| Acute myocardial infarction(AMI)is a common cardiovascular disease,which seriously endangers human survival and health due to its high morbidity and mortality.At present,drugs and interventional therapy have been used clinically to treat myocardial infarction,but they can only relieve symptoms and cannot repair damaged myocardial tissue.Stem cell transplantation therapy has been attracted much attention in recent years.Adipose-derived stem cells(ADSCs),a kind of adult stem cells,are extracted from adipose tissue.ADSCs are one of ideal seed cells for AMI because they are easily obtained and expanded,low immune rejection,and multi-potent differentiation properties.However,oxidative stress after myocardial ischemia and hypoxia leads to poor microenvironment,and the cell survival and engraftment rate of the stem cells transplanted are generally low,so the therapeutic effect is not ideal ultimately.Therefore,it is important to improve the therapeutic effect of AMI by using drugs to enhance stem cell viability and its antioxidant ability,or using biological material to prevent stem cells from spreading and provide good three-dimensional growth microenvironment.BIO((2 'Z,3' E)-6-bromoindirubin-3 '-oxime),a small molecule with cellular permeable,could promote proliferation of mammalian cardiomyocytes,inhibit proliferation of cardiac fibroblasts,regulate the post-infarction microenvironment,and improve the therapeutic effect of myocardial infarction,but the mechanism was not fully understood.It was reported that BIO could play an important role in the differentiation of stem cells,but it was not clear whether it had a protective effect on stem cells and improved the effect of stem cell treatment for myocardial infarction.Matrigel was an injectable,temperature-sensitive hydrogel,and was rich in extracellular matrix.Previous studies had shown that Matrigel could improve cardiac function through intramyocardial injection.Howerver,the effect of Matrigel in combination with stem cells was rarely reported.Therefore,based on the above considerations,our study used H2O2 induced oxidative stress injury to simulate the injury microenvironment after myocardial infarction.Then we explored the protective effect and underlying mechanism of BIO on ADSCs and H9c2 cells.The BIO-loaded thermo-sensitive nanomicelles(BIO-N)were prepared from nanomaterials,which could be used as the drug sustained-release system to prolong the release of BIO.A model of acute myocardial infarction was established by ligating the left anterior descending branch of coronary artery in SD rats.The survival of ADSCs was evaluated.The combined synergistic therapeutic effect and underlying mechanism of BIO-N,Matrigel and ADSCs were elucidated in order to provide theoretical basis for clinical treatment.Part ? Protective effect of BIO on ADSCs and H9c2 cells against H2O2-induced injury and its mechanismObjective:To investigate the protective effect of BIO on ADSCs and H9c2 cells against H2O2-induced injury,and elucidate its underlying mechanism.Methods:1.ADSCs were isolated from the adipose tissue in the bilateral inguinal region of SD rats,and cell surface markers CD29,CD34,CD45,and CD90 were detected by flow cytometry.ADSCs were cultured with induced differentiation solution,and the multi-lineage differentiation ability of ADSCs was detected.2.ADSCs were pretreated with different concentrations of BIO for 24h,then H2O2(800?mol/L)was added for another 24h.Cell viability was detected by CCK-8 kit.Intracellular ROS was detected by flow cytometry with DCFH-DA staining.Mitochondrial membrane potential was detected by flow cytometry with Rho123 staining.3.In the presence of H2O2,H9c2 cells were treated with BIO,and then cell viability was detected by CCK-8 kit.The level of ROS in cells was detected by flow cytometry with DCFH-DA staining and digitized as the mean fluorescence intensity.The mRNA expression of cyclin D1,CDK1,CDK2,and cyclin B in H9c2 cells was detected by qRT-PCR.Protein expression of MAPK signaling pathway(ERK1/2,JNK,p38)was detected by Western blotting.These indicators were to explore the effect of BIO on H9c2 cells and its underlying mechanism in the presence of H2O2.Results:1.ADSCs could express CD29 and CD90 as the surface markers of mesenchymal stem cells,but they did not express CD34 and CD45 which were the surface markers of hematopoietic stem cells.ADSCs could be differentiated into adipocytes,chondrocytes,and osteocytes,which indicated that ADSCs shared multi-potent differentiation properties.2.BIO could promote the proliferation of ADSCs.Compared with the control group(0 group),the cell viability of the BIO(1,2.5,5,and 10?mol/L)groups increased by(11.8±7.7)%,(39.9±4.2)%,(21.2±6.6)%,and(8.3±13.2)%after cells were treated with BIO for 3 days,respectively,among which 2.5?mol/L and 5?mol/L groups had statistical