| Background:Chronic obstructive pulmonary disease(COPD) is a major public health problem. Its pathogenesis has not been yet completely clear till now, mainly concerned with inflammation, proteinases-antiproteinases imbalance, oxidative stress and functional disorder in autonomic nervous system function(such as anomaly distributing of cholinergic nerve receptor). Recent findings have manifested that apoptosis may destroy alveolar wall cells, participate in emophysematous change. It plays an important role in pathogenesis of COPD.COPD pathology damage belongs to irreversible changes and lack of an ideal clinical treatment. Therefore an effective method is required to repair damaged organization.In recent years, bone marrow derived mesenchymal stem cells(MSCs) have becoming the hot spot of study of stem cells. Those not only have differentiation function but also have secretory function. One idea that bone marrow derived MSCs intervention may repair COPD is put forward.For this reason, this topic was aimed to investigate the effect of bone marrow derived MSCs intervention in lung tissue of papain and Cobalt-60-induced emphysema, to find out the formation mechanism of COPD and the repaired mechanism of bone marrow derived MSCs intervention on the apoptosis of alveolar wall cells of emphysema, and to provide experiment date of treatment of COPD with stem cell.Objective:1. To observe the biological characteristics of rat bone marrow derived MSCs.2. To investigate the apoptosis of alveolar wall cells in the papain and Cobalt-60-induced emphysema rats.3. To investigate the function of rat bone marrow derived MSCs intervention in the apoptosis of alveolar wall cells and the development process of emphysema in emphysema rats.Methods:1. Primary culture: Mononuclear cells from 8-week-old male rat bone marrow were isolated under sterile conditions by using density gradient centrifuge method. The cells adhered to the wall of flasks were preserved for passaging. The growth of bone marrow mesenchymal cells were observed.2. Cells analysis: Cell growth curve, cell immunophenotype and cell cycle was measured. Differentiation to osteoblasts and adipocytes cells were investigated in vitro.3. Animal grouping:12-week-old femal rats were randomly divided into four groups(n=6 in each group):normal control group(group A), papain only group(group B), papain+irradiation group(group C), papain+irradiation+MSCs intervention group(group D). Animal modeling: Group A:Rats were instilled intratracheally with phosphate buffer(PBS), then they were injected with PBS into tail vein.Group B:Rats were instilled intratracheally with papain, then they were injected with PBS into tail vein. Group C:Rats were instilled intratracheally with papain and exposed to Cobalt-60 irradiation, then they were injected with PBS into tail vein. Group D:Rats were instilled intratracheally with papain and exposed to Cobalt-60 irradiation, then they were injected with MSCs into tail vein. After 8 weeks animals were executed.4. Detection of indexes: Histopathological change of lung was assessed. The apoptotic index of alveolar wall cells was determined by TUNEL assay. The number of type-â…¡alveolar epithelial cells was detected by Immunohistochemistry. Sry on Y chromosome was detected by PCR. The level of vascular endothelial growth factor(VEGF) in bronchoalveolar lavage fluid(BALF) was inspected by ELISA. Expression of VEGF and VEGF receptor 2(VEGFR2) protein in lung tissue was analysed by Western blotting.Results:1. Rat bone marrow derived MSCs had proliferative and multilineage differentiation potential in vitro.2. Group B, group C and group D all showed emphysema changes. Compared with group A, there were significant differences in group B, group C and group D in the mean linear interval(MLI),the mean alveoli area (MAA) and the number of alveolar counted per unit area(MAN), respectively(P<0.01). However, the emphysematous change in group D was improved compared with group C. There were significant differences in the MLI,MAA and MAN between group D and group C(P<0.01).3. The apoptotic indexs(AI) of the alveolar wall cells in group B, group C and group D were significantly higher than that in group A, respectively(P<0.05). However, the percentages of surfactant protein-C(SP-C) positive cells were significantly lower in group B, group C and group D compared with group A(P<0.05).The AI of the alveolar wall cells in group D was lower than that in group C(P<0.05). However, its the percentage of SP-C positive cells was significantly higher compared with group C(P<0.05). 4. DNA fragment sequence, which was concordant with normal male rats, was amplified by PCR technique in group D lung tissues.5. The levels of VEGF in BALF, the levels of VEGF and VEGFR2 protein expression in lung tissue were significantly lower than that in group B, group C and group D compared with group A(P<0.05).The level of VEGF in BALF was significantly higher in group D compared with group C(P<0.05).And the levels of VEGF and VEGFR2 protein expression in lung tissue were significantly higher in group D compared with the group C(P<0.05).Conclusions:Rat alveolar wall cells, especially type-â…¡alveolar epithelial cells apoptosis may participate in formation of emphysema. Reduced level of VEGF expression in lung tissue may be involved in alveolar wall cells apoptosis in emphysema rats.The degrees of alveolar wall cells apoptosis and emphysemous changes in emphysema rats were decreased after bone marrow derived MSCs intervention possibly due to that MSCs divided into alveolar wall cells and reversed the levels of VEGF and VEGFR2 expression. |
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