Treatment Of Bronchopulmonary Dysplasia Of Rats Using Bone Mesenchymal Stem Cells Carried With VEGF Gene

BackgroundBPD (Bronchopulmonary dysplasia, BPD) is a chronic pulmonary disease that complicated by the use of mechanical ventilation or oxygen therapy in preterm children with acute respiratory failure, is due to immaturity and other factors,so that alveolar and pulmonary vascular development disrupt, BPD children expressed as oxygen dependent, secondary pulmonary infection, heart and lung failure, pulmonary hypertension and pulmonaryheart disease and so on, survivors addition to have pulmonary dysfunction, but can be left intellectual, psychomotor retardation and other aspects of the situation.It is the first named by Northway in 1967. Along with improved survival in preterm children, the incidence of BPD has gradually increased, as the important complications of neonatal intensive care and the most common chronic respiratory diseases of infant and early childhood. The incidence of BPD has a great relationship with the immaturity of lung and mechanical damage, including oxygen toxicity, capacity wounds, pressure wounds and infections. The "alveolar simplification", pulmonary vascular developmental disorders and vascular density decreased are as the main features, and pathology shows the small number and large alveole, and pulmonary intervals widened. Currently, the pathogenesis of the disease is not yet clear, clinical treatment including respiratory, circulatory, nutrition and other supportive care are palliative measures,lack of basic treatment, therefore, to deeply study the pathogenesis of BPD,and to find effective control measures, it is of great significance to improve the survival rate of premature children, reduce childmorbidity and enhance the quality of our population.The study found that the process of alveolar is closely related to lung angiogenesis, the autopsy for children death of BPD found that the density and number of lung small blood vessels were significantly lower than that of normal newborns. Vascular endothelial growth factor (VEGF) receptor inhibitor SU5416, or a special anti-angiogenesis drugs, can effectively reduce pulmonary capillary formation, blocked the alveolar process, the numbers of alveolar and the areas of gas exchange were significantly reduced, showing striking similarities with the pathological features of BPD,so these findings suggest that VEGF has a close relationship with pulmonary angiogenesis and the incidence of BPD.VEGF is a mitogen with broad activity secreted by typeⅡalveolar epithelial cells, and it effectively participates in the process of the development of secondary pulmonary interval and alveolar, can promote endothelial cell proliferation, increase vascular permeability, with a strong role in stimulating angiogenesis. Large number of experimental studies have shown that normal VEGF signaling pathway is essential for alveolar development,VEGF may also play a critical role in late fetal lung development and maturation of alveolar type II cells. Directly applied to experimental animal models of BPD, VEGF gene can accelerate the alveolar development process of hyperoxia lung injury of rat,and improve lung structural abnormalities and vascular density. VEGF gene therapy for BPD microvascular block recovery is indeed a road of hope, but many problems still exist, on the one hand,it is reported that VEGF interventions are liposome directly injected into the airway, great local irritation and inflammation, and a very short half-life of VEGF in vivo (less than 6min), seriously affect the treatment effect; the other hand, the normal development of the pulmonary vessels is not only dependent on the regulation of VEGF and other angiogenic factors also requires the involvement of precursor cells,such as endothelial cells, progenitor cells and so on, therefore, a simple VEGF gene therapy to promote lung angiogenesis, the formation of the vascular abnormalities may exist.Study shows that transgenic technology can import angiogenesis regulatory factor gene into target cells to express certain regulatory factor protein and contribute to angiogenesis.Bone mesenchymal stem cells (MSCs) are isolated from bone marrow mononuclear cells and can be expanded ex vivo. Under appropriate culture conditions, bone marrow mesenchymal stem cells have the capacity to differentiate into cells such as bone, cartilage, adipocytes, myocytes, and even cardiomyocytes. Recent reports that bone marrow mesenchymal stem cells also has a role on lung injury and repair, it can differentiate into type I alveolar cells and alveolar type II cells,differentiate into vascular endothelial cells, the core of the differentiation factor is induced by vascular endothelial cells growth factor.Bone marrow mesenchymal stem cells also have immunomodulatory and anti-inflammatory effects, but only evoke little immune reactivity. Moreover, bone marrow mesenchymal stem cells are amenable to genetic manipulation, thus, these cells are currently being tested for their potential use in cell and gene therapy. Bone marrow mesenchymal stem cell transplantation may be involved not only repair damaged alveolar tissue, and, probably as an endothelial source of progenitor cells,involved in the formation of normal structure of lung angiogenesis. Vascular endothelial growth factor is the strongest known angiogenesis promoting factor. In recent years the application of VEGF 165 gene transfection of bone marrow mesenchymal stem cells for myocardial ischemia or limb ischemic disease of experimental and clinical studies have achieved initial success. So far, the research about lung injury by transfection VEGF 165 gene into bone marrow mesenchymal stem cells has not been reported. Therefore, the use of gene transfer technology to transfected VEGF gene into bone marrow mesenchymal stem cells to the lung, so the bone marrow mesenchymal stem cells could endocrine VEGF protein in the local in short-term, while the VEGF gene therapy to