The Effect Of The Transplantation Of Rat Bone Marrow Mesenchymal Stem Cells On Rat Pulmonary Emphysema And Its Mechanisms

Chronic Obstructive Pulmonary Disease (COPD) is a chronic respiratory disease that is associated with an abnormal inflammatory response of the lungs to noxious particles or gases. This leads to chronic bronchitis-bronchiolitis and/or emphysema that cause airflow limitation that is not fully reversible. COPD is the fifth leading cause of death worldwide and the prevalence and mortality of COPD are expected to increase in the coming decades. The current view on the pathogenesis of COPD is that cigarette smoke induces the recruitment of inflammatory cells, which then release reactive oxygen species and proteolytic enzymes, causing the degradation of lung matrix and the death of structural cells. Apoptosis of structural cells in the lung might possibly be an important upstream event in the pathogenesis of COPD. There is an increase in apoptotic alveolar epithelial and endothelial cells in the lungs of COPD patients. Since this is not counterbalanced by an increase in proliferation of these structural cells, the net result is destruction of lung tissue and the development of emphysema. In several animal models of COPD, development of emphysema was observed despite a remarkable lack of pulmonary inflammation. These studies demonstrated that, at least in animal models, apoptosis of alveolar wall or endothelial cells is sufficient to cause pulmonary emphysema, even without the accumulation of inflammatory cells. Apoptosis interacts with all of these pathways of inflammation, proteinase/anti-proteinase imbalance and oxidative stress, adding to the complexity of the disease. More studies are needed to identify the most important apoptotic pathways involved in the development of COPD. Moreover, it has to be evaluated if apoptosis can be used as a therapeutic target to prevent further deterioration of the disease. Finally, as disturbance of the balance between apoptosis and regeneration of structural lung cells seems important in the destruction of healthy lung tissue, it would be interesting to evaluate the potential use of stem cell therapy for emphysema.Recently, research of stem cells has garnered great attention and has shown promise by changing the view of traditional therapeutics, with broad impact on tissue injury and regeneration. Stem cells are unique cells that have the capacity for limitless self-renewal and differentiation. In experiments, cells of MSCs origin differentiated into mesenchymal tissue, such as bone,cartilage and muscle, as well as non- mesenchymal tissue, such as endothelium,hepatocytes and neural cells. Respiratory stem cell research has been less active and has moved more slowly than that of many other organs, such as cardiovascular system and nervous system. This is probably due to the complexity of structures and functions of the lung and airway system. Ortiz et al have reported that bone marrow derived stem cells may be able to alleviate bleomycin induced fibrosis, this is the first study that has shown noticeable effect in stem cell derived cell therapy for pulmonary disease. The study of the effect of injury lung by stem cell transfer is carried out. The focus is if stem cell is essential to lung injury and which type of lung cell that stem cell can differentiate.The reports of differentiation potency and effect of stem cell in lung are different. This has been recently confirmed by Hashimoto et al who transplanted EGFP expressing MSC/HSC into mice that received bleomycin. Induction of pulmonary fibrosis in such chimera mice by intratracheal bleomycin injection caused large numbers of GFP cells to appear in active fibrotic lesions, while only a few GFP cells could be identified in control lungs. This indicates that in the fibrosis model, most fibroblasts are derived from BM precursor cells. Mauricio Rojas demonstrate the effect of marrow–derived mesenchymal stem cell following bleomycin and busulfan-induced lung injury in mice. The cells of GFP-MSCs differentiated into type I alveolar epithelium,type II alveolar epithelium, myofibroblasts and endothelial cells in injure lung. The variations of the differentiated capability of HSC/MSC in this regard may be due in part to differences in techniques and experimental models, such as the extract method of bone marrow