| Objective: With experimental methods of in vitro separation, culture, generation andidentification of rat bone marrow mesenchymal stem cells (MSCs), this study aims atproviding new ideas for myocardial infarction (MI) treatment through observation on therole of MSCs modified by recombinant lentivirus vectors (LVs) with Nrf2-siRNA andEGFP fusion gene in repairmen of rat myocardial infarction and its mechanism.Methods:In vitro experiment: femora and tibia of the SD rat were taken. The marrow cavity wasrinsed with L-DMEM medium repeatedly. And then the flushing fluid was transformedinto the cultivation bottles for culture. Adherence filtration method was used to separate,cultivate, extend and amplify MSCs. Cell morphological change and growth conditionwere observed under microscope, and the third generation (P3) cells were collected fortechnical appraisement with flow cytometry; EGFP-carried Nrf2siRNA lentivirus andempty vector were bought and transfected to MSCs, with or without a specificconcentration of H2O2or culture with hyopexia and serum free for further oxidative stressprocessing. The experiment was divided into four groups: MSCsNrf2+/-normoxia group,MSCsNrf2-/-normoxia group, MSCsNrf2+/-hypoxia group, and MSCsNrf2-/-hypoxia group.Fluorescence expression and FCM were respectively used to determine the optimal multiplicity of infection (MOI); The protein expression level of Nrf2and the downstreamtargets HO-1in each group was detected with WB method; proliferation, apoptosis anddifferentiation of the cells in each group were measured with CCK8method andimmunofluorescence; Cell scratch experiment was used to observe the migration ability ineach group.In vivo experiment: Coronary artery ligation was used to establish MI model in rats.60rats were divided into solvent control group, empty vector lentivirus group andNrf2-siRNA-EGFP/BMSCs recombined lentivirus group, with20in each group. Thegroups were subdivided into four subgroups according to the postoperative7,14, and28days.300ul1×PBS or2×106/cells were separately injected to the corresponding localinfarct myocardial tissue of the experimental animals immediately after the establishmentof MI model. Hematoxylin and eosin (HE) staining was used to observe the myocardialpathological changes after cell transplantation; Immunofluorescence was applied toobserve the survival, proliferation and differentiation of implanted cells; Western-blotdetection was used to detect the protein expression levels of Nrf2, HO-1, Bcl-2, Bax andMMP9in rat myocardial infarction area in each experimental group; Masson staining wasadopted to detect the collagen fibers changes in rat myocardial infarction area on28daysafter cell transplantation. And parallel echocardiography was given to observe the heartfunction.Results:In vitro experiment: Primary cultured MSCs were adhered to the wall and stretchedinto polygon within24hours. The first3days was the relative inhibitory stage, and thenthe cell proliferation rate was gradually accelerated in logarithmic form. It reached theperiod of plateau around7days, with cell coverage up to90%. For about2weeks when thecells generated to P3, the number of MSCs can reach106, which satisfied the requirednumber of cell transplantation. The signs of BMSCS were detected by the glow cytometry,and the results showed that CD29and CD90were positive, while CD45was negative, whichwas consisted with the characteristics of MSCs. The optimum MOI of MSCs transfectedwith the Nrf2siRNA-EGFP recombined lentivirus was30. And the green fluorescent protein expression was strongest at72hours after transfection. Virus transfection had nosignificant effect on the growth of MSCs. Compared with MSCsNrf2+/-hypoxia group,silencing Nrf2in MSCsNrf2-/-hypoxia group can significantly increase the apoptosis rate ofMSCs after hypoxia treatment(P<0.05)，, and has no obvious effects on its the proliferation,migration and differentiation ability(P>0.05); compared with MSCsNrf2+/-normal group,apoptosis rate in MSCsNrf2+/-hypoxia group is significantly reduced(P<0.05); There is nostatistically significant difference of cell biological characteristics between MSCsNrf2+/-normal group and MSCsNrf2-/-hypoxia group(P>0.05). Western bolt results show thatcompared with MSCsNrf2+/-hypoxia group, nucleoprotein Nrf2and HO-1proteinexpression are obviously decreased in MSCsNrf2-/-hypoxia group(P<0.05); compared withMSCsNrf2+/-normoxia group, nucleoprotein Nrf2and HO-1protein expression areobviously increased in MSCsNrf2+/-hypoxia group(P<0.05); There is no statisticallysignificant difference of nucleoprotein Nrf2and HO-1protein expression betweenMSCsNrf2+/-normal group and MSCsNrf2-/-hypoxia group(P>0.05).In vivo experiment: HE, Masson and TTC staining results show that on the14th dayand28th day after establishment of myocardial infarction (MI) model, compared withMSCsNrf2+/-, myocardial tissue of MSCsNrf2-/-group has severer pathologicalchanges(P<0.05) and larger infarction area(P<0.05); but there is no significant differencecompared with the PBS group(P>0.05). Immunofluorescence results show that on the7thday after establishment of MI model, compared with MSCsNrf2+/-, the survival cells inMSCsNrf2-/-are decreased(P<0.05), and the expression of apoptosis protein Caspase3isincreased; Western blot results show that compared with MSCsNrf2+/-, Nrf2, HO-1andBcl-2protein expression in infarction area of MSCsNrf2-/-group are decreased, while Baxand MMP9expression are increased, and the difference is statistically significan（tP<0.05）;But compared with the PBS group, there is no statistically significant difference（P>0.05）.Echocardiography results show that on the28th day after cell transplantation, comparedwith MSCsNrf2+/-, LVDd and LVDs in MSCsNrf2-/-group is increased(P<0.05), while EF isdecreased(P<0.05); but there is no statistical difference compared with the PBSgroup(P>0.05). Conclusion: Highly purified MSCs can be isolated with adherence filtration method,and lentivirus vectors can be successful transfected into rat MSCs and significantly reduceNrf2gene expression. Biological characteristics of MSCs were unaffected aftertransfection. Silencing Nrf2can increase MSCs apoptosis rate and decrease the hypoxiatolerance, but it has no obvious effect on the proliferation, migration rate anddifferentiation in vitro. Implantation of a certain number of MSCs into the rat heart withMI can improve the cardiac function to a certain extent, which is closely related with thenuclear transfer of Nrf2in transplanted MSCs. Silencing Nrf2can reduce the resistantability of transplanted MSCs to the oxidative stress environment of acute infarctionmyocardial, and cannot significantly improve the infarction myocardial inflammatorymicroenvironment, and cardiac remodeling and cardiac function in rats with MI. |
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