Tracking Endothelial Progenitor Cells In Rabbit Aneurysms And Post-Tubridge Neointima Construction

The rupture of intracranial aneurysm is the main cause for subarachnoid hemorrhage, which was a complied with high mortality, morbidity and serious complications.Both of the aneurysm recurrence, rupture and healing repair process and the cause of aneurysms were under understand.Traditional surgical clipping and endovascular treatment is the main treatment of intracranial aneurysms.With the technology and materials advanced, endovascular treatment of aneurysm embolization from a simple occluding aneurismal sac to to complete parent artery reconstruction. Particularly, the development of stents made the foundation for the vascular reconstruction of aneurysm.Of the parent artery revascularization procedure requires further study.Endothelial repair are considered in the recurrence, regeneration and the cause of of 1aneurysms.The discovery of the endothelial progenitor cells (Endothelial progenitor cells, EPCs) provided new clues for aneurismal researches.In this study, the stem cell induced andtracking technology, the rabbit elastase-induced aneurysm model and a new flow-oriented support (Tubridge) were used to explore whether or not, if so and how the EPCs participate in the process of neointima formation after aneurysm stent treatment.一,The Expansion and Identification of Rabbit BoneMarrow-derived Endothelial Progenitor CellsAbstract Objective Mononuclear cells were harvested from rabbit bone marrow which was cultured for endothelial progenitor cells in EGM-2MV medium, and the cells expansion efficacy was observed. Methods Bone marrow mononuclear cells of ten rabbits were cultured with EGM-2MV medium in flasks coated with fibronectin. Morphology was observed with phase contrast microscopy, and a growth curve was constructed to evaluate the efficacy of expansion. 12 days after bone marrow mononuclear cells cultured, the attached cells were identified with immunocytochemical staining for CD133, CD34, vascular endothelial growth factor receptor 2(VEGFR-2). Incorporation of Dil-ac-LDL and combined FITC-UEA-1 lectin was tested to evaluate the function. The attached cells were selected with CD133 immunomagnetic beads. The percentage of CD133, CD34, VEGFR-2, CD34/ VEGFR-2 and CD133/CD34/ VEGFR-2 antigen-positive cell with flow cytometry detected, which before and after immunomagnetic beads selection, were compared. Results The cluster-like or colony-like growth cells were found after bone marrow mononuclear cells 48 h cultured. The cells were spindle, triangle or polygon shaped. The cells growth curve showed "S" type. About(1.46±0.13)×106 attached cells were harvested from each rabbit for 12 days. The cells took up Dil-ac-LDL and combined with FITC-UEA-1 lectin. Immunocytochemistry showed that CD34, CD133, and VEGFR-2 were positive. The percentage of CD34+/ VEGFR-2+ and CD133+/CD34+/ VEGFR-2+ cells after immunomagnetic beads selected were 3.38 times and 6.14 times that of unselected cells, respectively. Conclusion Endothelial progenitor cells can be harvested from rabbit bone marrow in a simple and efficacy method.二,Tracking endothelial progenitor cells in rabbit aneurysms repairAbstract Objective EPCs were presumed to be involved in intracranial aneurysm repair. The aim of this study is to explore whether EPCs contribute to and how to participate in repair aneurysms. Methods EPCs were obtained from clean rabbit bone marrow mononuclear cells which were induced to differentiate by the EGM-2MV. EPCs were double labeled by Hoechst33342 and CFSE (carboxyfluorescein diacetate, succinimidylester. Elastase-induced rabbit model aneurysms were used and divided into in situ arterial EPCs transfusion group and intravenous EPCs transfusion group randomly (n = 5). In situ arterial EPCs transfusion group: labeled EPCs were injected in situ of aneurysm. One week later, labeled EPCs were observed the cells focal adhesion, growth and involved in aneurysm repair. Intravenous EPCs transfusion group: labeled EPCs were transfused by the ear vein. Two weeks later, labeled EPCs were observed homing to the neointima of aneurysm. Results In situ arterial EPCs transfusion group: Labeled EPCs were detected within the neointima in all the 5 model aneurysms one week after EPCs transfusion. The neointima of aneurysms shows no endothelial growth. Intravenous EPCs transfusion group: Labeled EPCs were detected within the neointima in 3 of the 5 model aneurysms after two weeks EPCs transfused. The neointima of aneurysms shows no endothelial growth either. Conclusion EPCs are involved in the neointima formation during the initial process of aneurysm healing. 三,The Participation of Endothelial Progenitor Cells in Neointima Construction of Tubridge Stent Treated Rabbit AneurysmsAbstract Objective There has been a focus on the aneurysmal healing process. EPCs were presumed to participate in the neointima construction after a new flow diverter (Tubridge stent) treatment,and those processes were tested in this study. Methods The EPCs were derived from the bone marrow of ten rabbits and expanded in vitro. The EPCs were double labeled with Hoechst33342 and CFSE (5,6- carboxyfluorescein diacetate, succinimidyl ester), and transfused via autologous intravenous transfusion from the 1st day onwards (for the Two Weeks after Stent Group (TWSG, n=5) and the 14th day onwards (for the Four Weeks after Stent Group (FWSG, n=5 ) respectively. Two weeks after the transplant of EPCs, double labeled EPCs were tracked in the neointima of aneurysms and parent arteries. Scanning electron microscopic (SEM), transmission electron microscopic (TEM), pathological and immunohistochemistry methods were used to identify cells in the neointima.Results Double labeled EPCs were detected within the neointima or around the stent in 3 out of 5 rabbits in TWSG, while in the intimal surface in 2 out of 5 rabbits in FWSG. Only a few endothelial cells covered the neointima in the first two weeks after stent, but more endothelial cells covered the neointima in FWSG. Conclusion EPCs do participate in the neointima construction after aneurysmal Tubridge stent treatment, but not as uniquely differentiated endothelium.