Intervention Of Bone Marrow Mesenchymal Stem Cells Transfected With VEGF Gene And Ang1Gene Mediated By Lentivirus On Oxygen Induced Neonatal Mice BPD Model

Bronchopulmonary dysplasia (BPD), a major cause of morbidity and mortality amongpremature infants, is still not full understood and lack effective treatment. Thepathologic picture of BPD is characterized by a simplified alveolar pattern, withdecreased numbers of alveoli and impaired lung vascular growth. Based on the“vascular hypothesis” of new BPD, we designed this study to investigate the treatmenteffect and possible mechanism of Mesenchymal stem cells modified with VEGF orangiopoietin-1by lentiviral vectors in a neonatal mouse model of BPD. In this study,Ang1plasmid and VEGF plasmid were constructed by Gateway technologyrespectively, then packaged with lentiviral virus and transfected into mice bonemesenchymal stem cells. The obtained stably transfected cell lines were infused to theBPD mice model by intraperitoneal injection. The alveolar structure and microvascularstructure of the BPD mice model after cell transplantation were observed throughmorphology method, immunohistochemistry, western blotting, Q-PCR, and electronmicroscope scan. The inflammation response degree in lung issues was evaluated bydetect the Macrophage count and neutrophil count in lung lavage fluid. Our study willbe helpful to further understand the role of vascular damage in mechanism of BPD, andto find more effective intervention approach. This study consists of the following fourparts.Part I：Construction of VEGF and angiopoietin1plasmidIn this part, four plasmids contained fluorescent protein gene（PLV.Ex2d.P/neo-CMV>VEGF/IRES/EGFP, PLV.Ex3d.P/puro-CMV> Ang-1>IRES/DsRed-Express2,PLV.EX2D.p/puro-CMV>DsRed Express2, PLV.EX2D. neo-CMV>EGFP） wereconstructed by using Gateway technology successfully and identified to be correct byPCR and gene sequencing. In order to achieve good cell tracer, in our study, EGFP andDsRed were constructed into VEGF vector and Ang1vector respectively. Greenfluorescent protein (GFP) and Red fluorescent protein (DsRed) were powerful tools foridentifyingthesubcellularlocalizationofproteins andtomonitorgeneexpression. Bothof GFPand DsRed have been described that can emit light with a different wavelength.The longer wavelength of the emitted light of GFP and DsRed could minimizesproblems associated with light scattering and auto-fluorescence of the cells. Furthermore，Fluorescent proteins with different emission colors are valuable for invivo multilabeling experiments, allowing comonitoring of several events. Successfulconstruction of plasmids contained fluorescent protein gene laid an foundation forfurther virus packaging and cell transduction.Part Ⅱ：Lentivirus package of VEGF＆Ang1plasmid and construction ofstably transfected MSCs lineIn partⅡ, the lentiviral vectors carrying VEGF or Ang1was co-transfected into the293FT cells with three helper plasmids (PLV/helper-SL3, PLV/helper-SL4andPLV/helper-SL5) to package lentivirus by Lipofectamine method respectively and thenthe viral titer was determined under fluorescence microscope. Finally, the mice MSCswere transduced with the VEGF-containing lentiviral vector or Ang1-containinglentiviral vector to obtain the cell line with stable expression of VEGF gene or Ang1gene through several subcultures by repeated colony picking. The distinct green or redfluorescence was seen under fluorescence microscope48h after virus packaging andthe virus titer of CMV-VEGF and Ang1-DsRed(puro) was1×108TU/ml and5×108TU/ml respectively. The vector was success fully transduced into MSCs as evidencedby the significantly increased VEGF or Ang1expression level determined by both Q-PCR and Western blotting. The MSCs cell line with stable transduction of VEGF orAng1containing vector was established by repeated colony picking and subcultures. Inadditional, we confirmed that cell culture supernatant of both MSCs-VEGF and MSCs-Ang-1could promote angiogenesis in chicken embryo allantoic sac, and VEGF andAng1have a synergistic role in angiogenesis.Part Ⅲ：Establishment of the oxygen-induced neonatal mice BPD model andobservation of the pulmonary vascular damage in this kind of model.In this part, one neonatal mice BPD model was established successfully by continuedhigh oxygen (60-70%) induced method and its alveolar and capillary structure wereobserved