| Background and objective: Stem cell transplantation for the treatment of acute myocardial infarction(AMI)has become a new trend in global research.Cardiac stem cells(CSCs)are believed to have great potential for the treatment of MI because of their ability to regenerate and repair infarcted myocardium,promote angiogenesis,reduce infarct size after myocardial infarction,and improve microcirculation in infarcted area.However,transplantation of CSCs still encounters some bottleneck problems,that is,the stem cell survival is very low and the differentiation and proliferation ability is inhibited after the transplantation of CSCs into the severe microenvironment after myocardial infarction,which can not effectively achieve the effect of impaired myocardial regeneration and repair.That CSCs mainly through the paracrine mechanism to improve the local myocardial infarction microenvironment,promote myocardial cell survival and local angiogenesis play a role.Exosomes are nano-sized,membrane-bound vesicles that contains a variety of proteins and nucleic acids.The key intermediary of information,and exosomes play an important role mainly through non-coding RNAs,of which circ RNAs have become more and more conservative,becoming the current research focus.With the deepening of research,it has been recently discovered that circ HIPK3 in circ RNAs has cardioprotection and regeneration of neovascularization.It has been confirmed that circ HIPK3 can promote diabetic retinal blood vessel formation.Another study reported that the overexpression of circ HIPK3 inhibited bladder cancer cell migration and neovascularization.So the circ HIPK3 can promote angiogenesis in the infarction area after acute myocardial infarction,improve the microcirculation,promote myocardial infarction area repair capacity and reduce myocardial infarction area? These problems have not been reported yet.Therefore,this study intends to investigate that the exosomes derived from cardiac stem cells can be used to regenerate myocardium and vascular network through the transmission of circ HIPK3 after myocardial infarction,which will lay a foundation for the non-cellular treatment of cardiac stem cells.Part one:Methods:(1)The left atrial appendage of 3-4-week-old SD rats was obtained.C-kit pos CSCs were isolated,cultured and sorted by enzyme digestion combined with MACS in vitro.MACS sorting for c-kit pos CSCs identification,the purity of CSCs was identified by flow cytometry,and the expression of C-kit in CSCs was detected by immunofluorescence.(2)Exosomes in c-kit pos CSCs were extracted by differential ultracentrifugation(UC).Exosomes concentrations were determined using the BCA kit.The identification of Exosomes derived from c-kit posCSCs.The size and morphology of Exosomes were observed by transmission electron microscopy(TEM).The size and distribution of Exosomes were detected by nanoparticle tracking analysis(NTA).Western blot was used to detect the expression of Exosomes surface marker proteins CD9,CD63 and HSP70.(3)Lentiviral vectors were used to transfect the overexpression of circ HIPK3,silenced circ HIPK3 and lentiviral vector to c-kit pos CSCs in vitro.The pre-infection experiments were divided into four groups:(1)Complete medium with virus infection,(2)complete medium containing 5ug / ml polybrene plus virus infection,(3)Enhance Infection Solution plus virus infection,(4)Enhance Infection Solution containing 5ug / ml polybrene plus viral infection.Multiplicity of infection(MOI)of 1,10,and 100 different gradients was set in each group.Fluorescence microscopy was used to observe the fluorescence expression after 4 days of lentivirus infection to confirm the transfection conditions and transfection efficiency of c-kit pos CSCs.(4)Experimental group:(1)c-kit pos CSCs derived-Exosomes without any treatment,(2)LV-exo group: Empty vector lentivirus transfected c-kit posCSCs derived-Exosomes;,(3)LV-circ-exo group: Carrying the circ HIPK3 gene Lentiviral Vector transfected with c-kitposCSCs and extracted Exosomes,(4)LV-Si R-circ-exo group: Carrying the Silent circ HIPK3 gene Lentiviral Vector transfected c-kitposCSCs and extracted Exosomes,the expression of circ HIPK3 in each group of cell-derived Exosomes were detected by RT-PCR.