Studies On Effect Of MiR-483-3p Regulation On Endothelial Progenitor Cells For Venous Thrombus Recanalization And Resolution

Deep venous thrombosis(DVT) is a common peripheral vascular disease. It may lead to post-thrombotic syndrome(PTS) and pulmonary embolism(PE) which can lead to the sudden death of patients. Currently, the anticoagulants, thrombectomy, transcatheter, thrombolytic therapy and other clinical treatments for DVT, have failed to remove the incidence, low future patency, easy recurrence and other defects of PE. Therefore, it is necessary to find a safer and more effective treatment for DVT.Recently, among the ischemic diseases, the discovery of endothelial progenitor cells(EPCs) has been widely discussed. Endothelial progenitor cells(EPCs) are endothelial precursor cells derived from bone marrow, which plays an important role in the process of neoangiogenesis. However, the application of EPCs has met many problems, because only a small amount of transplanted EPCs can be differentiated into vascular endothelial cells. Therefore, it has become an important research direction to improve the functions of EPCs.MicroRNAs are a class of non-coding RNAs about 22 nucleotides long, which can identify the 3’UTR zone of target genes by exactly/non-exactly matching methods, inhibit the protein translation or affect the stability of miRNAs, and suppress gene expression at the post-transcriptional level, which acts significant biological functions. In recent years, studies have confirmed the involvement of miRNAs in regulating EPCs functions, which plays an important role in vasculogenesis and angiogenesis. However, there are still some doubts to be resolved. And this topic is designed for people to have a further knowledge about whether there are any miRNAs expression differences of EPCs in peripheral blood between DVT patients and normal patients, as well as whether the changed miRNAs can control the EPCs functions and affect the venous thrombus recanalization and resolution.Mononuclear cells in peripheral blood collected from DVT patients and healthy people were isolated by density gradient centrifugation. The EPCs were induced and cultured in vitro; the miRNAs expression differences of EPCs between DVT patients and healthy people were screened by gene microarrays, the results of which were verified with a method of real-time fluorescence quantitative PCR(qRT- PCR). As to the miRNAs which have consistent results with those of microarrays, some were chosen according to relative literatures and bioinformatics to conduct a further verification on cell functions( 6 mi RNAs were selected), the results of which showed miR-483-3p can influence the EPCs functions. To conduct a further study on miR-483-3p, the possible target genes of mi R-483-3p were predicted through the bioinformatics; the influence of target genes, co-transfection and gene silencing on EPCs functions were further confirmed by using Western blot to detect the protein change following the luciferase experiment, up-regulation and down-regulation of miR-483-3 p, co-transfection and gene silencing.The influence of mi R-483-3p on EPCs homing as well as thrombus recanalization and resolution was observed in vivo experiments through the establishment of rat thrombus model, confocal laser scanning fluorescence microscope, HE staining and DSA. 。It turned out that miR-483-3p showed high expression for EPCs in DVT patients; when EPCs is down-regulated, miR-483-3p expression would promote EPCs migration and tube formation ability, inhibit EPCs apoptosis, and promote the EPCs homing as well as EPCs recanalization and resolution for thrombus. Our research may present a promising clinical therapy for ischemia diseases.This study shall be divided into the following five chapters Chapter I Cultivation and Identification of Endothelial Progenitor Cellsfrom Human Peripheral Blood and Rat Bone MarrowObjective:It is to culture the endothelial progenitor cells from human peripheral blood and bone marrow-derived endothelial progenitor cells from rats for further experiments.Methods: