| BackgroundPostnatal vasculogenesis has been reported through bone marrow derived EPCs incorporating into new intima and involving in the new capillaries,and EPCs promots the vasculogenesis of ischemic myocardium was approved by many reaserchers since1997.The effects of EPCs on ishchemic lower limbs are similar with that of ischemic myocardium,but was researched less than adequate. And the theory above has been challenged through bone marrow EPCs (BM-EPCs) pormoting the vasculogenesis but in other ways such as it never incorpearted into the intima of new vessels and involve in the new capillaries but promots vasculogenesis by paracrining and autocrining VEGF and HGF and other cytokines which aggregate the endothelial cells (ECs) and smooth muscle cells (SMC) which build up the new vessels in the final. Our research used autologous implantation to avoid the immue response and cultured bone marrow monoueuclear cells (MNCs) and induced them to be BM-EPCs to abserve whether BM-EPCs can significantly promote the vasculogenesis by incorpearting into the new intima of new capillaries.We hope to acquire more evidences before BM- EPCs'clinical application.ObjectivesCultrue BM-EPCs from bone marrows and implant them into the ischemic muscles of the rabbit hindlimbs and observe whether they incorporate into the intima of new blood vessels and promote angiogenesis and vasculogenesis significantly within two weeks.Method and materialTwenty-four New Zealand White Rabbits was assigned to 2 groups (each twelve) by random number law. A longitudinal incision was then performed, extending downward from the middle of inguinal ligament to the point just proximal to the upper edge of patella. The limbs in which the incision was performed on were left hindlimbs.Through this incision, the femoral artery was dissected free, along its entire length, all of branches of femoral artery, including the inferior epigastric, deep femoral, lateral circumflex, and superficial epigastric arteries, were also dissected free. After further dissecting the popliteal and saphenous arteries distally, the external iliac artery also as all of the above arteries were ligated (4.0 silk) Finally, the femoral artery was completely excised from its proximal origin as a branch of the external iliac artery, to the point distally where it bifurcates to form the saphenous and popliteal arteries. Ligated and cut off every visible branches (including deep femoral artery) between the two sites and freed and abandoned vessels which was cut and established the ischemic animal model. Bone marrow 4ml were extracted from upper extremity of tibia and mononuclear cells were separated from the bone marrow by gradient density centrifugation method. BM-EPCs were induced and cultured in EBM-2 medium with VEGF(vascular endothelial growth factor), bFGF(Basic fibroblast growth factor)and IGF(Insulin-like growth factor)and other components and 5%FBS(fetal bovine serum) and were identified by binding with both Ac-LDL ( Acetylated low-density lipoprotein ) and UEA-1 each coupled with Fluorescein, and checked by fluorescent microscope. The amount of BM- EPCs was expanded by cell passage based on its'quality of clone. EPCs were transplanted into the muscles of rabbit ischemic hindlimbs of group of treatment on tenth culturing day ie.the seventh day of after the establishemnent of ischemic model, and were marked with Brdu(5 - bromo-ODN Fu)by incubating with Brdu with a concentration of 10 umol/l for 24 hours in medium before they were transplanted. To the ischemic hindlimbs of control group only the equal volume medium was injected at the same time. Six rabbits of treatment group and control group were sacrificed randomly by overdose anesthesia after 7 days of transplantation, and the specimen were fixed with 10% neutral formaldehyde and performed immunohistochemistry with antibody of CD31and Brdu to mark capillary and BM- EPCs seperately. Then the capillary of each specimen was counted and we used SPSS 13.0 software package to analyse the data by two samples t-test, and the location of theBM- EPCs was observed as well. The similar procedure was performed after 14 days of transplantation on the rabbit left.ResultsBM-EPCs cultured and labeled by Brdu incorporated into the new intima of capillaries and the amount of capillaries and capillary muscle fibre rate per high folder field of the treatment and control groups as below: cappilaries counted and cappiliaries muscles fibro ratio of 7d treatment and control group( x±sd n=6 , the same as below ) :4.19±1.10vs2.47±1.08,P=0.021 ; 0.86±0.38vs 0.38±0.08,P=0.027。That of 14days: 5.3 9±1.58vs2.10±1.05 , P=0.002 ;1.12±0.58vs0.26±0.14,P=0.014。ConclusionsBM-EPCs cultured by our research directly incorporated into the new blood vessels of the ischemic hindlimbs of rabbits and BM-EPCs transplantation significantly promoted angiogenesis and vasculogenesis of the ischemic hindlimbs of rabbits within two weeks. |
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