by HE staining, immunohistochemistry, microvascular count and electronmicroscopic scan. The results showed that the lung pathology of BPD mice presentedtypical BPD alveolar simplification, accompany with decreased microvascular numberand abnormal vessel structure. Compared with air group, RAC was significantlyreduced in BPD group in d7, and more significant difference of RAC between twogroups was observed in d21. Furthermore, CD34, one specific marker of vesselendothelial cell, was used to detect the effect of high oxygen inhalation on lung microvascular density by immunohistochemical stain. The results show MVD ofhyperoxiagroupslightlylowerthan airgroupon d3andd7,butnosignificantdifferencewas found. On d14and d21, the MVD of the hyperoxia group significant lower thanthat of the normal air group. Obviously, continued inhaling high concentration ofoxygen could reduce the number of vessel endothelial cell and lead to angiogenesisdevelopment damage. In addition, in this study, expression changes of three keyangiogenic factor, Ang1, VEGF and EphrinB2, were observed by immunohisto-chemical staining in different time between two groups. The results showed highoxygen exposed continuously could down regulate the expression ofAng1, VEGF andEphrinB2in lung issue. In hyperoxia group,ultrastructure of lungs tissues showed thatalveolar type II epithelial cell were swollen along with the electron densitydecreased．Lamellarbodies，withaloosestructure，reducedevendisappeared．Swollenmitochondria were also viewed. Microvascular structure disorder, bleeding,deformability of red cells was found in lung issues. Obvious swollen of capillaryendothelial cells was observed. The capillary wall was coarse with more black granularcondensation and disintegrated red blood cells. The result showed that not only thestructure of alveolar and alveolar epithelial cells were confirmed to be abnormal,pulmonary microvascular structure and blood air barrier were also impaired inhyperoxia BPD model. These findings confirm our initial hypothesis that high oxygeninduced pulmonary circulation damage play one key role in the pathogenesis of BPD.Part Ⅳ：Intervention of MSCs Carrying VEGF and Ang1gene on BPD modelmiceIn thein vivo mouse model, administration ofMSCs-Ang1, MSCs-V+Aor MSCs aloneall significantly alleviated oxygen-induced pulmonary injury, of which MSCs-V+Ashowed most obvious effect. The administration of MSCs-V+A could effectivelyimprove the weight and RAC, increase lung angiogenesis, reduce pulmonaryinflammation caused by high oxygen inhalation, reduced lung collagen deposition andfibrosis. These finding showed that Ang-1, VEGF and MSCs have synergistic effect inthe repair of pulmonary vascular injury. Both of MSCs-Ang1and MSCs-VEGF wereconfirmed can home to lung in response to injury by immunofluorescence and GFPimmunohistochemistry observation, but fewer in number. The determination results ofWestern blotting and Q-PCR showed increased expression of Ang1and VEGF in lungissue were found after intraperitoneal injection of MSCs-VEGF or MSCs-Ang1. Thepulmonary vascular network of different groups was observed by the ink perfusion method.Theresults showed thevascularnetwork in hyperoxiagroup treated with salinewas more sparse and failed to form a complete system of three-dimensional vascularnetwork compared with those in air group treated with saline. While the vascularnetwork in hyperoxia group treat with MSCs slightly improved compared withhyperoxia group treated with saline. And vascular network was found to be denserfurther in MSCs-VEGF group and MSCs-Ang-1group. But the study also found thatusing MSCs VEGF alone cannot effectivelyreduce the lung injuryof BPD model mice,even make the lung injury get worse. We speculate that this result may be related toabnormal angiogenesis induced by VEGF. The generation of immature blood vesselsinduced by VEGF with higher permeability, can cause hemorrhage and infiltration ofinflammatory cells, leading to deterioration in lung function.In conclusion, these above results confirmed our initial assumption that administrationof MSCs carrying VEGF gene and MSCs carrying Ang1gene can play a synergisticeffect, effectively repair damaged pulmonary circulation, promote alveolardifferentiation, and reduce lung injury in BPD. This study may be developed as apotential novel strategy for the prevention and treatment of BPD.