Experimental Study Of Bone Marrow Mesenchymal Stem Cells In The Treatment Of Fibrosis Of Lung Induced By Bleomycin In Rats

Part One:A study on the isolation, culture, purification of bone marrow-derived mesenchymal stem cells in Sprague Dawley rats and identification of its biological characteristicsObjectiveBone marrow mesenchymal stem cells (BMSCs) has multiple potential differentiation, which can promote the regeneration of mesenchymal tissue. The content of BMSCs in bone marrow is very low, but tissue engineering requires a large number of seed cells, the difficulty of separating BMSCs from animal bone marrow has largely restricted the development of many experiments. Therefore, isolation and culture of high purity, vitality, and stability of the biological characteristics of BMSCs is essential. To explore the method of isolation, culture, purification of SD rat bone marrow mesenchymal stem cells in vitro and identification of its biological characteristics. To establish an ideal isolation and culture system of SD rat bone marrow mesenchymal stem cells for further study and clinic application.Materials and Methods1. Primary culture of BMSCs:10 clean-grade male SD rats with 6-8 weeks old were purchased. The rats were anesthetized by 2% pentobarbital and soaked with 75% ethanol for 10 minutes. The BMSCs were obtained by flushing the bone marrow cavity of rats with PBS to produce single-cell suspensions. Centrifugation, discard the supernatant and heavy suspension, The cell concentration of 3-4×105 cells were inoculated in 25cm2 culture bottle, placed it in 37℃, CO2 saturated humidity incubator.2. Cultivation, purification and passage of BMSCs:In the primary culture, the culture medium was changed every 2-3 days till the cells were confluent a single layer on the bottom of the bottle, digested with 0.25% trypsin, subcultured at the ratio of 1:2.3. The morphological observation of BMSCs:Characteristic morphology of cultured BMSCs was observed by inverted phase contrast microscope every day and took photos.4. Identification of BMSCs4.1 Differentiation of BMSCs in vitro:Select the third generation BMSCs, according to 5×103 cells/cm2 inoculated in a 6-well culture plate until the cells adhered to the wall to the cell density reached 80%, adding osteogenic induction medium or adipogenic liquid, cell culture hole without induction medium as control. Adipogenic inducers for 14 days, fixed by 10% formaldehyde, oil red O staining was performed on the differentiation of fat cells; Osteoblasts induced for 21 days, fixed by 10% formaldehyde, Alizarin Red S staining on osteoblasts differentiation.4.2 Flow cytometry was used to detect cell surface markers:Harvest P3 cells, digest with 0.25% trypsin,4℃ centrifugal,1200rpm for 5 minutes, washed the cell 2 times with PBS and resuspended, cell amount was counted, the tube were sequentially added monoclonal antibody CD29, CD90 and CD34. At the same time, each sample set up the same type of negative control tube. Avoid light incubated at room temperature for 30 minutes, washed cells 2 times with PBS to remove unbound antibody, with 100μl PBS cells resuspended and flow cytometry were detected and analyzed.Results1. BMSCs morphology observation Primary culture:After Bone marrow cells were seeded in culture flask, the cells were round and suspended in the culture medium. After 24 hours, the cells adhered to the wall, round, fusiform or polygon. With the build-up culture days, some colonies gradually emerged in a radial array. About 3-5 days later, cells fused on the whole, meanwhile, the morphologic change of cells colonies shows radiating or whirlpool-like.12-14 days later, cells were tightly arranged. Subculture:The morphology and growth rate of the cells could be stably and continuously for 10 passages. It was found that BMSCs were spindle shape and the size of BMSCs was homogeneous under microscope.2. BMSCs osteogenic differentiation and identification:P3 generation rats BMSCs induced by osteogenic medium for twenty-first days using alizarin red staining, the BMSCs could be induced and differentiated to osteoblasts.3. BMSCs adipogenic differentiation and identification:Revulsant for adipose could differentiate BMSCs to adipose cells, appearing adipose globelet and Oil Red O positive in cytoplasm.4. Flow cytometry showed that CD29, CD90 membrane antigen could be detected in 98% of the third generation of BMSCs, but not CD34 membrane antigen.ConclusionsAn effective and ideal isolation and culture system of SD rat bone marrow mesenchymal stem cells in vitro has been established. The BMSCs were isolated and purified by density gradientcentrifugation combined with