stem cell,the method of injury of lungs,the detection method of graft in lung. But now, it was not confirm the quantity of stem cell transfer following lung injury,opportunity,the effect of stem cell reparation in lung.MSCs secrete growth factors, such as interleukin-6(IL-6),IL-7,IL-8,IL-15,IL-11,IL-12,IL-14,leukaemia inhibitory factor(LIF),colony-stimulating factor (GM- CSF), stem cell factor and vascular endothelial growth factor (VEGF),basic fibroblast growth factor(bFGF),nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF). Chen demonstrate that intravenously administered male MSCs increase bFGF expression, reduce apoptosis, promote endogenous cellular proliferation, and improve functional recovery after stroke in female rats. Kocher demonstrate the transplantation of stem cell can decrease apoptosis of hypertrophied myocytes in the peri-infarct region and improve cardiac function. Stem cell not only have differentiated capability, but also can suppress apoptosis by some methods in injury position.The report is few,such as if the transplantation of MSCs can repair lung emphysema, which type of lung cell it can differentiate, which kinds of cytokine it can secrete under lung microcirculatory ,if it have the effect of anti-apoptosis, what path of anti-apoptosis is.This study was aimed to investigate the effect of MSCs transplantation in lung tissue of papain and Co60-induced pulmonary emphysema, find out the mechanism of emphysema,the mechanism of repair lung injure after MSCs transplantation and provide experiment date of stem cell therapy emphysema .Part 1 In vitro culture and differentiation of rat marrow-derived mesenchymal stem cellsAim: To establish the method of isolating and culture of rat marrow-derived mesenchymal stem cells (MSCs), investigate the biological characteristics of MSCs. Methods: Mononuclear cells from 2-month-old rat bone marrow were isolated by using density gradient centrifuge method .The cells adhered to the wall of flasks were preserved for passaging. Then the proliferation capability, cell cycle, immunophenotype, differentiation to adipocytes,osteoblasts cells were investigated.Results: Rat bone marrow derived MSCs have proliferative and multilineage differentiation potential.Conclusion: Using our method of isolating and culture of rat marrow-derived mesenchymal stem cells, MSCs possessing the potential of differentiation potential in vitro can be got,which can be used as the seed cells in the tissue engineering. Part 2 The effect of irradiation on the engraftment of rat bone arrow mesenchymal stem cells in different organs of recipient ratsAim:To investigate the effect of irradiation on the engraftment of rat bone marrow mesenchymal stem cells in different organs of recipient rats and possible mechanisms.Methods:The apoptosis in the irradiated rats'heart,kidney,liver and lung were assessed by TUNEL at day 14 after irradiation. MSCs cultured from male rats were delivered systemically into irradiated(60COγ)and unirradiated syngeneic female rat and sacrificed at day 28 after MSCs transplantation. Tracking of MSCs were determined by PCR and Y chromosome fluorescent in situ hybridization (Y-FISH).Results:We found that the number of apoptosis cells in the heart,kidney,liver and lung was significantly greater in the irradiated rats compared with the unirradiated controls. Y chromosome sequence was amplified in the irradiated recipient's heart,kidney,liver and lung. Moreover, we found cells with Sry gene in these organs by Y-FISH in the irradiated recipient's. However, no Sry gene sequence and cells with Y chromosome were found in the unirradiated recipients.Conclusion:Total body irradiation promotes the apoptosis in the rats'heart,kidney, liver and lung. Irradiation results in the engraftment of MSCs in the recipient's heart,kidney,liver and lung.Part 3 Apoptosis of alveolar wall cells is involved in rat papain-induced emphysematous changesAim:To observe the apoptosis of alveolar wall cells in the papin-induced rat emphysema, and explore the role of apoptosis of alveolar wall cells in the pathogenesis of emphysema.Methods:Rats were randomly divided into three groups: normal control group, papain only group, papain+irradiation group. Morphology of lung tissues was assessed. TUNEL assay was used to determine the apoptic cells. Immunohistochemistry was performed to determine