碱性成纤维细胞生长因子治疗下肢缺血剂量的实验研究
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摘要
目的:通过对30只兔下肢缺血模型的大体观察、DSA计数侧支血管数、微血管密度检测及微血管/肌纤维束比值测定,评价不同剂量rh-bFGF治疗兔后肢缺血的有效性,筛选rh-bFGF治疗兔下肢缺血的最佳剂量;通过肝、肾功能检测、动脉造影和病理学观察,评价不同剂量rh-bFGF治疗兔后肢缺血的安全性;为临床应用rh-bFGF治疗肢体动脉狭窄或闭塞性病变提供理论依据。
材料和方法:应用30只大耳白兔,采用切除左侧后肢股动脉及结扎其主要分支的方法建立下肢缺血模型。随机分为5组(每组6只),A组(对照组),经肌肉局部注射5ml Tris缓冲液/只;B、C、D、E组为治疗组,B组,经肌肉局部注射rh-bFGF 2.5μg+5ml Tris缓冲液/只;C组,经肌肉局部注射rh-bFGF 5μg+5mlTris缓冲液/只;D组,经肌肉局部注射rh-bFGF 10μg+5ml Tris缓冲液/只;E组,经肌肉局部注射rh-bFGF 20μg+5ml Tris缓冲液/只。各组在模型制作后第10天在术侧肢体股内侧肌群分5点注射给药,造模术后观察30天处死。各组于给药前、给药后10天、给药后20天抽取静脉血检测肝、肾功能;各组动物处死前均采用DSA技术观察肢体侧支血管形成,计数侧支血管数;处死动物后取股内侧肌肉制作标本,进行毛细血管密度、微血管/肌纤维束比值检测及病理学观察。
结果:1.大体观察:各组动物实验中均无死亡。模型制作后10天内动物左下肢有不同程度的跛行。模型制作10天以后A、B组跛行逐渐加重;C、D、E组用药后跛行逐渐缓解。各组手术创口愈合较好,无下肢溃疡坏疽。A组处死后取病理组织时发现一只愈合切口内有脓肿形成。2.下肢动脉造影:DSA影像显示各组左侧股动脉连续性中断,A组有少量纤细的侧支血管分布于大腿中段乏血管区,B、C组由同侧髂内动脉、股深动脉发出数支侧支血管,远端动脉未见显影,D、E组见缺血肢体有较多的侧支血管迂曲走行,缺血肢体远端动脉通过侧支循环显影,充盈速度较健侧稍慢;各组DSA均未见肿瘤样及畸形血管形成。D、E组侧支血管数均多于对照组及B、C组(P＜0.05),但D、E组间侧支血管数比值无统计学意义(P＞0.05);B、C组与对照组及B、C组间侧支血管数比较均无统计学意义(P＞0.05)。3.微血管密度和微血管/肌纤维束比值:B、C组与对照组微血管密度和微血管/肌纤维束比值差异无统计学意义(P＞0.05):B、C组间微血管密度和微血管/肌纤维束比值无统计学意义(P＞0.05);D、E组微血管密度和微血管/肌纤维束比值较对照组增高(P＜0.05);D、E组缺血肢体的微血管密度和微血管/肌纤维束比值与B、C组比较差异有统计学意义(P＜0.05);但D、E组间微血管密度和微血管/肌纤维束比值无统计学意义(P＞0.05)。4.病理学观察:A组骨骼肌纤维萎缩、变性,肌纤维间距增大,间质内血管稀少;B、C组骨骼肌纤维轻度萎缩,肌纤维间距增大,间质内见少量毛细血管;D、E组血管生成较多,管腔内可见红细胞,肌纤维未见明显萎缩;各组骨骼肌细胞在rh-bFGF的影响下未见异常增殖或肿瘤样生长等表现。5.血肝、肾功能检测:各组给药前、给药后10天、给药后20天抽取静脉血检测血ALT、AST、BUN、Cr,各组内不同时间各项指标检测值比较差异无统计学意义(P＞0.05)。
结论:1.rh-bFGF可有效促进兔缺血肢体血管新生、侧支循环建立:其疗效随着rh-bFGF剂量的增加而增强,在10μg时取得最好的治疗效果,20μg时疗效不再增加,10μg是rh-bFGF治疗肢体缺血的最佳剂量。2.应用实验剂量rh-bFGF治疗肢体缺血是安全的,对肝、肾功能无明显影响,不会造成血管平滑肌、骨骼肌细胞的异常增生和肿瘤样生长。
Objective:To screen the optimal dose of recombinant human basic fibroblast factor (rh-bFGF)and evaluate the effect of different doses of rh-bFGF in 30 rabbit models of hindlimb ischemia through observation in general,the number of collateral branch, microvessel density and microvessel density/myolin ratio.To evaluate the safety of different doses of rh-bFGF by testing the liver and kidney function,digital subtraction angiography and pathological observation.To provide theoretial basis for clinically practising rh-bFGF to treat low limb ischemia.
