Experimental Study On The Co-culture Of Nrf2 Gene Overexpressed By Bone Marrow Mesenchymal Stem Cells And Myocardial Microvascular Endothelial Cells Under Oxidative Stress

Background:Acute myocardial infarction（Acute myocardial infarction,AMI）is the most common clinical acute cardiovascular disease and one of the most important causes of sudden cardiac death.Although reperfusion has a certain effect on the treatment of myocardial infarction,due to the non-renewability of myocardial cells,ischemic cardiomyopathy is secondary to large-area cardiomyocyte necrosis,which leads to heart enlargement and cardiac function decline,and eventually develops heart failure.Therefore,seeking a more effective treatment method has become an important task for researchers.In recent years,stem cell transplantation for acute myocardial infarction has obtained extensive basic research,including bone marrow mesenchymal stem cells（Bone Mesenchymal Stem Cells,BMSCs）transplantation for myocardial infarction is quite popular.However,due to the microenvironment of ischemia and hypoxia after myocardial infarction,the survival rate and retention rate of transplanted BMSCs have been greatly limited.Previous studies have shown that genetic modification can improve cell survival rate and biological function.Nuclear factor-erythroid 2-Reated Factor 2（Nrf2）is one of the most important regulators of the body’s antioxidant response,under oxidative stress,it can activate a variety of downstream antioxidant genes and phase II detoxification enzymes Gene transcription and expression,such as Heme Oxygenase-1（OH-1）.This experiment intends to overexpress the level of Nrf2 in BMSCs under oxidative stress in an attempt to improve the survival rate of transplanted BMSCs under ischemic and hypoxic environment after myocardial infarction,and thus improve the biological function of transplanted cells.The formation of new blood vessels is essential for the recovery of cardiac function after myocardial infarction.Angiogenesis is the generation of new blood vessels from existing Vascular Endothelial Cells（VECs）,which involves the activation,proliferation and migration of endothelial cells,Cardiac Microvascular Endothelial Cells（CMECs）and myocardial angiogenesis closely related.The purpose of this study is to use hydrogen peroxide(H₂O₂)to induce apoptosis of BMSCs,observe the overexpression of Nrf2 gene in BMSCs,establish an in vitro co-culture model with CMECs,simulate the intracellular microenvironment,and explore the co-culture system.The effect of BMSCs on the angiogenesis ability of CMECs through paracrine effects provides new therapeutic targets and strategies for stem cell therapy.Part Ⅰ The role and mechanism of Nrf2/HO-1/NQO1 axis inthe apoptosis of BMSCs overexpressing Nrf2 geneObjective:（1）Culture the BMSCs required for the experiment and select P3 generation BMSCs to identify their surface molecules and construct a rat lentiviral vector Nrf2 gene overexpression and silent transfection of stable cell lines;（2）Observe the effect of overexpression of Nrf2 gene on the proliferation and apoptosis of BMSCs on the basis of oxidative stress induced by 300μmol/L H₂O₂ treatment of BMSCs for 4h to induceBMSCs apoptosis;（3）Under oxidative stress,explore the effect of Nrf2/HO-1/NQO1 signaling pathway on inhibiting apoptosis of BMSCs under oxidative stress.Methods:（1）Use the whole bone marrow adherent method to culture the BMSCs required for the experiment and use flow cytometry（FCM）to detect the surface marker antigens（CD90 and CD45）of BMSCs;（2）Successful detection of lentivirus transfection of Nrf2 gene to P3 generation BMSCs using inverted fluorescence microscopy,qRT-PCR and Western Blot method;（3）Establish the oxidative stress model of BMSCs through H₂O₂:FCM was used to detect the effect of different concentrations of H₂O₂（0,100,200,300,400μmol/L）treatment on BMSCs for 4hours,and the best H₂O₂ concentration was selected as the best inducer of BMSCs,Apoptosis model as a treatment condition for subsequent experiments.The experiment was divided into 5 groups:blank control group（Control