| Pulmonary circulation is a low resistance, low pressure and high flow system. Pulmonary arterial hypertension (PAH) occurs when the pulmonary arterial systolic pressure is higher than 30mmHg (or pulmonary arterial mean pressure higher than 20mmHg). PAH is a life-threatening disease with a high vitality rate. It is characterized by intimal fibrosis, medial hypertrophy, adventitial proliferation, obliteration of small arteries, which produce progressive pulmonary hypertension, right ventricular (RV) failure and ultimately, death. It has been demonstrated that eNOS deficiency in the lungs and impaired NO production have been linked to the development of pulmonary hypertension in this disease. Moreover, inhalation of NO has become recognized as a therapeutic breakthrough that can improve hemodynamics and survival without unwanted and dangerous systemic vasodilatation in patients with PAH. However, it is always cumbersome, expensive, and requires a fairly sophisticated delivery system. Furthermore, short half-life, easy drug resistance and thepossibility of the whole body side-effect have hindered it from further use. Janssens et al aerosolized AdCMVceNOS into rat airway during the inspiratory phase of ventilatory cycle, accomplished the eNOS gene transfer into rat lungs for the first time. The high pulmonary pressure produced by acute hypoxia was ameliorated with no change in systolic pressure. This demonstrated that gene transfer of eNOS in vivo with overproduction of endogenous eNOS may be a promising approach to ameliorate pulmonary hypertension without those tedious side effects. As we know, to treat PAH, the ways through which xeon gene is transferred include vascular endothelium, adventitia and airway epithelia. To provide data of gene therapy for PAH, study the transgene distribution, efficiency, dose-effect relationship, time curve and side effect of adenovirus mediated transgene expression in vivo and in vitro, MOI 30 of recombinant adenovirus AdCMVceNOS and different dose of AdCMVLacZ were administered to cultured vascular endothelial cells in vitro and rat lungs in vivo respectively.Experiment 1 Adenovirus-Mediated HumanEndothelial Nitric Oxide Gene Expression in HumanVascular Endothelial Cells in VitroObjective To investigate the transgene expression of adenovirus-mediated human eNOS gene transfer and secretion of NO in vascular endothelial cells.Methods (1) DNA of virus AdCMVceNOS was transferred into 293 cells by liposome, and packaged inside. After being confirmed by PCR analysis, the virus was amplified in 293 cells and purified by discontinuous CsCl gradient. Viral titer was determined with TCID50method. (2) HUVECs were cultured according to the improved Jaffe's procedure and were confirmed. (3) HUVECs were divided into Ad-eNOS group, Ad-LacZ group and blank control group, each with 15 culture wells. Cells were infected with AdCMVceNOS, AdCMVLacZ at MOI of 30 or culture medium only. After 2 hours, the viral suspension was removed, and cells were incubated with 10%FBS DMEM. (4) NO concentration in culture medium supernatant was messured on day 1, 3, 5. The presence of the eNOS gene product was detected by immunostaining with mouse monoclone antibody.Results (1) DNA of virus AdCMVceNOS was transferred into 293 cells and integrated virus was made. Then, it was confirmed, amplified. The titer was quantified to be 1.58xl010PFU/ml. (2) Transgene expression was found in 1 day after the virus infection in Ad-eNOS group, and achieved peak on day 3. The brown particles diffused in cell plasm indicated that AdCMVceNOS can generate large quantity of eNOS in vascular endothelial cells. (3) NO production of Ad-eNOS group was significantly higher than those of the other two groups on day 1 ( F=79.39, P<0.05). On the 3rd and 5th day, it achieved as high as 50-60 times than control.Conclusion (1) Adenovirus is highly affinitive to vascular endothelial cells and can mediate human eNOS gene expression efficiently. (2) After the transfection of Recombinant virus AdCMVceNOS, transgene expression in...
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