| Objective:Chronic obstructive pulmonary disease is a chronic lung disease which characterized by irreversible airflow limitation and cigarette smoking is the most important pathogenic factor of COPD.Increasing morbidity and mortality,as well as the insufficient treatment,have brought a large economic burden to our country and even the world.This experiment explored whether the BMSCs,which overexpressed of CXCR4,could delay establishment of apoptosis models of COPD by inhibiting apoptosis.Methods:1.Extracted and purified BMSCs from SD rat through the cell adherent method in vitro.2.After passage 3,BMSCs were identified by flow cytometry,and induced to differentiate to adipocyte-oriented,chrondrocyte-oriented and osteocyte-oriented cells.3.Retrovirus transfection methods were used to establish the CXCR4-overexpression model of BMSCs.The expression of CXCR4 was examined by WB and qRT-PCR.4.Apoptosis models of COPD were established by exposure to cigarette smoke.Seventy-two SD rats were randomly divided into control group,model group,CXCR4-BMSCs treatment group and pBABE-BMSCs treatment group.BMSCs were transplanted in vivo twice a month during building of the models.Six rats were sacrified in each group every month,observed histological alterations and detected the alveolar mean linear intercept(Lm)on the lung.TUNEL was used to detect the apoptosis signal of lung cells,and western blot was used to detect and compare the concentrations of related apoptosis proteins in the lungs of four groups.Results:1.The cells which isolated and purified through the cell adherent method in vitro,could be differentiated into adipocytes,osteocytes and chondrocytes.Flow cytometry(FCM)results showed that the positive rate of CD90 and CD29 is 97.15%,97.36% respectively,whereas the positive rate of CD45 and CD34 is 0.62%,0.43% respectively.The results above indicated that the cells which were isolated and purified were BMSCs.2.Both CXCR4 mRNA and protein expression of CXCR4-BMSCs group were significantly higher than pBABE-BMSCs group.(p<0.01).3.Continuous exposure to cigarette smoke could cause alveolar septal rupture: compare among all the model groups,the alveolar MLI of the first month was significantly lower than the third month(p<0.05).Comparing all the groups of the third month,the alveolar MLI of control group and CXCR4-BMSCs group of third month were both lower than the model group(control group p <0.01,CXCR4-BMSCs group p <0.05),and alveolar MLI of pBABE-BMSCs group was also lower than the model group,but the difference between them were not statistically significant(p>0.05).4.Expression of apoptosis proteins: There was a significant difference in the expression of apoptosis proteins between the groups in the third month.Compared with the control group,the apoptosis proteins about Cleaved-Caspase-8,Cleaved-Caspase-3 and Cleaved-PARP-1 both markedly increased in the model groups(p<0.05).The expression of apoptosis proteins in CXCR4-BMSCs group were significantly lower than that in the model group(p<0.05).And the expression of the apoptosis proteins in the pBABE-BMSCs group also decreased comparing to the model group,but only the difference in the expression of Cleaved-Caspase-8 was statistically significant(p<0.05).5.TUNEL stain showed that the main apoptosis cells were alveolar parenchymal cells,and the apoptosis rate of control group and CXCR4-BMSCs group were both significantly lower than the model groups(p<0.01).The apoptosis rate of pBABE-BMSCs group was also lower than that of the model group(p<0.05).ConclusionBone marrow mesenchymal stem cells can be isolated from bone marrow,and purified by attachment method.CXCR4-overexpression models of BMSCs were successfully constructed by retrovirus transfection.The transplantation of CXCR4-BMSCs can reduce the apoptosis in COPD models,and delay the progression of COPD by inhibiting apoptosis and reducing the expression of apoptotic proteins.. |
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