the expression of PCNA,Bax and SP-C in the lung alveolar wall cells . SP-C immunofluorescence staining was performed to identify the type II alveolar cells in the TUNEL positive cells.Results:Destruction of alveolar wall and loss of the alveolar unit were observed in papain only group and papain+irradiation group. Compared with rats in papain only group there was significant difference in rats of papain+irradiation group in the mean linear interval(MLI),the number of alveolar counted per unit area (MAN)and mean alveoli area (MAA), respectively (P<0.01). The PI,AI and the percentage of Bax in the papain only group and papain+irradiation group were significantly greater than those of the normal control group(P<0.01). However, the percentage of SP-C positive cells was siginificantly lower in the papain only group and papain+irradiation group compared with the normal control (P<0.01). Moreover, the PI,AI and the percentage of Bax in the papain+irradiation group was higher than the papain only group. The percentage of SP-C positive cells in the papain+irradiation group was lower than those of the papain only group. Most of the TUNEL positive cells expressed the SP-C .Conclusion:Rat alveolar wall cells, especially type-II cell apoptosis may take part in the pathogenesis of emphysema.Upregulation of Bax expression may be involved in alveolar wall cells apoptosis in papain-induced emphysema.Part 4 Transplantation of rat bone marrow mesenchymal stem cells improves the rat pulmonary emphysemaAim:To study the effects of bone marrow MSCs transplantation on the apoptosis of alveolar wall cells , the expression of Bcl-2 and Bax in lung tissue of papain and Co60-induced pulmonary emphysema, and investigate the engraftment and differentiation of bone marrow MSCs in the recipient rat lung.Methods: Female Lewis rats were randomly divided into three groups: normal control group, emphysema group, emphysema+MSCs transplantation group. Rats were instilled with papain intratracheally and exposed to Co60 irradiation to induce pulmonary emphysema. Bone marrow MSCs were injected through tail veins.Rats were sacrificed at days 14 and 28 after treatment. Morphologic analysis of the rat lung was performed. The apoptosis of the lung cells was assessed by TUNEL staining. The expression of Bcl-2 and Bax were determined by immunohistochemical staining. The engraftment of male bone marrow MSCs in female recipient lung was determined by PCR and Y chromosome fluorescent in situ hybridization (Y-FISH). Surfactant protein C (SP-C) immunoflourescent staining and Y-FISH were performed on the same slide to determine whether the engrafted bone marrow MSCs differentiated into type II pneumocytes.Results: Emphysematous changes of the lung tissue were observed in emphysema group and emphysema+MSCs transplantation group. However, the emphysematous change in emphysema+MSCs transplantation group was improved compared with the emphysema group. There was significant difference in the number of alveolar counted per unit area (MAN),mean alveoli area (MAA) and mean linear interval(MLI) between emphysema group and emphysema+MSCs transplantation group. The apoptotic index of the alveolar wall cells in emphysema+MSCs transplantation group was less than that of the emphysema group. The percentage of Bcl-2 positive cells in emphysema+MSCs transplantation group was significantly higher than that of the emphysema group. The percentage of Bax positive cells in emphysema+MSCs transplantation group was significantly lower than that of the emphysema group. The ratio of Bcl-2/Bax of emphysema+MSCs transplantation group was significantly higher than that of the emphysema group. DNA fragment of Sry gene which is located on Y chromosome was amplified in the genomic DNA from the lungs of rats in emphysema+MSCs transplantation group. Y chromosome positive cells were observed in the lungs of rats from emphysema+MSCs transplantation group. Some of the Y chromosome positive cells also expressed SP-C, the marker of type II pneumocytes.Conclusion:Bone marrow MSCs transplantation inhibits the apoptosis of alveolar wall cells, upregulates the expression of Bcl-2 and downregulates of the expression of Bax, and can differentiate into type II pneumocytes.This may be part of the reason that bone marrow MSCs transplantation improves the papain and Co60-induced pulmonary emphysema.