| Background Using transplantation of stem cell for the therapy of myocardial infarction(MI) has become a global hot spot. In theory, stem cells can promote angiogenesis, restore blood supply, regenerate new myocardial tissue, and induce cardiac structural and functional recovery. According to the present report, the seed cells which could be selected include mesenchymal stem cells(MSCs), embryonic stem cells(ESCs), induced pluripotent stem cells(i PS), endothelial progenitor cells(EPCs), and cardiac stem cells(CSCs), etc. CSCs is considered as a huge potential seed cell in the treatment of MI, because it is much more easier than the other seed cells to differentiate into myocardial cell. Through several years of animal experiments and clinical trials research, it has been demonstrated that CSCs has the ability to promote restoration of damaged myocardium. However, there are some problems in using stem cell to treat MI, which are the poor ability of stem cell engraftment, survival and differentiation. CSCs also exists the generally problem. Therefore, in order to enhance the ability of CSCs repair damaged myocardium, many methods have been reported, including genetic modification, histone modification, hypoxic preconditioning, etc. In this study, we use BMSCs-Exosomes to stimulate the CSCs, in order to improve the ability of CSCs repair damaged myocardium. It has been confirmed that BMSCs-Exosomes could promote angiogenesis in infarction area, and reduce the area of myocardial infarction. In addition, a study found that the ability of migration/invasion in the breast cancer cells was significantly enhanced after BMSCs-Exosomes stimulation. In this study, we also use the BMSCs-Exosomes to stimulate CSCs. However, whether would this method activate the CSCs, promote CSCs differentiation, and improve the ability of myocardial repair? These problems still have not been reported. This study, by means of Exosomes, was designed to improve CSCs the function of myocardial repair, and further to analyze the mechanism of gene regulation about the improvement of CSCs function through the micro RNA gene chip detection.Part I 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 rat bone marrow. Experimental platform is builted for further extraction of exosomes.Method: Rat whole bone marrow cell were cultured. BMSCs were able to attach to the wall and could be purified through passages. Identification of BMSCs surface marker antigen expression by flow cytometry; Draw the BMSCs growth curve; Detect the cell cycle of BMSCs by flow cytometry.Results: BMSCs have basically covered dish after 14 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 4 to 6 days. Cells morphology is uniform, and like the spindle. Identification of flow cytometry: the results showed that over 90% of rat BMSCs express CD29, CD90 and CD44; but did not express the hematopoietic progenitor cell surface marker CD34. P3 MSCs 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. 3th passage BMSCs in G0/G1 phase cells was 88.15%, G2 phase cells was 1.73%, S phase cells(G3) was 10.12%, the cells in a quiescent, comply with the stem cell characteristics.Conclusion: Using rat whole bone marrow cells cultured, and BMSCs can be purified an 3rd passage. It is a effective method to cultivate purified BMSCs.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 process CSCs.Method: Application of exosomes extraction kit to extract exosomes from P3 BMSCs supernatant according to the instructions. Measure the concentration of extracting exosomes by BCA kit. Identify the shape and size of exosomes by transmission electron microscope(TEM); Identify the surface marker antigen CD63 expression by flow cytometry and protein CD63 expression by Western Blot.Results: The exosomes protein concentration was detected by BCA kit which is high up to 1896.7 mg/ml. Identification of exosomes by TEM: the results showed that the sizes were inhomogeneous, average diameter of 46.55±11.64(11-98nm), and the appearance was round or oval, saccate, and light in the low density area. Identification of flow cytometry showed that the rate of exosomes expressing CD63 was 96.7%. Exosomes positively expressed CD63 protein by western blot detection.Conclusion: Using Kit to extract exosomes, the concentration is high enough to further research. By flow cytometry, TEM 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 II Establishment of rats MI modelObjective: To confirm that MI model can be produced by ligating the left anterior descending(LAD) coronary artey with thoracotomy and tracheal intubation.Methods: 17 SD rats, 250g-300 g, were devided into MI group(10) and control group(10). For MI group, rats were under general anesthesia, intubated and then positive pressure ventilation was maintained. The left anterior descending(LAD) coronary artey was ligated. To confirm the success of MI, we examed rats with electrocardiogram and ultrasonic cardiogram. Four weeks after the surgery, we took out the