significance.ADSCs were treated with H2O2 at different concentrations for 24h.H2O2 could dose-dependently inhibited cell viability.According to the experimental results,800?mol/L of H2O2 was selected to establish the oxidative stress damage model.BIO pretreatment could improve the viability of ADSCs under H2O2-induced injury.Compared with the H2O2 group(0 group),the cell viability of the BIO(0.5,1,2.5,and 5?mol/L)groups increased by(12.9±4.1)%,(18.2±2.6)%,(18.3±1.8)%,and(6.8±5.4)%,respectively,among which 1?mol/L and 2.5?mol/L of BIO groups had statistical significance.BIO could reduce intracellular ROS induced by H2O2 in ADSCs.BIO could improve H2O2-induced mitochondrial membrane potential reduction.Compared with the H2O2 group(0 group)(100±3.1)%,the mean fluorescence intensity of the BIO(1?mol/L and 2.5?mol/L)groups were(120±9.6)%and(120±3.9)%,respectively.3.BIO could promote the proliferation of H9c2 cells.Compared with the control group(0 group),the cell viability of the BIO(1,2.5,5,and 10?mol/L)groups increased by(8.2±2.1)%,(26.2±8.7)%,(23.7±10.1)%,and(0.4±1.8)%after cells were treated with BIO for 3 days,respectively,among which 2.5?mol/L and 5?mol/L groups were statistically significant.BIO could inhibit the proliferation of cardiac fibroblasts in a concentration-dependent manner.In the presence of H2O2,H9c2 cells were treated with BIO for 24h.Compared with the H2O2 group,the cell viability of the BIO(0.5,1,2.5,and 5?mol/L)groups increased by(10.8±5.3)%,(17.6±5.8)%,(16.5±9.8)%,and(1.2±2.1)%,respectively,among which 1?mol/L and 2.5?mol/L groups were statistical significance.BIO could reduce intracellular ROS induced by H2O2 in H9c2 cells.There was no statistical significance about the different mRNA expression of cyclin D1,CDK1,CDK2,and cyclin B among the groups.In the presence of H2O2,BIO could promote the protein expression of p-ERK1/2 and p-p38,and inhibit the protein expression of p-JNK in H9c2 cells.Conclusion:1.BIO could promote the proliferation of ADSCs,and pretreatment with BIO had a protective effect on ADSCs against oxidative stress injury induced by H2O2.2.BIO could promote the proliferation of H9c2 cells,and play a protective effect on H9c2 cell damage caused by H2O2 probably through MAPK signaling pathway.Part ? Construction of sustained-release system and Its biocompatibility detectionObjective:To prepare the nanomicelles loaded BIO(BIO-N),and detect the biocompatibility of BIO-N,Matrigel and ADSCs,which can provide theoretical basis for experiments in vivo.Methods:1.PEG-PCL block copolymer could be self-assembled into blank temperature-sensitive nanomicelles in the process of water dialysis.When PEG-PCL was mixed with BIO for dialysis,BIO-loaded sensitive nanomicelles(BIO-N)were prepared to construct a drug sustained-release system.The cumulative release of BIO in vitro was detected by UV spectrophotometer.2.The particle size of blank nanomicelles and BIO-N were detected by DLS.The morphology of blank nanomicelles and BIO-N were observed by TEM.The microstructure of BIO-N,ADSCs,and Matrigel were observed by SEM.3.ADSCs were cultivated in Matrigel(PBS:Matrigel=1:1 dilution,protein content approximately 4mg/ml).The three-dimensional growth and proliferation of ADSCs in Matrigel were observed under laser confocal microscope with Live/Dead staining.The proliferation of ADSCs in Matrigel was detected by CCK-8 kit,and the effect of BIO-N on the proliferation of ADSCs in Matrigel was analyzed.4.The mRNA expression of VEGFa,EGF,and Ang-1 in ADSCs were detected by qRT-PCR.Results:1.The PEG-PCL block copolymer was self-assembled in water to form blank temperature sensitive nanomicelles with a kind of core-shell structure.After blend dialysis,the hydrophobic core PCL wraped the BIO inside and the hydrophilic shell PEG located outside,forming temperature sensitive nanomicelles loaded BIO(BIO-N).BIO was red,and the collected liquid was uniformly red without precipitation,indicating that the preparation of temperature-sensitive nanomicelles loaded BIO was successful.BIO was released in BIO-N for up to 5 days.The encapsulation rate of the drug was calculated to be about 61.4%.2.DLS results showed that the particle size of blank nanomicelles and BIO-N were about 134nm and 270nm,respectively.The morphology of the two nanomicelles was observed under TEM,and the results were consistent with DLS.The results of SEM showed that Matrigel formed a network structure,and ADSCs were wrapped in it,while BIO-N were distributed in Matrigel,and the particle size was basically consistent with DLS results.3.The results of After Live/Dead staining showed that only a few cells were in red(dead cells)while most were in green(live cells),indicating that ADSCs could grow well in Matrigel.CCK-8 results showed that BIO-N could promote the proliferation of ADSCs in Matrigel at 1 day compared