Material and methods:30 rabbits were used to establish the animal model of lower limb ischemia by removed femoral artery of left and deligated its branch vessels. Randomized at five groups(6 each group),A group was intramuscular injected 5ml Tris/each;B,C,D and E groups are treating groups.The four groups were respectively intramuscular injected bFGF 2.5μg,5μg,10μg and 20μg+5ml Tris/each. The animals of each experiment group were once intramuscular injected in five spots of its thigh in 10 days after operation,and were put to death in 30 days after operation. Before injection,10 days after injection and 20 days after injection,the venous blood was collected to detect the liver and kidney function.The number of collateral branch was counted by digital subtraction angiography before the rabbits were put to death. The sample of medial vastus muscle was analyzed and observed for the changes of pathologic structure with microscope.
Results:1.observation in general:Animals of each groups were all alive in the period of experiment,and showed claudecation in distinct degrees in 10 days after the models were made.The claudication in A group and B group became more serious gradually,But it improved gradually in C,D and E groups.The cut healed up well and no ulcer and gangrene in each group.Abscess was found in one of A group.
2.Digital subtraction angiography of lower limb:DSA showed that the left femoral artery broke off.A few slight collateral vessels were distributed over the middle of thigh in A group.In B group and C group,several collateral vessels originated from internal iliac artery and deep femoral artery,the distant artery didn't develop.However,many collateral vascular creeped in D group and E group,and the distant artery of operated hindlimb developed because of collateral circulation,the speed of blood was a little slower than the uninjured side.There are no malformed capillaries and tumorous vessels in each group.The number of collateral casculars in D group and E group were all higher than A group,B group and C group(P＜0.05);but D group and E group were not statistically significant(P＞0.05).Among A,B and C group,the numbers were not statistically significant(P＞0.05).
3.Microvessel density and microvessel density/muscle fiber ratio:microvessel density and microvessel density/muscle fiber ratio among A group,B group and C group was no statistical difference(P＞0.05).But it in D group and E group was higher than A group(P＜0.05).It was also higher than B group and C group(P＜0.05). But D group and E group have no significant difference(P＞0.05).
4.Pathological observation:In A group,many skeletal muscle fibers were atrophy,space between muscle fibers turn long,and there were little blood vessels in mesenchymal.In B group and C group,the muscular atrophy was slight,there were a few blood vessels.But there were a lot of angiogenesis and collateral vessels in D group and E group.Some red cells could be seen in the vessels and no signifivant muscular fibers atrophy.Besides,skeletal muscle cell and other cell can not abnormal proliferation.Tumorous development was not found.
5.Liver and kidney function test:Before injection,10 days and 20 days after injection,the venous blood was collected to test ALT,AST,BUN,Cr.There were no statistical difference of the result in different time among each group(P＞0.05).
Conclusion:1.bFGF could promote therapeutic angiogenesis and collateral circulation formation effectively in a rabbit model of hind limb ischemia.Its curative effect strengthen with the dose of bFGF added.The curative effect was the best in 10μg,but it didn't increase in 20μg.10μg was the best dose for bFGF to treat lower limb ischemia.2.Applying experimental dose of bFGF to treat lower limb ischemia was safe,it has no affact on liver and kidney function and can not lead to abnormal hyperplasia and tumorous growth of skeletal muscle cell and other cells.