supply sufficient angiogenesis precursor cells, it is possible to solve the short maintenance time of simple VEGF treatment and the major stimulus and the drawbacks of vascular structural abnormalities which contributing to BPD model for normalization of lung structure and function.Based on the above theory, specific research includes the following five parts: construction of animal model of BPD in neonatal rat, isolation and purify rat bone marrow mesenchymal stem cells and construction rat VEGF 164 eukaryotic expression vector and transfect VEGF 164 gene into rat bone marrow mesenchymal stem cells with liposome, MSCs carried VEGF injected into the rat lung in BPD, to observe the pathological structure of lung tissue, the expression of VEGF protein andⅧfactor,and its role in the reconstruction of lung structure and function and its prognosis, to explore new avenues and open up new prospects for the clinical treatment of BPD.Therefore, a series of experiment studies were performed as follows:Part I Construction of animal model of neonatal rat BPDObjective:To explore the establishment of SD rat model of hyperoxia-induced lung injury, and to observe neonatal rat lung tissue structure, blood vessel density and VEGF proteinexpression and correlation of them.Methods:24 Sprague-Dawley neonatal rats were randomly continually exposed to hyperoxia (FiO2=95%) or room air (FiO2=21%,control group). Histological study of the lung tissue and radical alveoli count (RAC) were carrided out, and VEGF protein and factorⅧexpression in the lungs were determinded by immunohistochemical methods respectively in14 days after birth.Results:Alveolarization gradually makes progress,radical alveolar counts and VEGF protein and factorⅧexpression increased with increasing postnatal age in the control group.In the hyperoxia exposure group alveolarization were arrested, RAC and VEGF protein and factorⅧexpression was significantly reduced,statistic were significant differences (P<0.05).There has always been correlaiton between VEGF and factorⅧ.Conclusion:Hyperoxia exposure decrease VEGF protein and factorⅧexpression and arrest lung development in the lungs of neonatal rats. VEGF might be involved in the pathogenesis of hyperoxic lung injury.Part II Isolation and purification of rat bone marrow mesenchymal stem cellsObjective:The aim of this study is to research on the method to isolate and punify rat bone marrow mesenchymal stem cells and provides basis for the amendment of tissue defects by tissue engineering.MethodsrRat bone marrow mesenchymal stem cells were isolated by combining density gradient centrifugation with plastic adherence. Morphological observations were performed with phase contrast microscope; cell phenotype were detected by flow cytometry.Results:Higher purity of rat bone marrow mesenchymal stem cells could be achieved by density gradient centrifugation. The positive expression rates of cell phenotypes were various as followings respectively:CD29,98.9%; CD44,98.7%; CD34,0.0%; CD45,0.3%.Conclusion:Higher purity of rat bone marrow mesenchymal stem cells can be isolated and cultured by combining density gradient centrifugation with plastic adherence.Part III Transfection and expression of vascular endothelial growth factor gene in rat bone marrow mesenchymal stem cellsObjective:To observe the protein expression of exogenous gene after rat bone marrow mesenchymal stem cells transfected with rat VEGF 164 gene by liposome 2000.Methods:The vector pcDNA3.1(-)/VEGF164 was transfected into rat marrow mesechymal stem cells by liposome mediated and the rat bone marrow mesecchymal stem cells were divided into three groups:plasmid group,the empty plasmid group, liposome group.The cells in the plasmid group were transfected with pcDNA3.1(-)/VEGF 164,while cells in the empty plasmid group were transfected with pcDNA3.1(-) and cells in the liposome group were transfected with Lipofectamine 2000. VEGF expression in vitro was assessed by immunohistochemistry and Western blot.Results:Using liposome pcDNA3.1 (-)/VEGF 164 transfected rat MSCs,immunohistochemistry showed that MSCs transfected by pcDNA3.1 (-)/ VEGF 164 can be seen scattered yellow granules, and empty plasmid control group and liposome control group there were no obvious yellow granules. Western blot showed 72 h after pcDNA3.1 (-)/VEGF 164 transfected MSCs,cell lysis products of VEGF were significantly higher than the empty plasmid group and the liposome group.Conclusion:Rat bone marrow mesenchymal stem cells were transfected with VEGF 164 gene successfully and deffecitively express target gene and protein,provide a novel gene therapentic strategy for repairing lung injury which can possibly apply for clinic usage.PartⅣIntratracheal injection the MSCs carrying VEGF to rat lung of BPDObjective:To study the effects of transplanted vascular endothelial growth factor gene expressing bone marrow mesenchymal stem cells on pulmonary alveolar structure and microvascular of bronchopulmonary dysplasia in rats.Methods:To divide BPD rats into transfected group (MSCs/VEGF group), the control group (MSCs group),and blank group (serum-free medium group),10 in each group, respectively, to inject 1×105 MSCs transfected by VEGF, MSCs and the same amount of simple serum-free medium by airway. To get rats’ lungs after transplantation 1w and 4w,observe lung tissue by hematoxylin-eosin staining to study lung structure and radial alveolar counts (RAC), evaluate VEGF protein expression and angiogenesis densities by immunohistochemistry.Results:Transfected group significantly increased more than other groups in alveolar development, VEGF and factorⅧexpression, after transplantation 1w and 4w, the difference was significant (P<0.05).Conclusion:Transplantating bone marrow mesenchymal stem cells transfected by vascular endothelial growth factor gene can significantly promote pulmonary alveolar structure and microvascular regeneration, and improve lung function, which provides a novel gene therapentic strategy for BPD.