| Objective:To study bFGF gene transfected MSCs influenceing the expression of IL-10、IL-4 and differentiation in COPD rats Method:Isolated by whole bone marrow adherent cell culturing.Import bFGF gene into MSCs by using lipsome transfection method. The rat model of COPD was induced by two intratracheal instillations of LPS and exposed to cigarette smoke for 1month.SD rats(n = 75) were randomly are divided into 5 groups, norrmal contol group(Group A):inject 1ml PBS into normal rats through the tail vein,COPD group(Group B): inject 1ml PBS into the rat model of COPD through the tail vein,MSCs Treatment Group(Group C): inject 1×106/1ml CM-Dil-labeled MSCs into the rat model of COPD through the tail vein,pcDNA3.1-MSCs group(Group D):inject 1×106/1ml CM-Dil-labeled pcDNA3.1-MSCs into the rat model of COPD through the tail vein,bFGF-pcDNA3.1- MSCs group(Group E): inject 1×106/1ml CM-Dil-labeled bFGF-pcDNA3.1-MSCs into the rat model of COPD through the tail vein. Five rats of each group were sacrificed at d7, d14, d28. To observe COPD pathologic change through HE staining. Peripheral blood and pulmonary tissues harvested for measurement of IL-10 and IL-4 levels by ELISA and qRT-PCR.To observe the implantation and differentiation of MSCs in the lungs of COPD rats.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. CM-Dil label combined immunofluorescence was used to identify the transdifferentiation of MSCs in the lung. Statistical comparison were tested with single-factor variance analyses.Results:1.To isolate the bone marrow derived mesenchymal stem cells and culture them in vivro. Flow cytometric analysis revealed that the rMSC were high positive forCD29、CD44 and negative for CD34、CD45.2. The detection of blank pcDNA3.1 plasmid or bFGF-pcDNA3.1 plasmid sequences is in line with the description of Nucleic Acid Database.Transfect the bFGF-pcDNA3.1 plasmid into the bone marrow stromal cell(MSCs) by liposome,so that to achieve the high and stable expression of bFGF in body.3. HE-stained sections revealed obviously pathologic improvement in Group C、D and E in comparison with Group B. HE-stained sections revealed relatively obvious improvement Group E in comparison with GroupB and C.It revealed that MSCs can improve pathology of COPD rats.4. At the same time, the IL-10 and IL-4 level of Group C 、 D and E were significantly higher than that of Group B(P<0.01).The IL-4 and IL-10 level of the PB and lung tissues in Group E higher than that of Group C and D(P<0.05)The IL-4 and IL-10 level of the PB and lung tissues have little difference in Group C and D. It revealed that bFGF can make MSCs strengthen the anti-inflammatory effects in COPD rats.5. Some CM-Dil positive cells showed immunofluorescence positivity of CC16(molecular marker for Clara cell) or SPC(molecular marker for type II alveolar epithelium) in the same time.Conclusions:bFGF gene transfected MSCs can improve pathology of COPD rats,increase the level of IL-10、IL-4,strengthen the anti-inflammatory effects, and differentiate into type II alveolar epithelium or Clara cell. |
