神经生长因子基因转染在小鼠治疗性血管生成中诱导缺血肢体骨骼肌纤维重塑的机制

Mechanism of nerve growth factor promotes angiogenesis and skeletal muscle fiber remodeling in a mouse hindlimb ischemic model

目的探索神经生长因子(NGF)在小鼠治疗性血管生成中诱导缺血肢体骨骼肌纤维重塑的机制。方法雌性ICR小鼠18只,无特定病原体级,6周龄,体重25~30 g。按随机数字表法分为假手术组(n=6)、空白对照组(n=6)和NGF基因治疗组(n=6)。对空白对照组和NGF基因治疗组小鼠进行后肢缺血造模,在术后第7天对假手术组注射生理盐水,空白对照组小鼠进行空质粒转染,NGF基因治疗组小鼠进行NGF基因转染。在术后第21天对患肢血流灌注评估后进行腓肠肌取材。对3组腓肠肌标本进行苏木精-伊红(HE)染色,CD31和增殖细胞核抗原(PCNA)免疫组织化学染色,实时荧光定量PCR检测肌球蛋白重链(MHC)-Ⅰ、MHC-Ⅱa及MHC-Ⅱb的mRNA表达,Western blot检测NGF和过氧化物酶体增殖物激活受体-β/δ(PPARβ/δ)的表达,酶联免疫吸附试验(ELISA)法检测细胞色素C氧化酶(COX)、异柠檬酸脱氢酶(IDH)和三磷酸腺苷(ATP)表达量。结果术后第21天,NGF基因转染组小鼠患肢血流灌注量为(195.70±9.99)PU,低于假手术组(312.15±17.32)PU(P=0.001),但高于空白对照组(82.11±8.55)PU(P=0.001)。NGF组的肌肉萎缩程度低于空白对照组,NGF基因转染组的毛细血管密度为(0.34±0.05),高于假手术组(0.11±0.03)和空白对照组(0.27±0.04)(均P<0.05)。NGF基因转染组的内皮细胞增殖指数为(0.39±0.19),高于假手术组(0.18±0.01)和空白对照组(0.25±0.14)(均P<0.05)。NGF基因转染组的NGF、PPARβ/δ、COX、IDH、ATP的表达水平,以及MHC-ⅠmRNA表达水平较假手术组和空白对照组均明显升高(均P<0.05)。结论NGF基因转染后能够促进小鼠缺血肢体毛细血管生成,增加其血流灌注,进而诱导骨骼肌肌纤维向Ⅰ型重塑,该过程可能与NGF诱导PPARβ/δ表达,促进骨骼肌细胞有氧代谢有关。

Objective To explore the mechanism of nerve growth factor(NGF)in the skeletal muscle fiber remodeling in ischemic limbs during therapeutic angiogenesis.Methods Eighteen female mice with SPF grade,6 weeks old and 25-30 g weighed were randomly allocated to sham-operated group(n=6),blank control group(n=6)and NGF gene transfection group(n=6).The left hindlimb ischemia models were established by ligating the femoral artery in blank control group and NGF gene transfection group.Seven days after the operation,mice in the three groups were separately injected with normal saline,empty plasmids,and NGF plasmids.Gastrocnemius of left hindlimbs was harvested after the blood perfusion assessment of the ischemic limb on the 21st postoperative day.The gastrocnemius muscle specimens were stained with HE,CD31 and proliferating cell nuclear antigen(PCNA)immunohistochemistry staining,the mRNA expressions of myosin heavy chain-Ⅰ(MHC-Ⅰ),MHC-Ⅱa and MHC-Ⅱb were measured by real-time PCR,and the protein level of NGF and peroxisome proliferator-activated receptors-β/δ(PPARβ/δ)were detected by Western blot.The expression of cytochrome C oxidase(COX),isocitrate dehydrogenase(IDH)and adenosine triphosphate(ATP)were examined by enzyme-linked immunosorbent assay(ELISA).Results On the 21st day after operation,the blood perfusion of the ischemic limb in NGF gene transfection group was(195.70±9.99)PU,which was lower than that in sham-operated group(312.15±17.32)PU(P=0.001),while it was higher than that in blank control group(82.11±8.55)PU(P=0.001).The degree of muscle atrophy in the NGF gene transfection group was lower than that in the blank control group.The capillary density of NGF gene transfection group(0.34±0.05)was higher than that of sham-operated group(0.11±0.03)and blank control group(0.27±0.04)(P<0.05).The endothelial cell proliferation index in NGF gene transfection group(0.39±0.19)was significantly higher than that in sham-operated group(0.18±0.01)and blank control group(0.25±0.14)(P<0.05).The expression of NGF,PPARβ/δ,COX,IDH,ATP,and MHC-ⅠmRNA in NGF gene transfection group were significantly higher than those in sham-operated group and blank control group(P<0.05).Conclusions NGF gene transfection can promote angiogenesis in the ischemic limbs of mice,increase the blood perfusion,and thus induce the remodeling of skeletal muscle fibers to typeⅠ.This process may be related to NGF-induced PPARβ/δexpression and promote the cellular aerobic metabolism in skeletal muscle.