| Background: Treatment strategies for acute myocardial infarction(AMI)such as traditional medicine,percutaneous coronary intervention(PCI)therapy and coronary artery bypass graft(CABG)are insufficient to stave off cardiomyocyte losses,prevent the remodeling of the left ventricle(LV)and terminate the progress of heart failure.Regenerative medicine has been developed in recent years,such as mesenchymal stem cells(MSCs)transplantation to repair myocardial infarction and restore heart function both in animal experiments and in patients.There is mounting evidence that MSCs help repair or regenerate damage tissues primarily by means of secreting paracrine factors including anti-apoptotic factors,pro-angiogenic factors,exosomes and so on,rather than via the differentiation into cardiomyocytes.Exosomes are nano-sized membrane vesicles with the diameter ranging from 30 to 150 nm.As the natural carrier,they transfer signal molecules such as protein,DNA,messenger RNAs(m RNAs)and micro RNA(m RNAs)from original cells to recipient cells to facilitate cell to cell communication.Compared with conventional carriers such as liposomes,nanospheres,micelles,microemulsion and different kinds of conjugates,exosomes afford all the desirable advantages such as low toxicity,low immunogenicity,high stability in circulation,biocompatibility and biological barrier permeability,which makes them promising carriers for efficient drug or therapeutic gene delivery.Nevertheless,both the conventional carriers and exosomes are apt to be trapped in nonspecific organs especially in the lung and the liver,leading to insufficiency in targeting myocardial ischemia area.Therefore,attempts to modify exosomes as effective carriers directly targeting ischemic myocardium have been considered.One method that has been harnessed is to restructure transmembrane proteins of exosomes to fuse with ligands or homing peptides which confers exosomes targeting capability to tissues or organs carrying the corresponding receptors.However,only a few researches concerning tumor or nervous system exploited engineered exosomes for targeted disease therapy.Recently,a new peptide sequence CSTSMLKAC that can preferentially target to ischemic region of the heart has been discovered via in vivo phage display technique.In our study,we clearly elucidated that fusion with ischemic myocardium-targeting peptide CSTSMLKAC(IMTP)could enhance the specificity and efficiency of exosomes directly targeting ischemic myocardium.Part ? Rat bone marrow mesenchymal stem cells were isolated,cultured and the exosomes were extractedThe first section Rat bone marrow mesenchymal stem cells were isolated,cultured and identifiedObjective: Bone marrow mesenchymal stem cells(BMSCs)were isolated from mouse bone marrow and bone chips.Experimental platform is builted for further extraction of exosomes.Method: Mouse whole bone marrow cell and bone chips digested with collagenase II were co-cultured.BMSCs were able to attach and could be purified through passages.The morphology of cells was observed.Identification of BMSCs surface marker antigen expression was conducted by flow cytometry and the growth curve of BMSCs was drawn.Results: BMSCs have basically covered dish after 5 days,and the integration of cells were more than 90%.The rate of cells proliferation accelerated significantly after passage,and passage was carried out every 2 to 3 days.From passage three,cells morphology was uniform and like the spindle.Identification of flow cytometry: the results showed that approximately 93.79% of mouse BMSCs expressed Sca-1,72.83% expressed CD44 and 31.82% expressed CD105.Meanwhile,there were few or no cells expressed CD31,CD45 and CD11 b.P3 BMSCs growth curve presented: incubation period for the first 2 days;entered into proliferation period at 3 days;then the phase of logarithmic growth at 4,5,and 6 days;at last the platform stage at 7 days.Conclusion: Using the new improved method which suggested co-culturing the whole bone marrow cells with bone chips can obtain purified BMSCs after the 3rd passage.The second section BMSCs-derived exosomes were extracted and identifiedObjective: Exosomes were extracted from P3 BMSCs culture supernatant,which had the certain shape,size and membrane markers.Experimental platform is builted for further application of exosomes to modify construction.Method: Application of exosomes extraction kit to extract exosomes from P3 BMSCs supernatant according to the instructions.The concentration of the extracted exosomes was measured by BCA kit.The shape and size of exosomes were identified by transmission electron microscope(TEM)and nanoparticle tracking analysis(NTA).The expression of surface marker antigen CD63 was detected by Western Blot.Results: The exosomes protein concentration was detected by BCA kit which is high up to approximately 1000?g.Identification of exosomes by TEM: the results showed that the sizes were in homogeneous and the appearance was round or oval,saccate,and light in the low density area.NTA showed exosomes were morphology homogenous with ranging in size from 30-150 nm and peaking at 134 nm.Exosomes positively expressed CD63 protein by western blot detection.Conclusion: Using Kit to extract exosomes,the concentration is high enough to further research.By TEM?NTA and Western