(5)Forty SD rats,200-250 g,were divided into sham group and myocardial infarction model group.Sham group underwent endotracheal intubation with ventilator-assisted ventilation only thoracotomy without ligation of left anterior descending coronary artery,and MI group underwent endotracheal intubation with ventilator-assisted ventilation thoracotomy ligation of left anterior descending coronary artery.The myocardial infarction model was established successfully.Before and after the coronary artery ligation,the electrocardiogram was detected before and after ligation of the coronary artery.The echocardiography was performed 4 weeks after the operation.Paraffin sections were obtained from the heart tissue at 4 weeks after operation.HE staining and Masson staining were used to analyze the myocardial infarction.Results:(1)The primary CSCs isolated and cultured in vitro by enzymatic digestion were uniform in size and shape,and adhered to the wall after 24 hours.They grew in long triangles and polygons with high refractive index.3 days after a small amount of adherent cells can grow,more relatively miscellaneous cells.After 5 days,most of the cells adhered well,showing a long triangular and polygonal growth.The cells began to proliferate,and the number of extracellular cells decreased compared with the previous ones.After 7-8 days,the cell morphology was uniform and the fusion was about 50%-60%.After 10 days,the entire bottom of the bottle was covered,the speed of amplification was obviously accelerated,and there was no obvious change in morphology.80%-90% of cell confluence was seen.CSCs obtained after MACS sorting were observed immediately under the microscopy can be seen in the small round or oval and higher refractive index of the target cells,scattered magnetic beads can be seen in the meantime.A few days after the visible triangular or polygonal cells adherent growth,part of the surface with a higher refractive index of the beads.After 3 days,the cell proliferation rate increased significantly,the morphology was still triangular or polygonal,cell density reached about 50%.After 5 days,the cells continued to proliferate,no obvious changes in morphology,cell fusion can be 80%-90%.After MACS sorting,the results of flow cytometry showed that CSCs expressed CD34 about 0.40%,CD45 about 1.28%,and c-kit about 91.62%.Immunofluorescence showed that CSCs expressed c-kit the molecules up to 90%.(2)BCA kit measured Exosomes concentration of 2.77mg/ml.TEM,NTA and Western blot were used to identify Exosomes.The results showed that different size,round or round cystic vesicles were observed by TEM.The NTA showed that the size of Exosomes particle size of about 93.6nm.Western blot showed that Exosomes surface marker proteins CD9,CD63 and HSP70 were positive.(3)In vitro transfection the overexpression of circ HIPK3,silenced circ HIPK3 and lentiviral vector to c-kit posCSCs by lentivirus vector showed that c-kitposCSCs were easily infected in complete medium and infected with 4 Days after the MOI of 100 can achieve more than 90% of the infection efficiency.Therefore,the complete medium and MOI value of 100 as transfection conditions to meet the needs of subsequent experiments.(4)The expression of circ HIPK3 in Exosomes of each group was detected by RT-PCR.The results showed that the expression of circ HIPK3 in LV-circ-exo group was significantly higher than that in Control group(P <0.01),Whereas the expression of circ HIPK3 in LV-Si R-circ-exo group was significantly decreased(P <0.01),there was no statistically significant difference between the Control group and the LV-exo group.(5)In the MI group,the successful rate of operation was 80% in rats.The postoperative mental state of the rats was sluggish,unresponsive,the activity was weakened,the hair was sparse and dull,the color was yellow-white,the respiration rate was faster and lighter,and the food intake was significantly reduced.Left coronary artery ligation ECG prompted I,II limb lead ST significantly elevated.4 weeks after the establishment of MI model echocardiography,MI group left ventricular cavity expansion,left ventricular wall segmental motion weakened or disappeared,infarct area(P <0.01),LVSd and LVDd increased significantly(P <0.01)in the sham-operation group.HE staining show that Myocardial cells were disordered,blurred in appearance,degenerated and necrotic in muscle fibers,normal myocytes were replaced by fibrous tissue In MI group.In Masson staining,a large amount of collagen fibers were observed in MI group.A small amount of red normal myocardium was seen in the infarct area.Conclusion:(1)C-kitpos CSCs with high purity and good growth status were obtained by enzymatic digestion combined with MACS sorting,in line with their cell morphology and characteristics.(2)The concentration of Exosomes extracted by UC was higher than that of Exosomes,which was identified by TEM,NTA and Western blot,which accorded with the morphology and characteristics of Exosomes.(3)The transfected circ HIPK3 into c-kitposCSCs can be successfully transfected with lentivirus,and the transfection efficiency of more than 90% can be achieved when the MOI value is 100 in complete medium to meet the needs of subsequent experiments.(4)Ligation of left anterior descending coronary artery with tracheal intubation and ventilator-assisted ventilation can successfully prepare MI rat model and prepare for the follow-up experiment.Part two:Methods:(1)Fresh Exosomes derived from c-kitpos CSCs transfected with 100 ul of Di I working solution labeled UC were collected.The MI model was successfully injected into the local area of myocardial infarction.