Reparation Of Mesenchymal Stem Cell Nsplantation On Airway And Lung Injury In Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease(COPD)is one of the most common disease and the 4th leading cause of death in the world. The morbidity has been rising even in recent years due to several factors such as environmental pollution. COPD is a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive. The pathogenesis of COPD is complicated and its pathobiology involve s multilevel structures in the lung, including central and peripheral airways, lung parenchyma,even blood vessel, which is regarded as inreversible. The injure of COPD can not be fully repaired by itself or drugs. Thus, we need badly to explore a new way of structural reparation and reestablishment of the lung.Recently, many studies have identified that bone marrow derived mesenchymal stem cells (MSC) and hematopoieticstemcells (HSCs) have the potency of multi-directional differentiation. Because of the abundant resource, convenient isolation, easy cultivation and vigorous proliferative ability, MSC has become increasingly common in stem cell research. By far, the MSC transplantation was widely used on the treatment study of different diseases and presented as a promising clinical application to them, including diabetes mellitus, myocardial infarction, graft-versus-host disease, and so on.Bone marrow derived MSC can differentiate into some lung parenchymal cells in vivo and in vitro, as confirmed by several studies, which observed that MSC could be guided to location of lungs by inflammation and injury. Exogenous MSC might repair the lesions in two ways: induction and cell differentiation into alveolar epithelium or tracheal epithelium under the local microenvironment, and immunological regulation of MSC for the advantageous repairing conditions.Nevertheless, studies on the application of MSC in COPD is few. Most researchs focused on the use of MSC in the acute pulmonary parenchyma injury or pulmonary fibrosis. Moreover, therapeutic efficacy of different stem cells transplantation program also need to be studied. This research was designed to explore the field planting and survivorship of exogenous bone marrow derived MSC in the lungs of the COPD model rats after transplantation, understanding the therapeutic efficacy of MSC on COPD models via systematical observation.The experiment consists of three parts described as follows:1. Isolation, culture in vitro and study of biological characteristic of the rat bone marrow derived mesenchymal stem cells.Objective To isolate the bone marrow derived mesenchymal stem cells and culture them in vivro. To study the biological characteristics of rMSC.Methods and materials Under aseptic condition, bone marrow cells were collected by flushing the femurs and tibias from 6-8-week-old healthy male Wistar rats with PBS. All the mononuclear cells from bone marrow were plated in the plastic culture dish. The cells was maintained in a incubator with humidified atmosphere of 95% air and 5% CO2 at 37℃, using DMEM-LG medium supplemented with 10% fetal bovine serum (FBS), and was splitted as 1:3 every 3 or 4 days. The morphological features were observed under the invert microscope; Cell growth curve was determined by MTT method; cell cycle and phenotype were taken by flow cytometry. Moreover, their abilities to differentiate along osteoplastic and adipocytic pathways were also investigated. Tumor genesis were observed in BALB/c-nu/nu nude mice which received rMSC subcutaneously.Results The cells cultured in vitro showed spindle-shaped appearance like fibroblast, spreading radiatly. Up to 20 passages of rMSC were observed and no visible morphologic or growth velocity change was detected. Cell cycle ananlyses revealed that 53.07% and 85.24% of the cells at G0/G1 phase, 46.93% and 14.76% at S+G2+M phase when they attached to 30%-40% and 90% confluences, respectively. Flow cytometric analysisshowed that the rMSC were high positive for CD29,CD44 and Sca-1 and negative for CD34,CD117,CD45, Flk-1 and MHC-II. The rMSC could be differentiated into adipocyte and osteoblast cells in vitro. No tumor formation were observed in nude mice during those 3 months.Conclusions The rMSC can be isolated and cultured by all bone marrow cells adherence successfully. The cell population we harvested expressed morphology and phenotype of mesenchymal stem cells, significant renewal capacity, biological stabilities, multi-potentiality and free from tumorgenesis in vitro.2. Establishment and assessment of the rat COPD model. Objective To explore an effective and reasonable way of establishing a rat model of chronic obstructive pulmonary disease(COPD).Methods and materials A total of 15 8-week-old healthy Wistar rats were divided into 2 model groups and the control group(Group C) randomly. The rat COPD models were established by two ways, intratracheal instillation of lipopolysaccharide (LPS) twice + exposure to cigarette smoke for 1 month(Group A), and cigarette smoke inhalation for 80 days only(Group B). The pathologic characteristics of animal models, including the mean lining interval(MLI) and the mean alveoli number(MAN), were determinated to assess the model quality. The general state of health of those rats were observed. Total cell counts and different cell percentage of bronchoalveolar