Studies Of Targeted Transplantation Of Endothelial Progenitor Cells Transfected By HeNOS Gene Into Hemodynamic Pulmonary Hypertension

Part 1 Construction of recombinate adenovirus vector carrying gene heNOSObjective To construct a recombinate adenovirus vector carrying gene heNOS as wellas a recombinate adenovirus vector carrying gene EGFP as a reporter.Methods heNOS gene were cloned into adenovirus shuttle vector PSUCMV bystandard procedure to produce recombinate plasmid PSUCMV-heNOS. AfterPSUCMV-heNOS was identified, it was transferred into adenoviral packaging cell (293cell) with adenovirus Pbhge3 by lipofectamine 2000 to construct vectorAd.CMV-heNOS. Then the virus were amplified and purified. At the same time,recombinate adenovirus vector Ad.CMV-EGFP carrying gene EGFP was established inthe same way.Results The recombinate PSUCMV-heNOS was correctly constructed and confirmedby restriction endonucleas analysis and DNA sequencing analysis. The result of PCRdemonstrated the vector Ad.CMV-heNOS was successfully constructed. And theconcentration of Ad.CMV-heNOS and Ad.CMV-EGFP reached to 5.15×l0⁹pfu/ml and3.8×l0⁹pfu/ml respectively.Conclusion The high-titer recombinate replication-defective adenovirus vectorcarrying gene heNOS and EGFP were successfully constructed and purified.Part 2 Isolation, culture and identification of endothelial progenitor cells from rat bone marrowObjective To investigate the methods of isolating and culturing endothelial progenitor cells(EPCs) from rat bone marrow, as well as their ability of differentiating into endothelial cells.Methods The mononuclear cells were isolated from rat bone marrow by density gradient centrifugation and cultured for 48h. Then the suspending cells were cultured in the selective culture medium(with the supplement of VEGF, bFGF, IFG-1 and EGF) for2w. The attached cells were stained with Dil-ac-LDL and FITC-UEA-1, and the expression of vWF and Flk-1 was also assessed by immunofluorescence. The percentage of EPCs was analyzed by fluorescence-activated cell sorting(FACS). Their function of producing nitric oxide(NO) was evaluated by quantification of NO level in culture medium.Results The secondarily attached cells stretched and exhibited the clonal morphology after 3 and 5 days' inducing culture respectively. The cells proliferated faster after 7-10 days' incubation when cord-like structure was observed. After 2 weeks' induction, most of the cells exhibited multangular morphology. More than 70% attached cells took up Dil-ac-LDL, bound FITC-UEA-1 (double positive fluorescence), and expressed vWF and Flk-1 (immunofluorescence). According to the results from FACS, the percentage of vWF+ and Flk-1+ cells were 77.93% and 81.50% respectively. The level of NO in the selective culture medium of secondarily attached cells was higher than that of the primarily attached cells but lower than that of the mature endothelial cells (P＜0.05). Conclusion EPCs were enriched in rat bone marrow and may exhibit some of the characteristics endothelial cells owned after inducing culture in vitro. Part 3 Establishment of hemodynamic pulmonary hypertension(HPH) in ratsObjective To explore the influence on the systemic and pulmonary artery pressure, right ventricular hypertrophy and pulmonary vascular structural remodeling in rats after removal of different proportion of lungs. So as to establish a new model of hemodynamic pulmonary hypertension in rats.Methods Sixty-eight Wistar rats were randomly divided into group I (total resection of right lung), group II (total resection of left lung), group III (resection of the upper and middle lobe of right lung), group IV (sham operated) and group V (control). Four weeks later, the systolic systemic artery pressure(sSAP) and systolic pulmonary artery pressure(sPAP) were measured. The ratio of right ventricular mass to left ventricular plus septal mass [RV/(LV+S)] was detected. The relative medial area(RMA) of small pulmonary muscularized arteries(SMA) and the percentage of SMA to all the small pulmonary vessels (SMA%) were measured and calculated.Results There were 66.7% rats survived 4 weeks after the operation(12/18) in group I. And the weight of group I is less than that of group IV and V(P<0.05). Group I hadobviously higher sPAP, RV/(LV+s), RMA and SMA%(P<0.01). There was no difference in all the indexes between group II and III(P>0.05) and between group IV and V(P>0.05). But group II had higher RMA than group IV and V(P<0.05) while group III had higher sPAP and RMA than group IV and V(P<0.05). There was no difference in sSAP among the five groups(P>0.05).Conclusion Four weeks after the total resection of right lung, pulmonary artery hypertension, right ventricular hypertrophy and small pulmonary artery structural remodeling were emerged. But sSAP was not influenced. Compared with other HPH models, this method was easily performed, the mortality of rats was relatively low and the pulmonary hypertension was quickly induced.Part 4 Transfection of Ad.CMV-heNOS to EPCs and its inhibition effects on the proliferation of smooth muscle