Density gradient centrifugation method was adopted to isolate the mononuclear cells in human peripheral blood and rat bone marrow, which were suspended in the EGM-2MV culture medium for two or three weeks. Morphological changes were observed under the microscope. The markers of CD34, CD133 and VEGFR-2 expression on cell surface were detected by a flow cytometry. And the AO/EB fluorescent staining method was used to detect cellular ability of absorbing DiI-acLDL and FITC-UEA-1.Results: The isolated peripheral blood mononuclear cells(PBMC) and bone marrow mononuclear cells are round and small. Two days later, a few cells are adhered. Three days later, the cell volume increased, and more cells are adhered. After five days, the adherent cells are in a fusiform shape, and cell colony emerges, with surrounding cells radially arranged; from 10 th day to 14 th day, cells were arranged like the road with pebbles and paving stones. The results of flow cytometry shows that the main expression of endothelial cell surface markers, VEGFR-2, CD34 and CD133, is low. And the AO/EB fluorescent staining detects that cells can devour DiI-ac-LDL and FITC- UEA-1.Conclusion: Under the induction of EGM-2MV medium, EPCs have been successfully cultured from human peripheral blood mononuclear cells and rat bone marrow mononuclear cells, which showed characteristics of late EPCs after the culturing for 2 or 3 weeks.Chapter II: Screening and Verification of Differential Expression Profile of mi RNAs in Deep Venous ThrombosisObjective: It is to screen the mi RNAs expression profile differences in peripheral blood EPCs from DVT patients and healthy people with microarrays, and to verify the reliability of the results through q RT-PCR.Methods: Peripheral blood samples from DVT patients and healthy people were collected, the mononuclear cells of which were isolated with density gradient centrifugation method for EPCs culturing. By means of mi RNAs gene microarrays, mi RNAs expression profile differences in EPCs from DVT patients and healthy people were screened, and the results were verified through q RT-PCR.Results: MiRNA microarrays displayed that expression of MiR-483-3p and other mi RNAs in peripheral blood EPCs was different between DVT patients and healthy people. The q RT-PCR results were in consistent with those of microarrays.Conclusion: Expression of Mi R-483-3p and other mi RNAs in peripheral blood EPCs was different between DVT patients and healthy people.Chapter 3 Effect of Mi R-483-3p on Endothelial Progenitor Cell Functions as well as Prediction and Verification of Target GenesObjective: It is to investigate the effect of mi R-483-3p on migration, proangiogenic properties and apoptosis of human peripheral blood EPCs as well as to predict and validate the target genes.Methods: By use of lipofectamine 3000, the mi R-483-3p agomir, antagomir and negative control were transfected into EPCs. The impact of mi R-483-3 on migration of EPCs was detected by transwell experiment. The impact of mi R-483-3 on EPCs proangiogenic properties was detected through the matrigel tube formation assay. By flow cytometry, the impact of mi R-483-3 on EPCs apoptosis was detected. The possible target genes of mi R-483-3p were predicted by use of bioinformatics, and the genes were confirmed through the luciferase experiments, q RT-PCR, western blots and other experiments.Results: The up-regulation of mi R-483-3p expression in EPCs would inhibit the migration and proangiogenic properties of EPCs, and promote the apoptosis of EPCs; while the down-regulation showed the opposite result. According to bioinformatics, serum response factor(SRF) may be a target gene. Moreover, the prediction on SRF was reconfirmed through q RT-PCR and western blots experiments.Conclusion: The up-regulation of mi R-483-3p expression in EPCs shall inhibit the migration and proangiogenic properties of EPCs, and promote the apoptosis of EPCs; SRF is the target gene of mi R-483-3p.Chapter IV Construction and Expression of MiR-483-3p Lentiviral VectorsObjective: It is to construct the mi R-483-3p/mi R-483-3p