| BackgroundWith the change of eating habit, improvement of life and life lengthening, the morbidity of peripheral arterial diseases (PAD) is higher than ever, and lower extremity ischemic diseases are threatening human health seriously. The symptoms of the lower limb such as dread cold, numbness, intermittent claudication, pain and gangrene come out when the main arteries became stenosis and occlusion. Now the treatments of lower extremity ischemic diseases include medical therapy, surgery and endovascular treatment. But there is always no opportunity of surgery for those who have serious stenosis of ourflow tract especially for those with diabetes, and there is no satisfied treatments yet. Improving the neovascularization and collateral circulation by using some new methods maybe feasible and effective.At present, Therapeutic angiogenesis (vessel growth factors and its gene therapy, stem cells therapy) are hot spots in the treatments of ischemic diseases. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) can stimulate the angiogenesis and relieve the state of ischemia . The advantage of stem cells is that it is easier to be modified compared with adult tissues, and it is a good target cell for gene therapy. So combined with stem cell and gene therapy may be promising for the treatment of ischemic diseases. We designed the experimental research on promoting collateral circulation and angiogenesis in limb ischemia treated with integrated Chinese and western medicine.ObjectiveTo respective evaluate the effect of local application recombinant human basic fibroblast growth factor (rh-bFGF), mobilized bone marrow stem cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF) and co-application of them on angiogenesis in ischemic limbs of rabbits.Materials and methodsAfter ischemia models were induced by surgical ligation and cutting off left femoral artery in each rabbit, rabbits were randomized to received intramuscular injection of recombinant human bFGF (n=10) in ischemic zone, hypodermic injection of recombinant human G-CSF (n=10) , co-application of rh-bFGF and rhG-CSF (n=10) or normal saline (n=10). Four weeks later, angiogenesis of ischemia models were observed and evaluated by abdominal aortography, histopathologic analysis and immunohistochemical studies with the antibodies of VEGF. All experimental data were processed by SPSS11.5.Results1 General observationOne rabbit died of anesthetic accident (bFGF+G-CSF group). One rabbit died of intestinal obstruction at the second day after operation (bFGF group). One rabbit died of diarrhea at the fourth day after operation (control group). Two rabbits died unexpected at the fifth and sixth day after operation (G-CSF group). Other rabbits all have vital sign steadily. There is no ischemic necrosis of left hind limb. Operative incisions are healed well in four days after operation, and operative incisions are no red swelling of the skin or effusion. Three rabbits of control group are lameness after operation. One rabbit of control group show up an ulcer at dorsum of foot. There is no ulcer or lameness in other groups. All experience rabbits have falling off of hair in left hind limb.2 Blood routineAll rabbits of G-CSF group and bFGF+G-CSF group inject hypodermic G-CSF 10μgfkg after operation. White blood cell of peripheral blood rise obviously. The number of white blood cell would almost get highest at the sixth day, that is 26.2×10~9/L-43.6×10~9/L, and the average is 35.3×10~9/L. The juvenile cells can be found at high power lens in the fourth day. White blood cell of peripheral blood of other group's rabbits have no marked change, that is 5.4×10~9/L-10.2×10~9/L, the average is 8.6×10~9/L.3 The compensatory circulation blood vesselsThe photographs of abdominal aortography appear that the femoral artery ware broken off and appeared some compensatory circulation blood vessels. The photographs of bFGF+G-CSF group appeared many compensatory circulation blood vessel reticulodromous conjunct the broken femoral artery. The photographs of bFGF group appeared some compensatory circulation blood vessel. The photographs of G-CSF group appeared a few compensatory circulation blood vessels. The photographs of control group appeared few compensatory circulation blood vessels. The number of bFGF+G-CSF group compensatory circulation blood vessels is more than that of bFGF group (p<0.05). The number of bFGF group compensatory circulation blood vessels is more than that of G-CSF group (p<0.05). The number of G-CSF group compensatory circulation blood vessels is more than that of control group (p<0.05).4 The capillariesThe tissue slices of adductor and gastrocnemius muscle were stained by HE and counted the number of capillaries at the 400 times micro. bFGF+G-CSF group appeared many capillaries at muscle bundle. bFGF+G-CSF group appeared many capillaries at muscle bundle. bFGF group appeared some capillaries at muscle bundle. G-CSF group appeared a few capillaries at muscle bundle. bFGF+G-CSF group appeared few capillaries at muscle bundle. The member of capillaries in bFGF+G-CSF group is the most (p<0.05), that of bFGF group is more than that of G-CSF group (p<0.05), and that of saline group is the least (p<0.05).5 The vascular endothelial cells that expressed VEGF proteinThe tissue slices of adductor and gastrocnemius muscle were also used for studying expression with the antibodies of VEGF by immunohistochemistry staining. And counted the number of the vascular endothelial cells that expressed VEGF protein at the 400 times micro. The member of the vascular endothelial cells that expressed VEGF protein in bFGF+G-CSF group is the most (p<0.05), that of bFGF group is more than that of G-CSF group (p<0.05), and that of saline group is the least (p<0.05).Conclusions1. Hypodermic injection of recombinant human G-CSF (mobilization of bone marrowstem cells) can improve the angiogenesis of the ischemic limbs.2. Intramuscular injection of recombinant human bFGF can improve the angiogenesisof the ischemic limbs.3. Both intramuscular injection of recombinant human bFGF and hypodermic injection of recombinant human G-CSF (mobilization of bone marrow stem cells) can stimulate the angiogenesis of the ischemic limbs. And the effect will be better if co-application of them.
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