Blot detection,these results conform to the morphology and characteristics of exosomes.The method of exosomes isolation kit is convenient,time saving,and effective.Part ? Establishment of mouse AMI modelObjective: To verify that mouse AMI model can be produced by ligating the left anterior descending(LAD)coronary artey with thoracotomy and tracheal intubation.Methods: Twenty C57BL/6 mice,20g-25 g,were randomly devided into AMI group(10)and sham operation group(10).For AMI group,mice were under general anesthesia,intubated and then positive pressure ventilation was maintained.The left anterior descending(LAD)coronary artey was ligated.Whether mouse AMI models were established successfully or not was depended on electrocardiogram and ultrasoni cardiogram.We monitored thesa mice for four weeks after the surgery,and then heart tissues were collected and analyzed with HE stain and Masson's Trichrome stain.Results: The survival rate of AMI group was 90% while all mice in sham operation group survived.After ligation,electrocardiogram showed ST-elevation on I,II,III,and a VF.Twenty-eight days after ligation,ultrasonic cardiogram showed EF and FS are lower in AMI group than those in sham operation group(P<0.01),LVEDD and LVESD were higher in AMI group than those in sham operation group(P<0.01).HE staining revealed that massive myocardial fibers were degenerated and necrosis in AMI group.Masson's Trichrome staining showed massive bule collagen deposition,only a small number of red myocardium were survivied in AMI group.Conclusion: Mouse LAD was directly ligated under endotracheal intubation and thoracotomy,which can successfully establish the mouse model of acute myocardial infarction(AMI).Part ? Construction of exosomes targeted to ischemic myocardiumObjective: To construct effective targeting of exosomes to ischemic myocardium by engineering exosomal enriched membrane protein Lamp2 b fused with ischemic myocardium-targeting peptide motif CSTSMLKAC(IMTP).Methods: Molecular cloning and lentivirus packaging techniques were used to introduce the IMTP identified by in vivo phage display fused with Lamp2 b on the external surface of exosomes.Clonal expansion of Lamp2 b was conducted using c DNA extracted from mouse skeletal muscle cells.Lamp2 b + IMTP gene sequence was synthesized and purified by the Gene Pharma(CHN).Lamp2 b + IMTP gene sequence was joined to the p CDH-CMV-MCS-EF1-cop GFP vector via T4 DNA ligase at 4°C for over 12 h.Then,transformation was conducted to drive the plasmid vector into the competent cells that would clone it.Plasmid was extracted and sequenced in order to confirm the correct insertion.Lentivirus packaging was conducted using 293 T cells.PEG 8000 was used to concentrate the lentivirus.After determination of virus titer,appropriate lentivirus was applied to infect BMSCs.Results: Lamp2 b was cloned with c DNA extracted from mouse skeletal muscle cells and Bam HI and Ecor I restriction sites were inserted after the signal peptide sequence together with glycine linkers using appropriate primers.IMTP was cloned into Lamp2 b between linkers after the signal peptide.The full gene sequence was then cloned downstream of CMV promoter with Bam HI and Ecor I restriction sites into the vector plasmid.Agarose gel electrophoresis of RT-q PCR products was performed and revealed that the IMTP was successfully cloned into Lamp2 b.GAPDH was employed as internal reference and was detected at similar levels in all groups.Total exosomes were isolated from transfected BMSCs culture supernatant using Total Exosomes Isolation Reagent in accordance with the reagent specification.Infection efficiency of lentivirus in BMSCs was approximately 90%.The purified exosomes were identified according to morphology and size by TEM and NTA,respectively.The IMTP exosomes were morphology homogenous with ranging in size from 30-150 nm and peaking at 135 nm as examined by NTA.Reconstruction did not affect the physical properties of the IMTP-Exos according to the TEM images and the NTA results.Conclusion: Ischemic myocardium-targeted exosomes can be obtained by engineering exosomal enriched membrane protein Lamp2 b fused with ischemic myocardium-targeting peptide motif CSTSMLKAC through molecular cloning and lentivirus packaging techniques.Part ? The effects of ischemic myocardium-targeted exosomes on acute myocardial infarctionObjective: To confirm that fusion with ischemic myocardium-targeting peptide CSTSMLKAC(IMTP)could enhance the specificity and efficiency of exosomes directly targeting ischemic myocardium.Methods: To investigate whether IMTP-Exosomes(IMTP-Exos)were able to target more efficiently and selectively to ischemic myocardium,IMTP-Exos or Blank-Exosomes(Blank-Exos)were labeled with Di I and co-cultured with the H9C2 cells which were induced hypoxia by culturing in DMEM-HG containing 3% FBS and incubating in a hypoxic incubator containing 94% N2,5% CO2 and 1% O2 for more than 24 h.After 2 h co-culturing,the percentage of H9C2 internalized exosomes was confirmed by flow cytometry.To verify whether IMTP-Exos fused with H9C2 cells,we labeled the exosomes with Di I and labeled hypoxia pretreated H9C2 cells with Di O.Then the exosomes were co-cultured with the H9C2 cells for 30 or 60 min at 37°C.Images of co-localized exosomes and the H9C2 