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Objective: To investigate the effect of cobalt chloride (CoCl2) on transgene expression and viral particle titers in tumor cells infected by conditionally replicating adenovirus expression vector with hypoxia response elements(HRE)-regulated E1AE1B expression (Ad-5HRE-E1AE1B-RFP) and non-HRE regulated replication-deficient adenovirus expression vector (Ad-EGFP, Ad-Luc) in vitro and in vivo. Methods: Ad-5HRE-E1AE1B-RFP had five duplicated HRE and mini CMV acted as a promoter to drive E1AE1B expression and constitutive expression of RFP as reporter. The hypoxia model was optimized by exposing tumor cells to different concentrations of CoCl2. The hypoxia-inducible factor 1 alpha (HIF-1α) protein expression was determined by Western blotting. Under the optimized hypoxia model, the positive expression of exogenous gene and virus replication of Ad-5HRE-E1AE1B-RFP or Ad-EGFP-infected tumor cells were examined by conversed microscopic observation, FACS analysis and plaques formation test. Furthermore, transgene expression induced by combined application of hypoxia-inducible replicative adenovirus and replication deficient adenovirus (Ad-Luc) was also evaluated by examining the lucifererse activity in xenografted tumor models in nude mice by micro PET. Results: Western blotting results showed that CoCl2 at 0.4 and 0.08 μg/mL could stabilize and acumulate HIF-1α protein in gastric cancer SGC7901 cells, which could better mimic hypoxia condition. The microscopic observation and FACS analysis showed that CoCl2 at 0.4 μg/mL could remarkably increase the transduction efficacy of Ad-5HRE-E1AE1B-RFP, which was verified by significant increase in the percentage of positive expression of exogenous gene RFP and fluorescence intensity. But plaques formation test showed that Ad-5HRE-E1AE1B-RFP had no replication. CoCl2 0.4 μg/mL augmented the tranduction efficacy and expression levels of non-HRE regulated replication deficient adenovirus Ad-EGFP and Ad-Luc. Combined intratumoral injection of Ad-5HRE-E1AE1B-RFP and Ad-Luc significantly increased the expression of Ad-Luc in nude mice.Conclusion: CoCl2 markedly enhances transgene expression of recombinant adenovirus. However, the underlying mechanism is not only related to the CoCl2-induced hypoxia, but also probably related to regulation of gene transcription.
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BACKGROUND: Genetically engineered cells have been used in the replacement therapy and gene therapy. However, how to select proper donor cells, target cells, and corresponding viral vectors is the most difficult in the therapy.OBJECTIVE: To compare the transduction efficiency of recombinant adenovirus Ad5 and AdSF35, adeno-associated virus rAAV1/2 and rAAV2, and lentivirus LV in bone marrow mesenchymal stem cells (BMSCs) and exogenous gene expression level so as to select the vectors, which can efficiently transduce BMSCs.DESIGN, TIME AND SETTING: Gene engineering controlled observation, performed in the Central Laboratory, Shanghai First People's Hospital between October 2006 and March 2007.MATERIALS: Ten Sprague Dawley rats of clean grade were used to prepare BMSCs. All recombinant viral vectors carded enhanced green fluorescent protein (EGFP) report gene. Ad5 was prepared by the Central Laboratory, Shanghai First People's Hospital. Ad5F35 was gifted by professor Qian Qi-jun from the Second Military Medical University of Chinese PLA. rAAV2 and rAAVI/2 were the products of Benyuan Zhengyang Gene Technique Co.,Ltd. LV was gifted by professor Cuo Li-be from Shanghai Institute of Biochemistry and Cytobiology, Chinese Academy of Sciences.METHODS: Rat BMSCs were in vitro isolated and cultured by density gradient centrifugation. BMSCs of passage 4 were inoculated into 24-well plate at lxlO5/well. One day later, ceils adhered to the wall and allocated to 5 groups. Ad5-EGFP [10, 100,1 000 multiplicity of infection (MOI)], Ad5F35-EGFP (10,10, 1 000 MOI), rAAVI/2-EGFP (1×104,1x10× vg), rAAV2-EGFP(1×104, 1x105vg), and LV-EGFP (30 