attachment culture method have high purity and proliferation activity. The biological properties are stabile. It applicates for further research on BMSCs.Part Two:Comparation of the model of blemy cin-induced pulmonary fibrosis in rats with different methodsObjectivePulmonary fibrosis (PF) is a severe chornic interstitium disease in the lung. The prognosis of this disease is unfavourable owing to the lack of effective drug therapies. So the new drugs researches of PF are eagerly requested. And making successful models of pulmonary fibrosis is the foundation of drug research. The main purpose of this study is to provide a satisfactory foundation for the drug researche of PF by sorting and comparing multiple methods of establishing vitro models and vivo models of pulmonary fibrosis. To provide a better model matching with human pulmonary fibrosis by studying a model of pulmonary fibrosis in rats.Materials and MethodsThe Sprague Dawley rats were administered with intraperitoneal or tracheal injection of bleomycin (BLM), and were sacrificed after treatment on days 7,14 and 21, respectively, and their lungs were harvested for histological studies. The ratio of wet to dry of lung tissue weight (W/D) was detected; Hydroxyproline (HYP) in lung tissue was assessed; Light and electron microscopic evaluation were also done on rat lung tissue and then the index of quantitative assessment of histologic lung injury (IQA) was detected. In addition, width of alveolar septa and pleura depth, and hydroxyproline in lung tissues homogenate were measured; The content of TNF-a and IL-6 in bronchoalveolar lavage fluid was determined by enzyme linked immunosorbent assay (ELISA) method; The expressions of transforming growth factorβ(TGF-β) protein were analyzed by immunohistochemistry.Results1. There was no difference in the starting weight of rats in each group. The weight of the rats become lower after given bleomycin (P<0.01).2. The ratios of W/D were obviously higher in model group than those of control groups (P<0.01). Compared with control group, a comparison of values of IQA in model group were clearly higher (P<0.01). However, the ratios of W/D and values of IQA in model B groups were evidently lower than that in model A groups ((P<0.05 or P<0.01).3. Compared with control group, The content of HYP in model group shown an increase tendency accompany with the time and reached a peak on the 21th day.4. The expression of IL-6 and TNF-a in model B group was significantly higher than that in model A group on each time point (P<0.05 or P<0.01)5. TGF-β protein were up-regulated in lung tissues of model group than that in control groups (all of P<0.01).6. The distribution of the lesions in the lung of the rat was dominated by peripheral small bronchus, the early stage of lung inflammation was heavy, and the distribution of the lesion in the abdominal cavity was mainly in the pleural and lung tissue, and the inflammatory reaction was relatively mild.7. Electron microscope showed there were serious injury imposed on lung tissue in model groups. Different degree damage occurred in basilar membrane and endodermis of pulmonary arteriole. Lots of neutrophils and RBCs seeped into alveoli, blood capillary and intra-alveolar space. Injured alveolar epithelial could be seen. Relatively, only lightly damage occurred in control group, the ultrastructure of lung tissue was well preserved.8. There was a significant correlation between expression of TNF-a、IL-6 and TGF-β and HYP (r=0.833,0.882,0.861; all of P<0.01), as also that of W/D and IQA (r=0.418,0.849; all of P<0.01).Conclusion1. Both two administration methods could be used to induce pulmonary fibrosis and show a typical pulmonary fibrosis disease.2. BLM induced pulmonary fibrosis in rats, which may be through the promotion of lung TNF-a, IL-6 and TGF-β,causing inflammation of bronchial alveolar epithelium and vascular endothelial cell injury and abnormal repair and destroy the integrity of the alveolar capillary membrane, stimulate fiber cell abnormal proliferation and collagen synthesis, promote the pathogenesis of lung inflammation and lung fibrosis.3. The process and histological changes of the model for pulmonary fibrosis in rats established by intraperitoneal administertion was similar to that of human IPF, possiblely. Intraperitoneal administration may be an ideal method for the reproduction of animal model.Part Three:Experimental study of bone marrow mesenchymal stemcells in the treatment of fibrosis of lung induced by bleomycin in ratsObjectivePulmonary fibrosis is a chronic progressive disorder characterized by the excessive proliferation of fibroblasts and deposition