cells (SMCs) in vitroObjective To explore the transfection efficiency of adenovirus vector to EPCs from rat bone marrow, the expression of heNOS after the transfection of Ad.CMV-heNOS to EPCs, and their inhibition effects on SMCs in vitro.Methods After the transfection of Ad.CMV-EGFP to EPCs with different MOI, we used FACS to evaluate the transfection efficiency. Then we transfected Ad.CMV-heNOS to EPCs with MOI 50:1. After transfection, heNOS mRNA transcription was detected by RT-PCR, heNOS expression was detected by immunohistochemical staining and Western-blot. The level of NO in culture medium was also measured. Then we divided EPCs into 3 groups including Ad.CMV-heNOS transfected group, Ad.CMV transfected group and PBS group. All the groups of EPCs were mixedly co-cultured with EGFP-labeled SMCs for 72 hours. Then SMCs% were detected by FACS. We used "inserts" containing PET membrane to establish a co-culture system in which EPCs and SMCs can influence each other without mixing. After 24 hours' co-culture, FACS measured the alteration of SMCs cell cycle and apoptosis.Results The transfection efficiency of Ad.CMV-EGFP to EPCs increased with MOI. When MOI reached 50, the transfection efficiency increased to more than 85%. From then on, as MOI increased, the transfection efficiency increased limitedly. So we choose50:1 as the most suitable MOI. Using RT-PCR, immunohistochemical staining and Western-blot, we found that heNOS gene could be correctly transfected into EPCs. After the transfection, the level of NO in the culture medium was also higher than control group. After the mixed co-culture of EPCs with SMCs for 72h, the SMC% of the Ad.CMV-heNOS transfected group was lower than that of the other two groups(P<0.05). After non-mixed co-culture of EPCs with SMCs for 24h, there were obviously more SMCs of Ad.CMV-heNOS transfected group still in G₁ stage(P＜0.01) than the other groups. What's more, about 1% SMCs showed up apoptosis in Ad.CMV-heNOS transfected group.Conclusion The most suitable MOI for transfection in the research is 50:1. heNOS gene could be successfully transfected into EPCs by Ad.CMV-heNOS, followed by correct expression and effects of catalyzing more NO. In the co-culture system in vitro, EPSc themselves could interfere with the cell cycle of SMCs and inhibit their proliferation. Furthermore, the transfection of gene heNOS could enhance this effects. Part 5 Targeted transplantation of heNOS-transfected EPCs into rats with hemodynamic pulmonary hypertensionObjective To explore the survival, disposition of EPCs and expression level of heNOS in vivo after injection of heNOS-transfected EPCs to HPH rats through jugular veins. To investigate if heNOS-transfected EPCs could relieve the pathologic changes of HPH and decrease the pulmonary arterial pressure.Methods After the HPH model was established, we transplanted EGFP-labeled EPCs to the rats through jugular vein. The rats were put to death and the organs such as lung, heart, liver and kidney were taken out and made into frozen section on 1 day, 3 days, 1 week and 3 weeks after the operation respectively. Then we observed the disposition of green fluorescence cells. Forty HPH rats were randomly divided into group I (Ad.CMV-heNOS transfected EPCs were transplanted), group II (Ad.CMV transfected EPCs were transplanted), group III (EPCs were transplanted) and group IV (only PBS was transplanted). Two weeks later, we measured sSAP, sPAP, RV/(LV+s), RMA and SMA%. We detected the heNOS expression by RT-PCR and Western-blot. For group I, we even compared the heNOS expression in lung with in heart, liver and kidney by Western-blot.Results 1 day and 3 days after the EGFP-labeled EPCs transplanted, a lot of fluorescence cells could be seen in the frozen sections of lung. One week later, the fluorescence intensity decreased. Two weeks after the transplantation, there were still a few fluorescence cells locating on the wall of small pulmonary arteries. And some of them had become a part of the vessel endothelium. However, there were very few fluorescence cells in heart, liver and kidney all the time. Two weeks after the intervention, group I displayed obviously lower sSAP, RV/(LV+s), RMA and SMA% (P＜0.01). There was no difference between group II and group III(P>0.05). And the index of group II and III were lower than that of group IV but higher than group I(P<0.05) with the exception of SMA%. There was no difference in sSAP among the four groups. The expression of heNOS in lungs was successfully proved by RT-PCR and Western-blot. At the same time, there was very little expression of heNOS in heart, liver and kidney.Conclusion After injecion of heNOS-transfected EPCs to HPH rats through jugular vein, EPCs could be targeted into lung, correctly express heNOS and survive for more than 2 weeks. Transplantation of untransfected EPCs could relieve the progression of pulmonary hypertension obviously. And heNOS-transfected EPCs could strengthen this effect. In addition, it could even partially reverse pathologic change of hemodynamic pulmonary hypertension.