芪芍复脉汤对大鼠下肢缺血模型血管新生作用的研究

发布时间：2018-04-24 11:12

本文选题：芪芍复脉汤 + 血管新生　；参考：《山西省中医药研究院》2017年硕士论文

【摘要】：目的:通过检测芪芍复脉汤对下肢缺血大鼠的血清酶学指标、CD34+、缺血骨骼肌毛细血管密度(MVD)、血管内皮细胞生长因子(VEGF),以及组织病理学检测,探讨芪芍复脉汤对大鼠下肢缺血模型血管新生的影响及其可能机制,为临床下肢缺血性疾病的治疗提供实验依据。方法:90只SD大鼠左下肢行股动脉结扎、分支剔除术,制作下肢缺血模型。随机分为模型组,芪芍复脉汤高、中、低剂量组和粒细胞集落刺激因子(rhG-CSF)组,每组18只,并设立假手术组18只,假手术组仅作皮肤切开缝合,不结扎股动脉。术后第2天开始给药,芪芍复脉汤高、中、低剂量组分别按7.6 g·kg-1、3.8 g·kg-1、1.9 g·kg-1灌胃给药,每天灌胃1次,连续14天;rhG-CSF组给予rhG-CSF皮下注射0.21ml/100g,每日1次,连续5天;假手术组和模型组不给药,仅予等体积蒸馏水灌胃。分别于术后3天、7天、14天,每组大鼠分别取6只,用全自动生化仪对大鼠外周血肌酸激酶(CK)、乳酸脱氢酶(LDH)、谷草转氨酶(AST)进行检测;用流式细胞仪检测血清中CD34阳性细胞比例;取术侧内收肌,用免疫组化方法检测VEGF观密度值和毛细血管密度(MVD);用HE病理染色法对术测腓肠肌病理改变进行观察,比较各组差异。结果:1.MVD检测结果:术后14天,芪芍复脉汤高、中剂量组MVD值显著高于模型组和低剂量组(P0.01);芪芍复脉汤高剂量组MVD值显著高于中剂量组(P0.05)。2.酶学检测结果:术后3天,各组LDH、CK、AST值均较假手术组显著升高(P0.05或P0.01);术后7天,各组(除假手术组)LDH、CK、AST值均较3天时显著下降,其中高、中剂量组和rhG-CSF组下降最为明显(P0.01);术后14天,各组酶学LDH、CK、AST指标较7天时下降缓慢。3.CD34+阳性细胞比例检测结果:术后各时间点,芪芍复脉汤高剂量组CD34+比例均高于模型组(P0.01);术后7天和14天,芪芍复脉汤高剂量组CD34+比例明显高于中剂量组和低剂量组(P0.01)。术后7天,芪芍复脉汤高、中剂量组及rhG-CSF组CD34+比例较3天时明显上升(P0.01),术后14天,各组(除假手术组)CD34+比例均下降(P0.05或P0.01)。4.VEGF蛋白表达:术后各时间点,芪芍复脉汤高剂量组VEGF观密度值较模型组显著上升(P0.01)。术后7天,除假手术组外,各组表达均上升;术后14天,除假手术组外,各组表达均降低。5.病理结果:芪芍复脉汤高剂量组肌细胞萎缩程度较轻,中剂量组次之,高剂量组肌纤维间血管数目较模型组显著增多。结论:芪芍复脉汤能够增加缺血骨骼肌毛细血管密度,促进血管新生,其作用机制可能与上调VEGF,动员骨髓中的内皮祖细胞向缺血组织迁移、归巢以促进内皮细胞的增生有关。
[Abstract]:Objective: to detect the serum enzyme index (CD34), capillary density of ischemic skeletal muscle (MVDX), vascular endothelial growth factor (VEGF) and histopathology in rats with lower extremity ischemia treated by Qishaifumai decoction. To explore the effect of Qishaifumai decoction on angiogenesis and its possible mechanism in rat model of lower limb ischemia, and to provide experimental evidence for the treatment of ischemic diseases of lower extremity. Methods 90 Sprague-Dawley rats were treated with femoral artery ligation and branch removal. The model group was randomly divided into three groups: Qishao Fumai decoction group (high, middle and low dose) and granulocyte colony stimulating factor rhG-CSF (rhG-CSF) group (18 rats in each group), and the sham-operation group (18 rats) was used for skin incision and suture without ligation of femoral artery. On the second day after operation, Qishaifumai decoction was given intragastrically at the dose of 7.6g kg ~ (-1) ~ 3.8g ~ (-1) g ~ (-1) ~ (-1) kg-1, respectively. RhG-CSF was given subcutaneously to 0.21ml / 100g, once a day for 5 days for 14 days in the rhG-CSF group. Sham operation group and model group were not given drugs, only equal volume of distilled water intragastric perfusion. Six rats in each group were taken from each group at 3 days and 7 days and 14 days after operation. The levels of creatine kinase (CK), lactate dehydrogenase (LDH) and glutamic oxaloacetic transaminase (AST) in peripheral blood of rats were detected by automatic biochemical instrument, and the percentage of CD34 positive cells in serum was detected by flow cytometry. The adductor muscle was removed and the VEGF and capillary density were detected by immunohistochemical method, and the pathological changes of gastrocnemius muscle were observed by HE pathological staining. Results 1. After 14 days, Qishaifumai decoction was high, the MVD value in the middle dose group was significantly higher than that in the model group and the low dose group, and the MVD value in the high dose group was significantly higher than that in the middle dose group. The results of enzymatic examination: on the 3rd day after operation, the values of LDHK CK AST in each group were significantly higher than those in the sham operation group (P 0.05 or P 0.01), and on the 7th day after operation, the AST values in all groups (except sham-operation group) were significantly lower than those in 3 days after operation, especially in the middle dose group and rhG-CSF group (P 0.01); on the 14th day after operation, there was a significant decrease in the AST value in all groups (except sham-operation group). The results showed that the proportion of CD34 in high dose group of Qishoufumai decoction was higher than that of model group at 7 days after operation, and on the 7th and 14th day after operation, the proportion of CD34 in high dose group was higher than that in model group, and the ratio of CD34 positive cells in each group was higher than that in model group at 7 and 14 days after operation. The proportion of CD34 in high dose group of Qishaifumai decoction was significantly higher than that of medium dose group and low dose group (P 0.01). On the 7th day after operation, Qishaifumai decoction was high, the CD34 ratio in the middle dose group and rhG-CSF group was significantly higher than that in the 3rd day. On the 14th day after operation, the proportion of CD34 in each group (except sham-operation group decreased P0.05 or P0.01).4.VEGF protein expression: at each time point after operation), The apparent density of VEGF in the high dose group of Qishaifumai decoction was significantly higher than that in the model group (P 0.01). On the 7th day after operation, the expression of protein increased in all groups except sham-operation group, and decreased in all groups except sham-operation group on the 14th day after operation. Pathological results: the atrophy degree of muscle cells in the high dose group of Qishaifumai decoction was less than that in the middle dose group, and the number of intermuscular vessels in the high dose group was significantly higher than that in the model group. Conclusion: Qishaifumai decoction can increase capillary density and promote angiogenesis of ischemic skeletal muscle, and its mechanism may be related to up-regulation of VEGF, mobilization of endothelial progenitor cells from bone marrow to migration of ischemic tissue, homing to homing to promote proliferation of endothelial cells.
【学位授予单位】：山西省中医药研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5;R-332

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