，为研究ＩＦＮ－γ基因转染的巨噬细胞过继回输疗法，以重组腺病毒为载体将小鼠ＩＦＮ－γｃＤＮＡ转染入小鼠腹腔巨噬细胞，观察了对其体外免疫功能的影响，结果表明，ＩＦＮ－γ基因转染１８ｈ后巨噬细胞上清中存在高水平ＩＦＮ－γ，ＩＦＮ－γ基因转染的巨噬细胞杀伤活性显著增高，其分泌ＴＮＦ、ＩＬ－１、ＮＯ的水平均有不同程度的升高，表明重组腺病毒载体能将ＩＦＮ－γ基因成功地转染入巨噬细胞并增强巨噬细胞免疫效应功能。