小崔说立波秀肝细胞生长因子对慢性深静脉血栓机化再通的实验研究
中文摘要
目的：通过构建肝细胞生长因子( hepatocyte growth factor，HGF)腺病毒重组体，并建立犬下肢深静脉慢性血栓模型探讨HGF是否促进血栓内血管新生，加快血栓机化和再通。
方法：1、取犬外周静脉血，分离其中白细胞，使用Trizol提取其中总RNA，反转录为cDNA后，PCR扩增目的基因HGF，将其克隆到腺病毒载体，并在293A细胞中包装腺病毒，经超速离心机离心纯化和浓缩病毒。2、选取正常家犬18只（15Kg-18kg），雌雄不限，利用介入Seldinger穿刺技术结合传统开放手术模拟髂静脉受压建立犬下肢慢性深静脉血栓形成动物模型。随机选取成功成模的18只下肢慢性深静脉血栓形成的家犬分为三组：对照组、抗凝组、治疗组，每组6只，分别注入生理盐水、低分子肝素、表达肝细胞生长因子的腺病毒，常规喂养28天后，经DSA造影观察血栓及侧支情况。应用实时荧光定量PCR技术检测HGF mRNA的表达水平，同时应用western blot检测静脉血栓内HGF蛋白表达变化；vWF免疫组化染色观察血栓再通毛细血管密度。
结果：1、成功包装带有目的基因肝细胞生长因子的腺病毒，显微镜下可见绿色荧光，滴度约为1.0×108。2、成功建立下肢深静脉血栓形成模型并分别于14天、28天经DSA造影证实下肢深静脉血栓稳定。治疗后通过观察各组和取组织显示，治疗组于DSA造影下显示其血栓再通并侧支血管数明显优于对
照组及抗凝组（p<0.05），荧光定量PCR显示治疗组HGF mRNA的表达水平明显高于对照组及抗凝组（p<0.05），从western blot结果可见治
I四级英语听力技巧
疗组HGF蛋白表达量较对照组及抗凝组升高（p<0.05），利用vWF免疫组化染色观察到其治疗组毛细血管数较对照组及抗凝组明显增多（p<0.05）。结论：1、肝细胞生长因子能明显促进慢性血栓内新生血管生成。
2、肝细胞生长因子能明显加快慢性血栓机化和再通。
关键词：肝细胞生长因子；深静脉血栓；血管新生；血栓模型；HGF I I
Hepatocyte growth factor experimental rearch of chronic deep vein thrombosis organization recanalization联合培养研究生
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（Abstract）
Objective:
联通异地销户
To investigate the role of HGF in promoting organization, angiogenesis, recanalization of thrombus by constructing adenovirus hepatocyte growth factor (HGF) recombinants and its u in the model of deep vein thrombosis of the lower limbs of hou dogs.
Methods:
1、The peripheral blood of dogs was isolated and the white blood cells were isolated. Total RNA was extracted with Trizol reagent and rever transcribed into cDNA. The target gene HGF was amplified by PCR and cloned into adenovirus vector. The recombinant virus was packed in 293A cells, which was further purified and concentrated by ultracentrifuge and dialysis.
避孕套如何正确使用2、A total of 18 normal hou dogs (15Kg-18kg) were lected(Male or female). By using the technique of interventional Seldinger puncture technique combined with traditional open surgery, animal model of iliac vein compression and chronic deep vein thrombosis was simulated. 18 dogs with chronic deep vein thrombosis were randomly divided into three groups: control group, anticoagulation group and treatment group, 6 dogs in each group. Dogs were injected with normal saline, low molecular weight heparin, Adenovirus of HGF respectively. 28 days after feeding, dogs were obrved by DSA to detect the thrombosis and
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collateral circulation. The expression of HGF mRNA in the thrombus was detected by real-time fluorescence quantitative PCR. The expression of HGF protein was detected by western blot. Immunohistochemical staining to vWF was performed to measure the density of angiogenesis.
Result：
1、Adenovirus with the target gene hepatocyte growth factor was successfully constructed. The green fluorescence could be obrved under the microscope. The titer of recombinant virus was about 1.0 × 108/ml.
托福综合写作2、Dog model of deep vein thrombosis was established. DSA angiography confirmed deep venous thrombosis of lower limbs. After treatment, the number of collateral vesls were significantly higher in the treatment group than in the control group and the anticoagulation group (p <0.05) detected by DSA. The expression of HGF mRNA of thrombus in the treatment group was significantly higher than that in the control group (P <0.05). WB showed that the expression of HGF protein in the treatment group was higher than that in the control group and the anticoagulation group (p <0.05). The number of capillaries measured by vWF density in the treatment group was significantly higher than that in th
e control group and the anticoagulation group (p <0.05).The success of the hepatocyte growth factor gene into the adenovirus, with the green fluorescent drops to 1.0 x 108 hepatocyte growth factor of the adenovirus recombinant.
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Conclusion:
1、 hepatocyte growth factor can obviously promote angiogenesis in chronic thrombus formation.
2、 hepatocyte growth factor can obviously accelerate the chronic thrombosis machine and recanalization.
Key words: H epatocyte growth factor; Deep vein thrombosis; Angiogenesis; Thrombosis model; HGF
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