材料和方法:应用30只大耳白兔,采用切除左侧后肢股动脉及结扎其主要分支的方法建立下肢缺血模型。随机分为5组(每组6只),A组(对照组),经肌肉局部注射5ml Tris缓冲液/只;B、C、D、E组为治疗组,B组,经肌肉局部注射rh-bFGF 2.5μg+5ml Tris缓冲液/只;C组,经肌肉局部注射rh-bFGF 5μg+5mlTris缓冲液/只;D组,经肌肉局部注射rh-bFGF 10μg+5ml Tris缓冲液/只;E组,经肌肉局部注射rh-bFGF 20μg+5ml Tris缓冲液/只。各组在模型制作后第10天在术侧肢体股内侧肌群分5点注射给药,造模术后观察30天处死。各组于给药前、给药后10天、给药后20天抽取静脉血检测肝、肾功能;各组动物处死前均采用DSA技术观察肢体侧支血管形成,计数侧支血管数;处死动物后取股内侧肌肉制作标本,进行毛细血管密度、微血管/肌纤维束比值检测及病理学观察。
结果:1.大体观察:各组动物实验中均无死亡。模型制作后10天内动物左下肢有不同程度的跛行。模型制作10天以后A、B组跛行逐渐加重;C、D、E组用药后跛行逐渐缓解。各组手术创口愈合较好,无下肢溃疡坏疽。A组处死后取病理组织时发现一只愈合切口内有脓肿形成。2.下肢动脉造影:DSA影像显示各组左侧股动脉连续性中断,A组有少量纤细的侧支血管分布于大腿中段乏血管区,B、C组由同侧髂内动脉、股深动脉发出数支侧支血管,远端动脉未见显影,D、E组见缺血肢体有较多的侧支血管迂曲走行,缺血肢体远端动脉通过侧支循环显影,充盈速度较健侧稍慢;各组DSA均未见肿瘤样及畸形血管形成。D、E组侧支血管数均多于对照组及B、C组(P＜0.05),但D、E组间侧支血管数比值无统计学意义(P＞0.05);B、C组与对照组及B、C组间侧支血管数比较均无统计学意义(P＞0.05)。3.微血管密度和微血管/肌纤维束比值:B、C组与对照组微血管密度和微血管/肌纤维束比值差异无统计学意义(P＞0.05):B、C组间微血管密度和微血管/肌纤维束比值无统计学意义(P＞0.05);D、E组微血管密度和微血管/肌纤维束比值较对照组增高(P＜0.05);D、E组缺血肢体的微血管密度和微血管/肌纤维束比值与B、C组比较差异有统计学意义(P＜0.05);但D、E组间微血管密度和微血管/肌纤维束比值无统计学意义(P＞0.05)。4.病理学观察:A组骨骼肌纤维萎缩、变性,肌纤维间距增大,间质内血管稀少;B、C组骨骼肌纤维轻度萎缩,肌纤维间距增大,间质内见少量毛细血管;D、E组血管生成较多,管腔内可见红细胞,肌纤维未见明显萎缩;各组骨骼肌细胞在rh-bFGF的影响下未见异常增殖或肿瘤样生长等表现。5.血肝、肾功能检测:各组给药前、给药后10天、给药后20天抽取静脉血检测血ALT、AST、BUN、Cr,各组内不同时间各项指标检测值比较差异无统计学意义(P＞0.05)。
结论:1.rh-bFGF可有效促进兔缺血肢体血管新生、侧支循环建立:其疗效随着rh-bFGF剂量的增加而增强,在10μg时取得最好的治疗效果,20μg时疗效不再增加,10μg是rh-bFGF治疗肢体缺血的最佳剂量。2.应用实验剂量rh-bFGF治疗肢体缺血是安全的,对肝、肾功能无明显影响,不会造成血管平滑肌、骨骼肌细胞的异常增生和肿瘤样生长。
Objective:To screen the optimal dose of recombinant human basic fibroblast factor (rh-bFGF)and evaluate the effect of different doses of rh-bFGF in 30 rabbit models of hindlimb ischemia through observation in general,the number of collateral branch, microvessel density and microvessel density/myolin ratio.To evaluate the safety of different doses of rh-bFGF by testing the liver and kidney function,digital subtraction angiography and pathological observation.To provide theoretial basis for clinically practising rh-bFGF to treat low limb ischemia.
Material and methods:30 rabbits were used to establish the animal model of lower limb ischemia by removed femoral artery of left and deligated its branch vessels. Randomized at five groups(6 each group),A group was intramuscular injected 5ml Tris/each;B,C,D and E groups are treating groups.The four groups were respectively intramuscular injected bFGF 2.5μg,5μg,10μg and 20μg+5ml Tris/each. The animals of each experiment group were once intramuscular injected in five spots of its thigh in 10 days after operation,and were put to death in 30 days after operation. Before injection,10 days after injection and 20 days after injection,the venous blood was collected to detect the liver and kidney function.The number of collateral branch was counted by digital subtraction angiography before the rabbits were put to death. The sample of medial vastus muscle was analyzed and observed for the changes of pathologic structure with microscope.