group）,Nrf2silent empty vector group（Nrf2-Vector group）,Nrf2 silent group（si-Nrf2 group）,Nrf2 overexpression empty vector group（Nrf2-Scramble group）,Nrf2overexpression group（LV-Nrf2 group）;（4）Edu and flow cytometry test the proliferation ability of BMSCs in each group;（5）Tunel and FCM detect the apoptosis of BMSCs in each group;（6）Western Blot detected the expression levels of antioxidant-related proteins Nrf2,HO-1 and NQO1 in BMSCs of each group.Results:（1）The primary cells of P0 generation BMSCs were cultured until the third day,the cells had antennae extended,most of the cells were polygonal,prismatic,and a few were striated,and the cells grew in clusters.There is a granular structure distribution in the pulp;after the cultivation to the 7th day,the primary cells have grown to 80%⁹0%,and the subculture can be started.After passage,the cells entered a rapid growth phase,and the third-generation cells had a uniform morphology,showing a fish-like shape.Identified by FCM,the third generation of SD rats BMSCs CD90 positive expression（97.18%）,while CD45 weak expression（3.36%）.（2）When MOI=150,48 hours after transfection of lentivirus into BMSCs,a small amount of green fluorescence expression can be seen under an inverted fluorescence microscope.After 72 hours of transfection,most cells showed green fluorescence;qRT-PCR and Western Blot detection:The expression of Nrf2 was significantly up-regulated in the LV-Nrf2 group（P<0.01）;the expression of Nrf2 was significantly down-regulated in the si-Nrf2 group（P<0.01）.（3）Treating BMSCs with 300μmol/L H₂O₂ for 4h is the best concentration and time for inducing apoptosis of BMSCs.It is used to simulate the oxidative stress microenvironment after myocardial infarction in vitro.（4）Edu and flow cytometry results showed that the proliferation rate of BMSCs in si-Nrf2group was significantly decreased（P<0.01）;the proliferation rate of BMSCs in LV-Nrf2 group was significantly increased（P<0.01）.（5）Tunel and FCM detected the apoptosis rate of each group.The results showed that the overall apoptosis rate of BMSCs in the si-Nrf2 group was significantly increased（P<0.01）;the overall apoptosis rate of BMSCs in the LV-Nrf2 group was significantly decreased（P<0.01）.（6）Western Blot results showed that the relative expression levels of antioxidant-related proteins Nrf2,HO-1 and NQO1 in LV-Nrf2 group were significantly increased（P<0.01）;antioxidant-related proteins Nrf2,HO-1 and NQO1in si-Nrf2 group The relative expression level of was significantly down-regulated（P<0.01）.Conclusion:（1）Using the whole bone marrow adherence method can effectively cultivate the BMSCs with higher purity required for the experiment.（2）When MOI=150 and transfection for 72h,lentivirus can transfect BMSCs to obtain stable transfected cells.（3）300μmol/L H2O2 treatment of BMSCs for 4h is the best concentration and time to induce BMSCs apoptosis.（4）Under oxidative stress,overexpression of Nrf2 gene promotes BMSCs proliferation and inhibits BMSCs apoptosis.（5）Under oxidative stress,overexpression of Nrf2 can effectively increase the expression levels of antioxidant-related proteins Nrf2,HO-1 and NQO1.PartⅡThe role and mechanism of Nrf2/HO-1/NQO1 axis in inhibiting apoptosis of bone marrow mesenchymal stem cells（BMSCs）and promoting angiogenesis of myocardial microvascular endothelial cells（CMECs）after co-cultureObjective:The purpose of this experiment is to establish an indirect co-cultivation system of BMSCs and CMECs in vitro using Transwell cells to explore the effect of BMSCs overexpressing Nrf2 gene on the biology of CMECs through their paracrine effects.With CMECs cultured in complete medium alone as a negative control group,we explored the effect of Nrf2 gene transfected BMSCs on CMECs angiogenesis and the role of Nrf2/HO-1/NQO1 signaling pathway in the co-culture mode.Methods:The experiment was divided into 6 groups,NC group（individual CMECs culture group）,Control-BMSCs group（co-culture group of non-transfected BMSCs and CMECs）,Nrf2-Vector-BMSCs group（Co-culture group of BMSCs and CMECs transfected with Nrf2 silent empty vector）,si-Nrf2-BMSCs group（co-culture group of BMSCs and CMECs transfected with silenced Nrf2 gene）,Nrf2-Scramble-BMSCs group（co-culture group of BMSCs and CMECs transfected with Nrf2 overexpression empty vector）and LV-Nrf2-BMSCs group（co-culture group of BMSCs and CMECs transfected with Nrf2 gene）,Using Transwell chamber（membrane pore diameter0.4μm）to establish BMSCs-CMECs co-culture system in vitro.