heart tissue and analyzed with HE stain and MT stain.Results: The rate of survival is 80%. After ligation, electrocardiogram showed ST-elevation on I, II, III, and a VF. Four weeks after ligation, ultrasonic cardiogram showed EF and FS are lower in MI group than those in control group(P<0.01, P<0.01), LVEDD and LVESD are higher in MI group than control group(P<0.01, P<0.01). HE staining showed muscle fiber degeneration and necrosis in MI group. MT staining showed bule collagen deposition, hardly see the survival of red myocardium in MI group.Conclusion: Left anterior descending coronary artery(LAD) was directly ligated under endotracheal intubation and thoracotomy, the method of which can successfully establish the rat model of myocardial infarction(MI).Part III Sorting and culture of CSCsObjective: CSCs were isolated from newly born SD rat’s heart by magnetic bead cell sorting for further study.Methods: Take out the heart from newly born SD rats, isolate c-kit+ CSCs with collagenase II digestion and MACS. Using flow cytometry to identify the positive rate of c-kit on P0 CSCs. Then we observed the expression of c-kit and Ki67 on CSCs with immunofluorescence. After induced the differentiation of CSC, we use immunofluorescence to observe the expression of c Tn I, Desmin, Connexin-43, α-SMA, and CD31.Results: CSCs was isolated by MACS and collagenase II digestion, which was monomorphic and grow slowly. The positive rate of c-kit is 91.5%. The expression of c-kit and Ki67 were positive on CSCs, and c Tn I, Connexin43, Desmin, α-SMA, and CD31 were positive expression on CSC after induced differentiation.Conclusion: we can successfully isolate CSCs from new born rats with MACS. The degree of purity is very high. The isolated CSCs have the ability to self-renew and differentiate into myocardial cell, which correspond to the characteristics of CSCs.Part IV The effects of Rat BMSCs-derived exosomes on cardiac stem cells function and myocardial repair capacityObjective: After exosomes processing CSCs, to observe CSCs the change of function in migration, angiogenesis and proliferation, and the change of ability in myocardial repair after CSCs transplantation in MI rats.Method: Firstly, adding the Di I labeled exosomes into CSCs culture medium, to observe the fluorescence on CSCs. Secondly, using exosomes in different concentration(0μg/ml, 100μg/ml, 200μg/ml, 400μg/ml, 800μg/ml) process CSCs, CCK8 method was applied to observe the effects of CSCs expansion ability under different exosomes concentration; Application of transwell chambers, to observe the effects of CSCs migration ability under different exosomes concentration; Using Matrigel matrix, to observe the effects of CSCs angiogenesis ability under different exosomes concentrations. At last, the pretreatment of CSCs with 400μg/ml exosomes and normal culture CSCs were applied through local myocardial injection to treat MI rats, and then to observe the survival CSCs-Di I in border zone and infarction zone at short-term after MI; The newborn blood capillary density and the differentiation of GFP-CSCs into vessels in the border zone were observed by Immunofluorescence; Heart function were calculated through Doppler echocardiography; MT dyeing was used to observe myocardial infarction area.Results:(1) CSCs surface appeared red Di I fluorescent after exosomes was added into culture medium, which instructed that CSCs internalized Di I-exosomes.(2) Using exosomes in different concentration(0μg/ml, 100μg/ml, 200μg/ml, 400μg/ml, 800μg/ml) process CSCs, CCK8 method was applied to observe the effects of CSCs expansion ability, and the results showed that OD value changed little under a low concentration exosomes(0μg/ml, 100μg/ml); OD values were significantly higher in high exosomes concentration group(200μg/ml, 400μg/ml, 800μg/ml) than those in low concentration group(0μg/ml, 100μg/ml), but OD value had no significant difference in high concentration exosomes group. Transwell chambers was applied to observe the effects of CSCs migration ability under different exosomes concentration, and the results showed that the migration ability had no significant difference between the 0μg/ml group and 100μg/ml group; but the migration ability of CSCs increased significantly with the concentration increased. Using Matrigel matrix, to observe the effects of CSCs angiogenesis ability under different exosomes concentrations, and the results showed that the angiogenesis ability had no significant difference between the low concentration exosomes(0μg/ml, 100μg/ml); However, the angiogenesis ability increased significantly with the concentration increased; the angiogenesis ability had no significant difference between 400μg/ml group and 800μg/ml group.