Results:1.observation in general:Animals of each groups were all alive in the period of experiment,and showed claudecation in distinct degrees in 10 days after the models were made.The claudication in A group and B group became more serious gradually,But it improved gradually in C,D and E groups.The cut healed up well and no ulcer and gangrene in each group.Abscess was found in one of A group.
2.Digital subtraction angiography of lower limb:DSA showed that the left femoral artery broke off.A few slight collateral vessels were distributed over the middle of thigh in A group.In B group and C group,several collateral vessels originated from internal iliac artery and deep femoral artery,the distant artery didn't develop.However,many collateral vascular creeped in D group and E group,and the distant artery of operated hindlimb developed because of collateral circulation,the speed of blood was a little slower than the uninjured side.There are no malformed capillaries and tumorous vessels in each group.The number of collateral casculars in D group and E group were all higher than A group,B group and C group(P＜0.05);but D group and E group were not statistically significant(P＞0.05).Among A,B and C group,the numbers were not statistically significant(P＞0.05).
3.Microvessel density and microvessel density/muscle fiber ratio:microvessel density and microvessel density/muscle fiber ratio among A group,B group and C group was no statistical difference(P＞0.05).But it in D group and E group was higher than A group(P＜0.05).It was also higher than B group and C group(P＜0.05). But D group and E group have no significant difference(P＞0.05).
4.Pathological observation:In A group,many skeletal muscle fibers were atrophy,space between muscle fibers turn long,and there were little blood vessels in mesenchymal.In B group and C group,the muscular atrophy was slight,there were a few blood vessels.But there were a lot of angiogenesis and collateral vessels in D group and E group.Some red cells could be seen in the vessels and no signifivant muscular fibers atrophy.Besides,skeletal muscle cell and other cell can not abnormal proliferation.Tumorous development was not found.
5.Liver and kidney function test:Before injection,10 days and 20 days after injection,the venous blood was collected to test ALT,AST,BUN,Cr.There were no statistical difference of the result in different time among each group(P＞0.05).
Conclusion:1.bFGF could promote therapeutic angiogenesis and collateral circulation formation effectively in a rabbit model of hind limb ischemia.Its curative effect strengthen with the dose of bFGF added.The curative effect was the best in 10μg,but it didn't increase in 20μg.10μg was the best dose for bFGF to treat lower limb ischemia.2.Applying experimental dose of bFGF to treat lower limb ischemia was safe,it has no affact on liver and kidney function and can not lead to abnormal hyperplasia and tumorous growth of skeletal muscle cell and other cells.
引文
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13.Nakamura Y,Nishizawa T,Hagiya M,et al.Molecular cloning and expression of human hepatocyte growth factor[J].Nature,1989,342:440-443.
14.Hayashi S,Morishita R,Nakamura S,et al.Potential role of hepatocyte growth factor,a novel angiogenic growth factor in peripheral arterial disease:downregulation of HGF in response to hypoxia in vascular cells[J].Circulation,1999,9:301-308.
15.Morishita R,Nakamura S,Hayashi S,et al.Therapeutic angeogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy[J].Hypertension,1999,33(6):1379-1384.
16.Miller KJ,Thaloor D,Matteson S,er al.Hepatocyte growth factor affects satellite cell actiation and differentiation in regenerating skeletal muscle[J].Am J Cell Physiol,2000,278(1):174-181.
17.哈小琴,李元敏,毕建进等.质粒pUDKH基因治疗大鼠肢体动脉闭塞病的实验研究[J].中华医学杂志,2003,83(17):1521-1524.
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20.张鸿坤,张楠,吴丽花,等.自体骨髓CD34~+干细胞移植治疗严重肢体缺血的实 验研究[J].中华外科杂志,2005,43(19):1275-1278.
21. Kalka C , Masuda H , Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization[J]. Proc Natl Sci U S A ,2000; 97(7):3422-3427.
22. Saigawa T,Kato K,Ozawa T,et al. Clinical application of bone marrow implantation in patients with arteriosclerosis obliterans ,and the association between efficacy and the number of implanted bone marrowcells[J] . Circ J, 2004; 68(12):1189-1193.
23. Asai J,Takenaka H,Ichihashi K, et al. Successful treatment of diabetic gangrene with topical application of a mixture of peripheral blood mononuclear cells and basic fibroblas t growth factor[J]. J Dermatol, 2006; 33(5):349-352.