with the control group,and the effect was more obvious at 3 and 7 days.4.qRT-PCR results showed that the mRNA expression of VEGFa,EGF and Ang-1 in ADSCs were improved in BIO-N/Matrigel group,which was conducive to the therapeutic effect of ADSCs in vivo.Conclusion:PEG-PCL,Matrigel and ADSCs had good biocompatibility,providing important experimental basis for the combined treatment(BIO-N-Matrigel-ADSCs)of acute myocardial infarction.Part ? Effects of combined therapy of BIO-N,Matrigel and ADSCs on the repair of acute myocardial infarctionObjective:To investigate the therapeutic effect of combined therapy of BIO-N,Matrigel,and ADSCs on the repair of acute myocardial infarction and explore the underlying mechnism.Methods:1.Establishment of acute myocardial infarction model.Male SD rats(200±15 g)were subjected to permanent MI surgery by ligating the left anterior descending coronary artery with a 7-0 silk suture,and the myocardium in the infarction area was turned from red to white gradually.At the same time,the ECG showed ST-segment elevation.2.The treatment of AMI.Experimental groups:(1)MI group,intramyocardial injection with PBS;(2)ADSCs group,cells were suspended in PBS and injection;(3)BIO-N+ADSCs group,ADSCs and BIO-N were suspended in PBS;(4)BIO-N/Matrigel+ADSCs group,ADSCs,BIO-N,and Matrigel were thoroughly mixed(operated on ice),and then injected into myocardium.ADSCs-GFP+were extracted from transgenic SD rats for cell tracking ADSCs in vivo.After treatment for 3,7,and 14 days,the rats of the last three groups were anesthetized and sacrificed.Heart samples were taken out,fixed with 4%paraformaldehyde,dehydrated with sucrose gradient,and embedded in OCT.Frozen tissue sections were performed to observe the survival of ADSCs-GFP+ in vivo.EGF and VEGF were detected by immunofluorescence at different time points(Day 3,7,and 14).3.Histological observation.After 4 weeks treatment,hearts were removed,fixed with 4%paraformaldehyde,embedded in paraffin,and cut into 5 ?m sections.The ratio of myocardial fibrosis and infarct size were observed by HE staining and Masson staining.The angiogenesis was observed with vWF staining by immunohistochemistry.4.LVIDd,LVIDs,LVEF,and LVFS were quantitatively analyzed by echocardiography to evluate cardiac function after AMI.Results:1.The results of ADSCs-GFP+tracking in vivo showed that the survival of ADSCs decreased as time went on.At 3 days,there were many cells in myocardium.At 7 days,cell survival decreased sharply.At 14 days,the rate of ADSCs-GFP+survival was very low.By quantitative analysis of the green fluorescence area,the results showed that the green fluorescence area of the BIO-N+ADSCs group and BIO-N/Matrigel+ADSCs group at 3 days were 1.6 times and 2.4 times to that of the ADSCs group,respectively.At 7 days,the two groups were 1.8 times and 2.4 times higher to the ADSCs group.At 14 days,the fluorescence area of the ADSCs group was extremely low;the two groups were 4.1 and 6.5 times higher to that of the ADSCs group.Compared with ADSCs,the fluorescence area of BIO-N+ADSCs group was larger,while the largest was the BIO-N/Matrigel+ADSCs group at three different time points.After treatment for 3 days,no EGF expression was observed in each group.At 7 and 14 days,there was no EGF expression in MI group,but different levels of EGF were observed in treatment groups.There was no VEGF expression in MI group.However,different levels of VEGF expression were observed in treatment groups after 3,7 and 14 days.2.The results of HE staining and Masson staining showed that after 4 weeks treatment,compared with the MI group,the ratio of myocardial infarct size reduced by 40%and 55%in BIO-N+ADSCs group and BIO-N/Matrigel+ADSCs group,respectively.Compared with MI group,the ratio of collagen content lowered by 39%and 60%in BIO-N+ADSCs group and BIO-N/Matrigel+ADSCs group,respectively,indicating that the degree of myocardial fibrosis alleviated obviously after treatment.3.After 4 weeks of treatment,vWF staining results showed that the mean number of vessels in BIO-N+ADSCs group and BIO-N/Matrigel+ADSCs group were 2.8±0.7 and 4.2±0.7 times to that of the MI group.The most angiogenesis was in the BIO-N/Matrigel+ADSCs group.4.The results of echocardiography showed that after 4 weeks of treatment,compared with the MI group,the LVEF improved by 23%in BIO-N+ADSCs group,which indicated that the combination of BIO-N and ADSCs could improve the cardiac function;while it had the best therapeutic effect in BIO-N/Matrigel+ADSCs group because the LVEF increased by 34%.Conclusion:The drug sustained release system,constructed with nanomicelles loaded BIO(BIO-N),could improve the therapeutic effect of AMI to a certain extent,but the most effective way was the combination of BIO-N,Matrigel,and ADSCs in repairing AMI. |
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