of extra-cellular matrix, which destroy the architecture and function of normal lung tissue. The precise pathologic mechanisms of pulmonary fibrosis are not fully understood, and the current managements for it are not satisfactory. Therefore, it is crucial to find new therapeutic strategies for pulmonary fibrosis.Oxidative stress is an important molecular mechanism underlying fibrosis in a variety of organs, including the lungs. Oxidative takes part into the process of fibrosis by damaging cellular macromolecules such as DNAs, lipids, and proteins via oxidative stress-induced tissue injury which is caused by excessive levels of reactive oxygen species (ROS). Nrf2-ARE pathway plays an important role in the defense against oxidative stress. Recently, the functions of Nrf2 and its downstream genes have been shown to be important for protection against oxidative stress and chemical-induced cellular damage in various type cells, tissues and organs. Therefore, we hypothesized that the Nrf2-ARE pathway may be involved in the mechanism of cytoprotection in bleomycin-mediated oxidative stress of pulmonary fibrosis, and the activation of Nrf2 can decrease the sensitivity of bleomycin-induced oxidative damage in pulmonary fibrosis.Bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells which are capable of self-renewal and can differentiate into a variety of lineages under suitable conditions. BMSCs are now extensively applied in the fields of regenerative medicine, gene therapy and tissue repair. Therefore, BMSCs are thought to be an ideal candidate seeding cells for cell therapy and represent an excellent clinical implication prospect. In this study, we used in vivo bleomycin-induced lung fibrosis model to investigate the protective role of Nrf2 against the development of bleomycin-induced pulmonary inflammation and fibrosis and assess the potential therapeutic effect of BMSCs.Materials and MethodsIn vitro, BMSCs of SD rats were cultured and subcultured to the fourth generation was used in the experiment. Forty rats were randomly divided into four groups with ten animals in each. The control group animals received an intraperitoneal injection of normal saline alone. The bleomycin group animals were subjected to intraperitoneal injection of bleomycin (15mg/kg) for 5 days. The bleomycin plus BMSCs group of animals received the same dosage of bleomycin as that of bleomycin group animal and were treated with the suspension of BMSCs (2×106/1.5 ml) via the tail vein for 5 days. The BMSCs alone group received an intraperitoneal injection of normal saline and were then treated with BMSCs (2×106/1.5 ml) via the tail vein for 5 days. Twenty-one days after the bleomycin treatment, the animals were sacrificed. Take lung tissue for detecting the wet/dry (W/D). The pathological observation of lung tissue was obtained with HE and Masson stain. Hydroxyproline (HYP) in lung tissue was assessed. Malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were assessed by Thibabituric Acid (TBA) and xanthine oxidase respectively. The expressions of Nrf2、NQO1、HO-1 and γ-GCS protein were analyzed by western-blotting.Results1. A significant increment in the W/D and HYP was observed in the bleomycin-treated animals, compared to saline-treated control animals (P<0.01). BMSCs administration decreased the W/D and HYP in animals treated with bleomycin. Simultaneously, the increased W/D and HYP of the bleomycin-treated animals were prominently reduced when treated with BMSCs (P< 0.01).2. HE and MASSON staining showed that BMSCs significantly reduced the degree of alveolar inflammation and pulmonary fibrosis in rats.3. Western-blotting demonstrated that BMSCs could increase the expressions of Nrf2, NQO1, HO-1 and y-GCS protein (P< 0.05, P< 0.01)4. BMSCs significantly increased superoxide dismutase (SOD) activity and inhibited malondialdehyde (MDA) production in the injured lung.5.There was a significant correlation between expression of NQO1、HO-1、γ-GCS and Nrf2 (r= 0.847,0.924,0.957; all of P< 0.01).Conclusion1. In this study, we demonstrated BMSCs attenuates the histological abnormal changes and reduces the levels of hydroxyproline in pulmonary fibrosis induced by bleomycin in rats, significantly increases the activities of antioxidant enzymes (SOD) and decreases MDA levels in lung tissues.2. BMSCs attenuated bleomycin-induced oxidative stress, histological alterations and collagen depositions via the activation of NQO1, y-GCS, and HO-1 expression and the Nrf2 pathway. BMSCs is a promising treatment in protecting early lung tissue damage induced by bleomycin exposure or might be utilized in combination therapy for PF in clinics.