TU) were respectively added into the 5 groups. BMSCs were transduced for 2 days with Ad virus and separately for 6 days with rAAV and LV virus.MAIN OUTCOME MEASURES: EGFP-positive expression and fluorescence intensity.RESULTS: After twenty-four hours of Ad5-EGFP transduction, EGFP-positive cells were visible under the microscope. With virus dose increasing, EGFP-positive cells increased and fluorescence intensity strengthened. Twelve days later, EGFP-positive cells gradually reduced and fluorescence intensity weakened. For Ad5F35-EGFP, its transduction was basically similar to Ad5-EGFP, but EGFP-positive cell number and fluorescence intensity were increased. After 6 days of rAAV1/2-EGFP or rAAV2-EGFP transduction, EGFP-posidve cell number and fluorescence intensity were decreased. For LV-EGFP transduction, a small amount of EGFP-positive cells could be visible on the second day, and then EGFP-positive ceils and fluorescence intensity were gradually increased until the sixth day.CONCLUSION: Ad5, Ad5F35 and LV could effectively transduce BMSCs cultured in vitro and express exogenous gene.Furthermore, transduction efficiency was correlated with virus dose in dose-dependent manner, rAAVI/2 and rAAV2 had poor/in vitro transduction efficiency.
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Objective:To explore the influence of blood transfusion on cellular immunofunction in patient with laryngeal carcinoma.Method:EPICS-XL flow cytometry was used to measure T cell subgroup,NK cell and CD28 in 36 patients with laryngeal carcinoma pre-operation and 2 weeks post-operation.Patients were divided into allotransfusion group and non-transfusion group.Comparison was conducted between the 2 groups.Result:①Comparsion with normal population,decreasing of CD3,CD4,NK cell and CD28 in the 36 patients pre-operation was statistically significant (P＜0.01).②Decreasing of CD3,CD4,NK cell and CD28 was statistically signficant post-operation(P＜0.05).③In the non-transfusion group change of CD3,CD4,CD8,NK cell and CD28 post-operation was statistically insignificant (P＞0.05).Conclusion:While cellular immunofunction is generally low in patients with laryngeal carcinoma,allotransfusion will reduce further.It makes contribution to spreading and metastasis of carcinoma easier.
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Objective To study the correlation between vitamin D receptor(VDR)polymorphism and psoriasis in the Chinese patients.Methods VDR genotypes were determined by PCR-RFLP in 112unrelated psoriatic patients as well as 108unr elated healthy controls.Results AA and Bb genotypes were found to be ex-pressed more frequently in psoriatic patients(19.6%and 32.1%)than those in normal controls(7.4%and14.8%),respectively.However,no signifi cant correlation was identified between VDR polymorphismand sex,age at onset,family history of psoriasis in psoriatic patients.In addition,there was no significant difference in the fre-quency of A,a alleles and B,b alleles between normal controls and psoriatic patients.Conclusions There is a significant difference in VDR genotypes between psoriatic patients and normal controls.The presence of hom ozygote AA or heterozygote Bb might contribute to the susceptibility of psoriasis.
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AIM: To study the effect of Yupingfeng Powder(YPFP) on rat's allergic rhinitis induced by stimulating ovalbumin.and reveal the influence on Th1,Th2 and Th1/Th2 proportion. METHODS: 8-week-old BALB/c mice were sensitized by means of intranasal and systemic intraperitoneal injection application of OVA,6 subjects were administered including 2,500 mg/kg extracts of YPFP for 7 days in early stage(interference group);6 mice were administered YPFP in after challenge(therapy group);the placebo group were given saline.After 7 days,Th1 and Th2 in splenocyte were detected by flow cytometry(FCM) and nasobuccal mucosa pathology were observed. RESULTS: When compared with Th2 of animal model group(9.86?1.40),there was a significant decrease expressing Th2 after PFP treatment(3.41?0.72,P