lavage fluid(BALF) were determined. The comparison between the two model groups were also carried out.Results In the two model groups, the rats presented cough or breathlessness periodically, and amplification of weight were reduced than that of control group(P<0.01). Rats in Group A and B shared specific pathological features in tracheobronchial and lung tissues with that of human chronic bronchitis and obstructive emphysema. Significant increase in total white blood cells and neutrophils in BAIF and peripheral blood was found in Group A and B (P< 0.01) than those of Group C. MLI in Group A and B was significantly higher in comparison with Group C, while MAN was reversely much lower( both P<0.01). But there was no statistical difference between Group A and B on those measurements. The histology of Group A showed more often inflammatory cells infiltrating in the bronchial and lung tissue and the secretion of airway than Group B. The latter was charactered on the ruptured and enlarged alveoli.Conclusions By intratracheal instillation of LPS twice in addition to exposure to cigarette smoke and by cigarette inhalation only for more time both successfully established the rat COPD models, which shared many characteristics of human COPD including the pathological and pathophysiological feature.3. Implantation and differentiation of rMSC in the lungs of COPD model rats and its therapeutical effect.Objective To observe the implantation and differentiation of rMSC in the lungs of COPD rats, to study the therapeutical effect of rMSC.Methods and materials The rMSC from the bone marrow of male rats were cultured in vitro and labeled with CM-Dil before implantation into rats. The recipient Wistar rats, totally 36 female rats, were divided randomly into five groups: low-dosed/one-timed transplantation group (Group A, n=12, COPD model rats, intravenous infusion of 1×106/1ml CM-Dil-labeled rMSC after model establishment); large-dosed/twice transplantation group (GroupB, n=12, COPD model rats, intravenous infusion of 1×106/1ml CM-Dil-labeled rMSC twice in the same day after model establishment); rMSC control group (Group C, n=4, the same amounts of rMSC as Group A were injected into the normal female rats); COPD model rats group (Group D, n=4, COPD model rats, intravenous infusion of 1ml NS after model establishment) and normal control group (Group E, n=4). The COPD models were established by smoke inhalation + LPS perfused intratracheally. The rats in Group A and B were sacrificed at d1, d7, d15 and d30 after the intravenous infusion of rMSC. The frozen sections of fresh lung tissues samples were viewed under a fluorescence microscope to observe the implantation, distribution and survivorship of the CM-Dil positive cells. Hybridization in situ(ISH) and histopathological analysis were carried out to detect the localization, implantation efficiency and morphology of foreign cells in the lungs and other organs. CM-Dil label combined immunohistochemistry was used to identify the transdifferentiation of rMSC in the lung. Peripheral blood(PB)and bronchoalveolar lavage fluid(BALF)were harvested for cell counting and classification and the measurement of IL-10, TNF-αand G-CSF levels by ELISA. Statistical comparison were tested with single-factor variance analyses. The reported P value was 2-sided(α=0.05). Calculations were performed using the software SSPS13.0.Results (1)Many CM-Dil positive cells(red fluorescence)were observed in the lung tissues of Group A and B, mainly located in the interstitium, pulmonary alveoli and airway walls, even 30 days after the intravenous infusion of rMSC. Only a few CM-Dil positive cells were found in Group C, and none was detected in Group D and E,which were infused NS instead of rMSC. (2) The results of ISH were similar as those above. Moreover, some ISH positive cells presented with the morphology of type II alveolar epithelium, bronchial epithelium or vascular smooth muscle cell. None or few foreign cell was detected in other organs like the heart, the skeletal muscle and the liver. The implantation efficiency of rMSC in Group B were close to that in Group A. (3) Some CM-Dil positive cells also showed immunohistochemical positivity of SPC(molecular marker for type II alveolar epithelium) or CC16(molecular marker for Clara cell) in the same time. (4)The total white blood cells and neutrophil percentage in the PB and BALF of Group A and B were significantly lower, comparing to those of Group D(P<0.01),but still higher than those of Group Cand E(P<0.01). (5)The IL-10 level of the PB and BALF in Group A and B were significantly higher than that of Group D, while the level of TNF-αand G-CSF were higher(all P<0.01), even the IL-10 level of Group A and B were still much lower than those of Group C and E, and the TNF-α, G-CSF levels were higher reversely (all P<0.01). (6)HE-stained sections revealed obviously pathologic improvement in Group A and B in comparison with Group D, which implied the therapeutic effect of rMSC.Conclusions The rMSC infused from caudal vein can implant into the lungs of COPD model rats and be permanent planting there, improving the pathology of the recipient rats and regulating the local and systemic inflammatory factor level. The therapeutic efficacy of rMSC transplantation showed dose-independent to some extent.