(2)The experiment was divided into five groups:(1)Sham group: only thoracotomy,ligation of the left anterior descending coronary artery,(2)myocardial infarction control group(Control group): myocardial infarction regional injection of PBS solution,(3)transfected the lentiviral vector c-kitposCSCs derived-Exosomes group(LV-exo group)were injected with lentiviral vector to c-kitposCSCs derived-Exosomes by intramyocardial injection,(4)Transfection of lentiviral vector c-kitposCSCs derived-Exosomes group(LV-circ-exo group): myocardial infarction area by local injection transfection carrying circ HIPK3 gene lentiviral vector to c-kitposCSCs derived-Exosomes,(5)transfected with silenced circ HIPK3 lentiviral vector c-kitposCSCs derived-Exosomes group(LV-si R-circ-exo group):the infarct area was injected with Exosomes containing silenced circ HIPK3 gene lentiviral vector to c-kitposCSCs.Rats were compared with LVEF value,LVFS value,LVDd value and LVSd value between the groups on the preoperation,post-7 days,post-14 days and post-28 days.The MI rats were sacrificed at 4 weeks after operation and the hearts were removed to make frozen sections.The myocardium was observed by immunofluorescence Internalization of Exosomes-Di I.TTC staining was used to compare the myocardial infarct size among the groups.The area of myocardial fibrosis was observed by HE staining.The area of myocardial infarction was observed by Masson staining.The density and number of newborn capillaries were compared between the groups by immunohistochemical staining.Results:(1)Di I-labeled Exosomes were injected into the local area of myocardial infarction,the red fluorescence was observed in the myocardium by fluorescence microscope,indicating that the myocardium endocytosed Exosomes.(2)Exosomes transplantation after treatment of myocardial infarction,echocardiography results show that the LV-circ-exo group LVEF values and LVFS values were significantly higher than in the Control group(P <0.01),the LV-circ-exo group LVDd value and LVSd values were significantly lower than that in the Control group(P<0.01).The LVEF values and LVFS values in the LV-Si R-circ-exo group were significantly lower than those in the Control group(P<0.01).The LVDd values and LVSd values in the LV-Si R-circ-exo group were significantly lower than those in the Control group(P<0.01).The values of LVSd and LVSd were significantly higher than that of Control group(P<0.01).Compared with LV-circ-exo group,the LVEF value and LVFS value were significantly decreased in LV-Si R-circ-exo group(P<0.01),the LVDd value and the LVSd were significantly increased(P<0.01).The results of TTC staining showed that the non-infarcted myocardium was stained with red brick and the infarcted area was not stained.Compared with the Control group,the myocardial infarcted size in the LV-circ-exo group was significantly reduced(P<0.05).The myocardial infarcted size in the LV-Si R-circ-exo group was significantly increased(P<0.05).The infarcted size in the LV-circ-exo group was significantly smaller than that in the LV-Si R-circ-exo group(P<0.05).HE staining showed that compared with the Control group,the area of myocardial fibrosis area in the LV-circ-exo group was significantly reduced(P<0.05),and the myocardial fibrosis area in the LV-Si R-circ-exo group was significantly increased(P<0.05).The myocardial fibrosis area in LV-circ-exo group was significantly lower than that in LV-Si R-circ-exo group(P<0.05).Masson staining showed that the normal myocardium was stained red in the Sham group,and the myocardial infarcted area in the MI group stained blue.Compared with the Control group,the myocardial infarcted size in the LV-circ-exo group was significantly reduced(P<0.01),the myocardial infarcted size in the LV-Si R-circ-exo group was significantly increased(P<0.01).The infarcted size in the LV-circ-exo group was significantly lower than that in the LV-Si R-circ-exo group(P<0.01).Immunohistochemistry results showed that neonatal microvascular endothelial cells were often stained brown-yellow and nuclei stained blue.Compared with the Control group,the density of neonatal microvessels in the peripherial region of the LV-circ-exo group was significantly increased(P<0.05).In the LV-Si R-circ-exo group,the density of neonatal microvessels in the peripheral area of the myocardial infarction was significantly decreased(P<0.05).Compared with the LV-circ-exo group,the density of the neonatal microvessels in the peripherial region of the myocardial infarction was significantly decreased in the LV-Si R-circ-exo group(P < 0.05).Conclusion: In vivo experiments,the overexpression of circ HIPK3 has a role in neovascularization,improve myocardial repair capacity,reduce myocardial infarct size,improve cardiac function. |
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