| Stroke is the major disease which leads to death in the worldwide. At present, it have become the third largest cause of death only less than heart disease and cancer. Among all strokes,87%is ischemic, which is the main type of stroke with high morbidity, disability, mortality, recurrence, and so on. Ischemic stroke harms to patients and their families seriously, also imposes a significant economic burden on our healthcare system.Ischemic stroke brought about cerebral lack of blood, and the secondary reperfusion also can induce to severe brain damage. To return the blood supply of injuried brain as soon as possible and save the dying neurons, glial cells and vascular endothelial cells, which is the key to reduce injury of ischemic stroke. How to induce angiogenesis to promote the establishment of ischemic peripheral vascular or collateral circulation, is the most important for reducing tissue damage and promoting the functional reconstruction.In a series of experiment, we found that electroacupuncture can increase endothelial progenitor cells (EPCs) of bone marrow and peripheral blood in rat after cerebral ischemia and reperfusion injury, also can enhance the expression of VEGF, VEGFR2in the brain. While EPCs has close correlation with angiogenesis, also can participate in endothelial repair; VEGF is the most potential factor for EPCs in the process of which mobilization, homing, migration. It showed that acupuncture can stimulate the body to produce growth factors which related to EPCs, and promote the angiogenesis and endothelial repair. In addition, other studies have reported that bFGF and VEGF play a synergistic role in the process of angiogenesis, which can promote endothelial cell proliferation, migration, also had a positive effect on neuron; CD34is a surface antigen of EPCs, also can be used to mark the microvascular density to observe the angiogenesis of tissue. In order to research further the effect of acupuncture on EPCs expression and repairment of blood vessels in the region of cerebral ischemia-reperfusion, we intend to observe the expression of CD34+, bFGF in different time points.Our previous experiment belonged to the animal experiment. In order to observe the effects of acupuncture more clearly, and explore its mechanism, we intend to clture EPCs and use the "acupuncture serum" to intervene the EPCs for observing the effect of acupuncture on EPCs proliferation, migration, adhesion and so on. The "acupuncture serum" refers to the serum collected from the human or animal body which were treated by acupuncture or moxibustion.Through this research, we will further study the effects of acupuncture on the angiogenesis and EPCs after the cerebral ischemia-reperfusion, which may enable us to clarify the mechanism of promoting angiogenesis after injury by means of acupuncture promoting the move qi and blood and dredging collateral channels, also can play an important role for further exploring the mechanism of acupuncture treatment.PART ONE The effect of acupuncture on expression of bFGF, CD34in rats with cerebral ischemia reperfusionObjective: To observe the effect of electroacupuncture on expression of bFGF and CD34in ischemic cerebral cortex of cerebral ischemia-reperfusion rat, to study the influence of electroacupuncture on promoting angiogenesis after cerebral ischemia.Methods: male SD rats were randomly divided into normal group, sham-operated group, model group, electroacupuncture group and pre-electroacupuncturc group, later three groups manufactured middle cerebral artery occlusion (MACO) model. The pre-electroacupuncture group and electroacupuncture group cured separately before or after modeling using "Housanli(ST36)" and "Quchi(LT1L)" point. bFGF and CD34expression was observed in ischemic cerebral cortex of MACO rats after reperfusion12hours,24hours,48hours using immunohistochemistry.Result.(1) In normal group and sham operation group, the positive of CD34and bFGF was less.(2) The positive bFGF in model group was at a low level after cerebral ischemia-reperfusion12hours, later it increased with change of Lime, after cerebral ischemia-reperfusion24hours, it reached the peak, then gradually decreased; CD34was at a low value after cerebral ischemia-reperfusion12hours, with the extension of time, the rise began, and it still rose after cerebral ischemia-reperfusion48hours. bFGF and CD34in model group were different obviously from the normal group and sham-operated group at each time point.