Study On BFGF Gene Transfected MSCs Repairing COPD And Its Mechanism

Objective: To study the repair effect and mechanism of exogenous basicfibroblast growth factor (bFGF) gene transfection to bone marrow mesenchymal stemcells (BMSCs) on COPD molding ratsMethod: Isolated by whole bone marrow adherent cell culturing: cultureBMSCs of SD rats for4-5weeks and take CM-Dil as fluorescent markers. bFGFgenes are transfected into BMSCs in liposome transfection method and qRT-PCR andWestern-blot are used to test the expression quantity of the bFGF genes and the targetprotein after transfection. Smoked and LPS are dripped via trachea to establish COPDrats model in composite method. This topic has5experimental groups, where50ratsare included and evenly randomized into Normal Controlled Group (Group NC),COPD Controlled Group (Group PC), MSCs Treatment Group (Group M),pcDNA3.1-MSCs Treatment Group (Group P), bFGF-pcDNA3.1-MSCs TreatmentGroup (Group B). Group NC: Inject3ml of PBS liquid via caudal vein, as negativecontrol; Group PC:: Inject3ml of PBS liquid via caudal vein immediately aftermolding, as positive control; Group M: Inject labeled the3rdgeneration MSCs1×107/3ml of PBS liquid via caudal vein immediately after molding; Group P: Injectlabeled the3rdgeneration of pcDNA3.1-MSCs1×107/3ml of PBS liquid, via caudalvein immediately after molding; Group B: Inject labeled the3rdgeneration ofbFGF-pcDNA3.1-MSCs1×107/3ml of PBS liquid, via caudal vein immediately aftermolding. For all rats in each group described above, arterial blood is collected at30days after administration for blood gas analysis, and bronchoalveolar lavage isperformed to get the WBC and N%in BALF, and rats’ lung tissues are taken andmade into frozen section to observe the BMSCs engraftment status, HE is stained toobserve its pathological morphology and measure the MAN, MAA and MLI values.Result:1. BMSCs of rats were successfully isolated; CD44, CD29tested byflow cytometry phenotypic are positive, CD34is negative and the identificationsucceeded.2. RT-PCR was used to test the gene expression quantity after bFGF genetransfected BMSCs. The result showed that BMSCs transfected by bFGF-pcDNA3.1 plasmid expressed great quantities of bFGF genes, level of which is much higher thanthat on BMSCs transfected by blank pcDNA3.1plasmid or untransfected BMSCs andthe difference is statistically significant. It can be concluded that bFGF gene wassuccessfully transfected into BMSCs and could express the target gene in largeamount.3. Western-blot was used to test the protein expression quantity after bFGFgene transfected BMSCs. The result showed that BMSCs transfected bybFGF-pcDNA3.1plasmid expressed great quantities of bFGF target protein, level ofwhich is much higher than that on BMSCs transfected by blank pcDNA3.1plasmidor untransfected BMSCs and the difference is statistically significant. It can beconcluded that bFGF gene was successfully transfected into BMSCs and couldexpress the target protein in large amount.4. Red dot fluorescence distributed inclusters could be observed in the lung tissue pathology28days after labeled BMSCswere transplanted into rats. The conclusion is that BMSCs was successfully engraftedin rats’ lung tissue.5. Blood gas analysis result: rats in Group PC, M, P and B hadmild hypoxemia, no carbon dioxide retention and no acidosis. PO2and SaO2valuesof rats in Group PC, M, P and B were lower than that in Group NC-this difference isstatistically significant, among the4groups, the values of Group M, P and B werehigher than that of Group PC and the difference also has statistical significance. Thevalues of Group M, P and B had no statistically significant difference, but the PO2value of Group B had trend of increase compared with that of Group M and Group P.6. WBC and N%in BALF: WBC and N%of Group NC, M, P and B is significantlylower than those of Group PC and difference has statistical significance; but there isno significant difference among WBC and N%of Group NC, M, P and B. WBC andN%of Group B had no decreasing trend compared with those of Group M and P.WBC and N%of Group M, P and B were slightly higher than those of Group NC.7.MAN and MAA-parameter of lung tissue pathology morphological: MAN and MAAvalues of Group PC, M, P and B were lower than those of Group NC and thedifference was statistically significant. Besides, MAN and MAA of Group M, P andB were higher than those of Group PC and the difference was also statisticallysignificant. There was no statistically significant difference among Group M, P and B,but MAN, MAA values of Group B had increasing trend in comparison with those of Group M and P.8. MLI value-parameter of lung tissue pathology morphological:MLI values of Group PC, Group M, Group P and Group B are higher than that ofGroup NC and the difference from Group NC has statistical significance. Amongthem, MLI values of Group M, Group P, and Group B are lower than that of GroupPC and the difference from Group PC has statistical significance, but the differenceamong Group M, P, B is not statistically significant. MLI value of Group B has notrend to decrease in comparison with that of Group M and Group P.Conclusion:1. Exogenous bFGF gene could be transfected into BMSCs of ratsand also could express large quantities of its target gene and target protein;2. Theexogenous BMSCs could reach lung tissues of COPD molding rats after beinginjected via caudal vein and could achieve local engraftment.3. Repair effect oftransplantation therapy with exogenous bFGF pcDNA3.1-BMSCs on COPDmolding rats is clear, and COPD is clearly improved compared to pure molding group,and there is increasing trend in comparison with pure BMSCs transplant treatmentgroup, but the difference is not statistically significant. Therefore, it cannot beconcluded that bFGF has obvious effect on COPD.4. Pure BMSCs transplanttreatment has obvious effect on COPD molding rats and COPD is significantlyimproved compared with pure molding group.