membrane were observed under fluorescent microscopy.After AMI mouse models were successfully established,Di R-labeled exosomes derived from BMSCs were intravenously injected via the tail vein(50 ?g /100 ?l PBS /mouse).Internal fluorescent signals were monitored using the IVIS system at 0.5,2,24,48 and 72 h after the injection to acquire serial fluorescence images.At the last time point,heart,liver,spleen,lung and kidney from each mouse were harvested for ex vivo imaging.Fluorescence signal intensity was analyzed using the IVIS? Spectrum and Living Image? Software to determine tissue distribution of Di R-labeled exosomes.Transthoracic echocardiography was performed to assess cardiac function in response to AMI and /or exosomes therapy.Hematoxylin and eosin(H&E)staining,immunofluorescence staining of macrophages and reverse-transcription quantitative polymerase chain reaction(RT-q PCR)were applied to evaluate inflammatory response.TUNEL assay,Masson trichrome(MT)staining and immunohistochemistry were used to measure the degree of ventricular remodeling.Results: Flow cytometry illustrated that IMTP-Exos bound to injured H9C2 cells more efficiently than Blank-Exos(43.96±1.21% vs.38.66±0.86%),indicating that the IMTP augmented the binding ability of exosomes to injured H9C2 cells.Fluorescent microscopy analysis observed relatively low levels of interaction were observed in Blank-Exos treated cells up to 60 min.By contrast,merging of red and green fluorescence on the cells surface appeared within 30 min and increased over time(up to 60 min)in the IMTP-Exos treated group,approved the targeting ability of IMTP-Exos to injured H9C2 cells.More IMTP-Exos were internalized by injured H9C2 cells than Blank-Exos,anti-apoptosis function of IMTP-Exos did not achieve the desired effect.AMI mice were optically imaged at 0.5,2,12,48 and 72 h after injection of exosomes.The strongest signal was observed in the mouse-tail immediately after injection and a gradually enhanced fluorescence was also detected in the abdomen and chest.Animals were sacrificed and organs were separated at the time point of 72 h.We detected the signals of the liver,spleen,lung and kidney at the same time,almost no marked between-group difference was achieved in terms of passive exosomes transfer.Data showed that the signal detected in the MI region treated the IMTP-Exos was significantly stronger than that of the Blank-Exos treatment group(P<0.05).More Di I-labeled IMTP-Exos were accumulated in the MI region contrasted with Blank-Exos.Quantification results of fluorescence signals in ischemic myocardium confirmed that signal in the IMTP-Exos group was significantly higher than the Blank-Exos group.The m RNA levels of IL-6,TNF-? and IL-1? in the mice treated with Blank-Exos were statistically higher than those treated with IMTP-Exos(P<0.0001).TNF-?+ M1 macrophages was dramatically increased in the PBS group and Blank-Exos group,which was attenuated in IMTP-Exos group.Moreover,CD206+ M2 macrophages were slightly increased in IMTP-Exos group compared to the other two groups.Inflammatory cells in the PBS group were remarkably elevated compared to the other two groups,and the IMTP-Exos group exhibited the slightest inflammation.The IMTP-Exos group exhibited significantly increased capillary density in the ischemic border zone compared to the Blank-Exos group(65.0±5.5 vs.40.5±5.0/HPF,P<0.001)and the PBS group(65.0±5.5 vs.24.5±4.2/HPF,P<0.001).The number of arterioles were highest in the IMTP-Exos group compared with the Blank-Exos group(11.5±1.3 vs.5.8±1.5/HPF,P<0.01)and the PBS group(11.5±1.3 vs.3.5±1.3/HPF,P<0.001).A marked increase in the number of TUNEL positive cells was observed in the PBS group,indicating the induction of apoptosis,and such an increase was mitigated following treatment with Blank-Exos and IMTP-Exos.Quantitative results showed that compared with the PBS group,Blank-Exos and IMTP-Exos significantly inhibited myocardial apoptosis(IMTP-Exos: 4.5 ± 1.3/HPF;Blank-Exos: 11.3 ± 2.6/HPF;PBS: 28.3 ± 3.8/HPF;IMTP-Exos and Blank-Exos vs.PBS,P<0.001).IMTP-Exos were more effective in apoptosis resistance than Blank-Exos(P<0.05).Fibrosis length was remarkably reduced in the IMTP-Exos treated mice than in the Blank-Exos treated mice(31.8±1.9% vs.47.9±1.1%,P<0.001).The PBS group had a significantly broader infarct length compared with the other two groups(55.9±2.0%,IMTP-Exos vs.PBS,P<0.001;Blank-Exos vs.PBS,P<0.01).Cardiac function was examined using echocardiography.On postoperative day 0 and day 3,absolute values of EF and FS were basically the same among the PBS group,the Blank-Exos group and the IMTP-Exos group.Heart function of each group shared a similar downward tendency.However,in the following days till the mice were sacrificed,the group treated with IMTP-Exos resulted in a significantly upward tendency in EF and FS while the Blank-Exos group only had a slight increase in cardiac function.The heart function of PBS group even got worse.Conclusion: We creatively discovered that exosomes engineered with IMTP can preferentially target ischemic myocardium.BMSCs derived IMTP-Exos can effectively attenuate inflammation and apoptosis,reduced fibrosis,enhanced vasculogenesis and cardiac function in ischemic heart area. |
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