(3) After CSCs were transplanted to treat MI rat, the survival CSCs-Di I were significantly more in CSCsExo group than those in CSCs group at short-term(10 days), either in BZ or in IZ. The EF value were significantly higher in both CSCsExo group and CSCs group than in the Control group in 28 days after transplantation; The newborn blood capillary density and the number of GFP-CSCs/Lectin 1 cells in the border zone were both significantly more in CSCsExo group than those in CSCs group. The length and area of MI fibrosis were both significantly lower in CSCsExo group than those in CSCs group.Conclusion: In vitro experiments, BMSCs-derived exosomes are able to enhance the capacity of CSCs proliferation, migration and angiogenesis, the role of which obviously dependents on the concentration of exosomes; and the effect of exosomes on the amplification and vascular formation ability of CSCs has saturability, which will reach saturation when the concentration of exosomes is 400μg/ml. In vivo experiment, exosomes can promote CSCs survival, new capillaries born, differentiation into blood vessels, and improve CSCs the ability of myocardial repair, reduction of the area of MI fibrosis.Part V Microarray analysis of rat cardiac stem cells and the role of mi R-326-5p in CSCs angiogenesisThe first section Microarray analysis of rat cardiac stem cells Objective: Through the gene chip to detect two kinds of different status of CSCs, between CSCs group and CSCsExo group, to find out the significantly different mi RNA and further to analyze the results.Method: Using Affymetrix gene chip to detect the two different status of CSCs between CSCs group and CSCsExo group(pretreatment with 400μg/ml exosomes), and repeat the experiment 3 times. Different mi RNAs were identified and further analyzed through bioinformatics.Results: By mi RNAs gene chip detection, significantly different expression of mi RNAs were 23, 5 of which were significantly lower, and 17 of which were significantly raised. mi R-326-5p had the most significant change, by reduction of 7.14 times. By GO analysis, the target genes of these different mi RNAs involved a wide range of cellular biological functions, including signal transduction, positive regulating cell amplification, endocytosis, positive regulate cell migration, cell adhesion, heart development, blood vessel formation, etc. By Pathway analysis, the target genes of these different mi RNAs involved several signal transduction pathways, including metabolic pathways, endocytosis, Wnt signaling pathways, Hippo signaling Pathway, PI3K/Akt signal Pathway, VEGF signaling pathways, chemotactic signaling pathways, etc. By mi RNA-Gene-Network analysis, mi R-326-5p, mi R-328a-5p, mi R-207, mi R-760-3p, and mi R-702-5p presented higher Degree in the Network diagram, respectively with 166, 177, 188, 177, 188.Conclusion: By mi RNAs gene chip detection, 23 mi RNAs have significantly different, 5 of which are significantly lower, and 17 of which are significantly raised. Through bioinformatics analysis, the target genes of these different mi RNAs involved a wide biological function including endocytosis function, VEGF signaling pathway and regulating cell amplification, regulating cell migration, angiogenesis and so on, and the prediction of these biological functions are accordance with the change of CSCs function observed in our experiment. mi R-326-5p, mi R-328a-5p, mi R-207, mi R-760-3p, and mi R-702-5p are most likely dominant in regulating the function of CSCs in this experiment, and the target genes of which are the most.The second section The role of mi R-326-5p in CSCs angiogenesisObjective: To further verify the mi RNA gene chip, we check the expression level of mi R-326-5p, which is the interest and the most significantly different mi RNA. And to explore whether the promotion of the blood vessel formation in CSCs is related with the changes of mi RNA-326-5p expression after Exosomes preconditioning?Method: Through qRT-PCR technology, the expression level of mi R-326-5p was verified. Application of liposome transfection technique, mi R-326-5p inhibitors was transfected into the CSCs, and then the transfection efficiency was verified through q RT-PCR technology. Using Matrigel matrix, the tube formation of CSCs was observed among the three groups, including CSCs group, CSCsExo group(pretreatment with 400μg/ml exosomes), and CSCsmi R-326-5p i group.Results: By q RT-PCR, the relative expression of mi R-326-5p in CSCsExo group was 0.18±0.05, which was significantly suppressed. After transfected with mi R-326-5p inhibitors in CSCs, the relative expression of mi R-326-5p was 0.32±0.08 by q RT-PCR, the inhibition efficiency of which was significant more than 50%. Tube length was significantly greater both in CSCsExo group and CSCsmi R-326-5p i group than that in CSCs group, but which had no significant difference between CSCsExo group and CSCsmi R-326-5p i group.Conclusion: By qRT-PCR detection, the significant reduction of mi R-326-5p in the gene chip was exactly verified. We confirm that exosomes is able to enhance the ability of CSCs angiogenesis through down regulating the expression of mi R-326-5p in the CSCs. |
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