ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Forty-six patients with intractable urticaria were treated by acupuncture plus garlic-partitioned moxibustion. The results were compared with those of simple acupuncture in a control group of 30patients. The total effective rate were 91.3%, in the observation group and 73.3%, in the control group. It is showed that acupuncture plus garlic-partitioned moxibustion has a better effect on intractable urticaria than simple acupuncture.