sponge lentiviral vectors, to infect EPCs in rats, and to verify the expression of mi R-483-3p in EPCs, thus laying the foundation for subsequent in vivo experiments.Methods: p GLV3-H1-GFP-mi R-483-3p and p GLV3-H1-GFP-miR-483-3p sponge were generated with connection of mi R-483-3p precursors sequence and lentiviral vectors through double digestion, which were transfected into 293 T cells with secondary packaging carriers. After the collected virus supernatant was infected with EPCs, the transfection efficiency of which was observed under a fluorescence microscope. And the mi R-483-3p expression in infected EPCs was detected through q RT-PCR.Results: Lentiviral vectors, p GLV3-H1-GFP-mi R-483-3p/p GLV3-H1-GFP-mi R-483-3p sponge, were successfully established. The transfected EPCs can effectively up-regulate and down-regulate the mi R-483-3p expression in EPCs.Conclusion: Lentiviral vectors, p GLV3-H1-GFP-mi R-483-3p/p GLV3-H1-GFPmi R-483-3p sponge, which are carried with the target gene mi R-483-3p, were successfully constructed.Chapter V Effect of Mi R-483-3p Regulation on Endothelial Progenitor Cell for Venous Thrombus Recanalization and ResolutionObjective: It is to investigate the effect on EPCs homing, the venous thrombus recanalization and resolution after the regulation of mi R-483-3p on EPCs in rats.Methods: Lentiviral vectors, p GLV3-H1-GFP vector, p GLV3-H1-GFP-mi R-483-3p and p GLV3-H1-GFP-mi R-483-3p sponge, were transfected into EPCs. The rat model of deep vein thrombosis was constructed by ligating the postcava at the bottom of the left renal vein. And then the transfected EPCs were transplanted into thrombosis model through caudal vein of rats. On deep venous thrombosis after IVC ligation, the alive rats were divided as 4 groups for cells transplantion via tail intravenous injection: A(n=10), blank control group(blank control), which received 1 ml PBS. B(n=10), EPCs/ p GLV3-H1-GFP vector group(EPCs/vector), which received 1.0×106 EPCs transfected with lentivirus particle of p GLV3-H1-GFP vector. C(n=10), EPCs/p GLV3-H1-GFP-mi R-483-3p group(EPCs/ mi R-483-3p), which received 1.0×106 EPCs transfected with lentivirus particle of p GLV3-H1-GFP-mi R-483-3p. D(n=10), EPCs/p GLV3-H1-GFPmi R-483-3p sponge group(EPCs/ mi R-483-3p sponge), which received 1.0×106 EPCs transfected with lentivirus particle of p GLV3-H1-GFP-mi R-483-3p sponge. Specimens were collected seven days after the transplantation. The EPCs homing in thrombosis was detected by fluorescence microscopy. The venous thrombus recanalization and resolution were observed through the HE staining and digital subtraction angiography(DSA).Results: GFP fluorescence-labeled EPCs emerged in venous thrombosis, and the numbers of positive cells in all experimental groups were: EPCs/mi R-483-3p sponge group> EPCs/vector group> EPCs/mi R-483-3p group, suggesting that mi R- 483-3p restricted the EPCs homing into the venous thrombosis.The thrombosis weight in different experimental groups was: blank control group> EPCs/mi R-483-3p group> EPCs/vector group> EPCs/mi R-483-3p sponge group, suggesting that mi R-483-3p inhibited the thrombolytic ability of EPCs.The thrombosis recanalization and resolution observed through HE staining in all experimental groups were: EPCs/mi R-483-3p sponge group> EPCs/vector group> EPCs/mi R-483-3p group> blank control group;The thrombosis recanalization and resolution observed through DSA in all experimental groups are: EPCs/mi R-483-3p sponge group> EPCs/vector group> EPCs/mi R-483-3p group> blank control group, suggesting overexpression of mi R-483-3p inhibited the recanalization and resolution of EPCs for thrombosis, and down-regulation of mi R-483-3p promoted thrombolytic ability of EPCs.Conclusion: The transplanted and transfected EPCs can be homed to venous thrombosis transplantation and transfection. The up-regulation of mi R-483-3p can inhibit homing ability of EPCs, as well as recanalization and resolution of EPCs for thrombosis; while down-regulation of mi R-483-3p can promote the homing ability and thrombolytic ability of EPCs.