(3) The positive bFGF in the electroacupuncture group had no significant difference with the model group after cerebral ischemia-reperfusion12hours, then it rapidly increased and had significant difference with the model group after cerebral ischemia-reperfusion24and48hours; the positive CD34had a rapid rise after cerebral ischemia-reperfusion12~24hours, while after cerebral ischemia-reperfusion24~48hours, it rose in parallel to model group, after cerebral ischemia-reperfusion12hours, the positive CD34had no significant difference with the model group, but after24and48hours, it higher than in model group and had significant difference;(4) the bFGF in pre-electroacupuncture group rose rapidly and descended slowly, which was different obviously with the model group and electroacupuncture group at each time point; the expression of CD34rose persistently and was different obviously with the model group and electroacupuncture group at each time point. Conclusion: Electroacupuncture and the pre-electroacupuncture can obviously increase the expression of bFGF and CD34in ischemic cerebral cortex of cerebral ischemia-reperfusion rat, contribute to angiogenesis.PART TWO SD rat bone marrow-derived endothelial progenitor cells isolation, culture and characterizationObjective:To study the isolation, culture and identification of Sprague Dawley (SD)rat bone marrow-derived EPCs.Methods:Bone marrow mononuclear cells (BMMNCs) were isolated"from bone, marrow of SD rats (4weeks old) by density gradient centri fugation, and cultured by EGM-2MV medium. Then identified the cell by observing the morphology, the assay of "ptaking Dil-Ac-LDL and combining with FITC-UEA-1,the expression of surface (?)igens CD133and VEGFR2by fluorescent immunocytochemistry, and evaluating the formation capillary ability through the tube formation assay on matrigel.Results:(l)Morphology:After culturing of the separated BMMNCs, the shape was different in the early (about8th day) and later (about10th day) of growth stage. In the early, they were spindle, triangle and round;In the later, they showed round and short spindle appearance(2)The assay of uptaking Dil-Ac-LDL and combing with FITC-UEA-1:Both the8th and21th cells were all positive;(3)Fluorescent immunocytochemistry:The8th cells expressed CD133and VFGFR2;(4)Tube formation assay:FPCs formed capillary-like formations after about15hours when plated on matrigel.Conclusion: We can isolate the rat BMMNCs using density gradient centri fugation and then culture them with EGM-2MV medium. The aquired colls were proved to be consistent with the characteristics of EPCs. This method is simple, quick, reliable to get enough EPCs.PART THREE Effects of acupuncture serum on proliferation, migration, adhesion of rat bone marrow-derived EPCsObjective: To study and investigate the effect of electroaeupuncture scrum and pre-electroacupuncture serum on the proliferation, migration, adhesion of cultured EPCs.Methods: In experiment one, we got the serum after cerebral ischemia-reperfusion48hours from the model group,the electroacupuncture group and the pre-electroacupuncture group, meanwhile obtained the normal serum. Adding different serum to the cultured rat bone marrow-derived EPCs, and set the control group, using MTT method to determi ne the su itabl e concent ration of serum in vitro intervention, then using the MTT assay, migration assay, adhesion assay to observe the effect of electroacupuncture.Results:(1) In vitro, the most significant proliferal ion of EPCs was the concern rat ion of20%which came from pre-electroacupuncture(2) Pre-electroacupuncture serum had the most sigrnficanl effect on EPCs proliferation, migration, adhesion, which was different significantly from the normal group, model group, and electroacupuncture group;(3) Electroacupuncture serum can enhance the proliferation, migration, adhesion of EPCs in vitro. The migration and adhesion was di fferent significantly from the normal group and model group; The proliferation was different significantly from the normal group.(4) Model group serum can enhance the proliferation, migration, adhesion of EPCs in vitro, which was significantly different with the normal group;(5) In normal group, EPCs prol i ferat ion, migration, adhesi on abilit y was higher than the control group, also had obvious difference compared with control group.Conclusion: ElecLroacupuncture and pro-electroacupuncturc serum can obviously enhance the proliferation, migration, adhesion of EPCs, this may be one of the factors that the Electroacupuncture therapy can promote tissue repair and neovascularization after injury and ischemia, also may be the effect of acupuncture can " move the Qi and blood, smooth t ho main and col lateral channels". |
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