（1）Test the angiogenesis ability of CMECs in each group,namely Transwell cell（membrane pore diameter 8.0μm）to detect the migration ability of CMECs;（2）Matrigel detects the tube forming ability of CMECs;（3）Edu and flow cytometry to detect the proliferation ability of CMECs;（4）Tunel detects the apoptosis of CMECs;（5）Western Blot detected the expression levels of antioxidant-related proteins Nrf2,HO-1 and NQO1in CMECs of each group.Results:（1）The results of the Transwell chamber showed that the migration ability of CMECs in the five-group co-culture system was significantly higher than that of the CMECs alone culture group（P<0.01）;Compared with the Control-BMSCs group,the number of CMECs in the LV-Nrf2-BMSCs group that was positive for crystal violet staining across the Transwell cell membrane was significantly increased（P<0.01）;the si-Nrf2-BMSCs group was positive for crystal violet staining across the Transwell cell The number of CMECs migrated by the membrane decreased significantly（P<0.01）.（2）Matrigel results showed that the tube formation ability of CMECs in the five-group co-culture system was significantly higher than that of the CMECs alone culture group（P<0.01）;compared with the Control-BMSCs group,the tube formation ability of the LV-Nrf2-BMSCs group was significant Increased（P<0.01）;the tubule forming ability of the si-Nrf2-BMSCs group decreased significantly（P<0.01）.（3）The Edu results showed that the proportion of Edu purple fluorescent positive cells in CMECs in the five-group co-culture system was significantly higher than that in the CMECs alone culture group（P<0.01）;compared with the Control-BMSCs group,the LV-Nrf2-BMSCs group Edu The proportion of purple fluorescent positive cells was significantly increased（P<0.01）;the ratio of Edu purple fluorescent positive cells in si-Nrf2-BMSCs group was significantly decreased（P<0.01）.（4）The cell cycle results showed that the proliferation rate of CMECs in the five-group co-culture system was significantly higher than that of the CMECs alone culture group（P<0.01）;compared with the Control-BMSCs group,the CMECs proliferation in the LV-Nrf2-BMSCs group was significantly enhanced（P<0.01）;CMECs proliferation rate in si-Nrf2-BMSCs group was significantly reduced（P<0.01）.（5）Tunel results showed that the apoptosis rate of CMECs in the five-group co-culture system was significantly lower than that of the CMECs alone culture group（P<0.01）;compared with the Control-BMSCs group,the CMECs apoptosis rate of the LV-Nrf2-BMSCs group was significantly reduced（P<0.01）;CMECs apoptosis rate in si-Nrf2-BMSCs group was significantly increased（P<0.01）.（6）Western Blot results showed that the expression levels of antioxidant-related proteins Nrf2,HO-1 and NQO1 in CMECs of the five groups of co-culture systems were higher than those of the CMECs alone culture group（P<0.01）;compared with the Control-BMSCs group,LV-The expression levels of antioxidant-related proteins Nrf2,HO-1 and NQO1 in CMECs of Nrf2-BMSCs group were significantly up-regulated（P<0.01）;the expressions of antioxidant-related proteins Nrf2,HO-1 and NQO1 in CMECs of si-Nrf2-BMSCs group The level was significantly lowered（P<0.05）.Conclusion:（1）Immunofluorescence of CMECs identified the positive expression of CD31 and factor VIII.（2）Co-culture with BMSCs can promote the proliferation,migration and angiogenesis of CMECs,and reduce the apoptosis of CMECs.（3）Compared with untransfected BMSCs,Nrf2 gene transfection can enhance the effect of co-culture on the proliferation,migration,angiogenesis and anti-apoptosis ability of CMECs.（4）Overexpression of Nrf2 can effectively increase the expression levels of antioxidant-related proteins Nrf2,HO-1 and NQO1.