【作者】 周芃；

【导师】 吴开云；

【作者基本信息】 苏州大学 ， 人体解剖与组织胚胎学， 2009， 硕士

【摘要】 研究目的:探讨毛葡萄提取物的抗血栓形成作用及其机制,为进一步评估和研发毛葡萄的抗血栓作用提供实验依据。实验方法:(1)动-静脉旁路血栓形成模型:实验随机分为①对照组(control):蒸馏水灌胃;②毛葡萄大剂量组(VRE-LD):毛葡萄提取液(含生药4g/kg)灌胃;③毛葡萄中剂量组(VRE-MD):毛葡萄提取液(含生药2g/kg)灌胃;④毛葡萄小剂量组(VRE-SD):毛葡萄提取液(含生1g/kg)灌胃;⑤丹参组(CDDP):CDDP溶液(67.5mg/kg)灌胃;每天一次连续灌胃10天。末次给药后30min以4%水合氯醛(1ml/100g)麻醉动物,分离右侧的颈总动脉和左侧的颈外静脉。颈外静脉先结扎静脉远心端,剪开一个小口,将事先准备好塑料管(管中放置一根6cm的4号线,并将50U/ml肝素生理盐水溶液充满聚乙烯管腔)向近心端插入并结扎固定。右侧颈总动脉先用血管夹夹住近心端,剪开一个小口,将管向近心端插入并结扎固定。然后放开血管夹,计时15min后将线取出称取重量,HE染色观察血栓形态学改变。从腹主动脉取血3ml, 3000 rpm/min 4℃离心15min,取上清血浆用放射免疫法检测TXB2和6-keto-PGF1α的含量。(2) FeCl3诱导血栓形成模型:实验随机分为①假手术组(sham):蒸馏水灌胃;②对照组(control):蒸馏水灌胃;③毛葡萄大剂量组(VRE-LD):毛葡萄提取液(含生药4g/kg)灌胃;④毛葡萄中剂量组(VRE-MD):毛葡萄提取液(含生药2g/kg)灌胃;⑤毛葡萄小剂量组(VRE-SD):毛葡萄提取液(含生1g/kg)灌胃;⑥丹参组(CDDP):CDDP溶液(67.5mg/kg)灌胃;每天一次连续灌胃10天。末次给药后15min以4%水合氯醛麻醉动物,分离两侧颈总动脉,右侧颈总动脉备采血用,左侧颈总动脉分离长度为1.5cm,其下置一小片塑料薄膜(4cm×1.2cm),2～5组在颈总动脉上放置一吸有10μl FeCl3溶液的小滤纸片(1cm×0.6cm),作用20min后去掉滤纸片,纸片去除后90min从右颈总动脉取血, 3000rpm/min 4℃离心15min,取上清血浆用放射免疫法检测TXB2、6-keto-PGF1α和ET-1的含量。取有血栓段的血管称其重量,HE染色观察血栓形态学改变。(3)急性血淤模型:实验随机分为①正常组(normal):蒸馏水灌胃;②对照组(control):蒸馏水灌胃;③毛葡萄中剂量组(VRE-MD):毛葡萄提取液(含生2g/kg)灌胃;④丹参组(CDDP):CDDP溶液(67.5mg/kg)灌胃;每天一次连续灌胃10天;末次给药后1 h,除正常组外,其余各组皮下注射1mg/ml肾上腺素0.08ml/100g,2 h后将大鼠浸入冰水中5min进行冷刺激,间隔2 h后再注射等量肾上腺素。大鼠禁食不禁水,次日腹主动脉抽5ml,EDTA-Na2抗凝。用TYXN-96多功能智能血液凝聚仪测定全血粘度。结果:(1)对动-静脉旁路血栓形成模型的影响:与对照组相比,毛葡萄各剂量组可明显抑制血栓形成,降低形成血栓的重量(P<0.05),其中毛葡萄中剂量组效果最好,也好于丹参组;毛葡萄各剂量组能明显降低血浆TXB2、TXB2/6-keto-PGF1α和升高6-keto-PGF1α水平,其中也是毛葡萄中剂量组效果最好。(2)对FeCl3诱导血栓形成模型的影响:毛葡萄各剂量组血管腔血栓量明显小于对照组,其中以中剂量组最为显著。丹参组与对照组对比,形成的血栓稍有减小,但与毛葡萄组比较,其血栓明显大于毛葡萄组;毛葡萄各剂量组能明显降低血浆TXB2、ET-1和升高6-keto-PGF1α水平,其中也是毛葡萄中剂量组效果最好。(3)对血粘度的影响:与对照组相比,毛葡萄组能明显降低全血粘度(P<0.05),并毛葡萄组的效果好于丹参组。结论:1.在大鼠体外动-静脉旁路模型和FeCl3诱导血栓形成模型上证实毛葡萄提取物具有抗血栓形成作用。并其抗血栓效果好于丹参。2.通过检测血浆TXB2、6-keto-PGF1α、ET-1和血粘度的变化,发现毛葡萄提取物有降低血浆TXB2和ET-1和升高6-keto-PGF1α水平,以及降低血粘度的作用,这途径可能是毛葡萄抗血栓形成作用的主要机制。更多还原

【Abstract】 Objective: To investigate the antithrombotic effects and mechanisms of Vitis quinquangularis Rehd extract and provide an experimental evidence for the further evaluation and research of the anti-thrombosis of Vitis quinquangularis Rehd .Methods:(1) Arteriovenous shunt thrombosis model: The rats were randomly divided into five groups:①Control group: with distilled water intragastric infusion;②VRE large dose group (VRE-LD): with VRE intragastric infusion, equivalent to crude drug by 4g/kg dose;③V RE medium dose group (VRE-MD): with VRE intragastric infusion, equivalent to crude drug by 2g/kg dose;④V RE small dose group (VRE-SD): with VRE intragastric infusion, equivalent to crude drug by 1g/kg dose;⑤C DDP group: with CDDP solution intragastric infusion by 67.5mg/kg dose. After giving prophylactic intragastric infusion once a day for ten continuous days, rats were anaesthetized by chloral hydrate(1ml/100g) at 30 minutes after the last administration in each group. The left external jugular vein we separated was ligatured at the distal end and in which a narrow opening was cut. Then the previously prepared polyethylene pipe, which was filled with heparin sodium(50U/ml) and with a 6cm size 4 medical wire in it, was inserted toward the proximal in the narrow opening followed by ligation fixed. At the common carotid artery end, the right common carotid artery we separated was clamped with a arterial clip at the proximal. Then the other side of polyethylene pipe was inserted toward the proximal in the narrow opening which was previously cut in the right common carotid artery followed by ligation fixed. Timing when the right common carotid artery was unclamped and the blood flow keep unobstructed for 15minutes. The medical wire was taken out to be measured the thrombus weight (either wet or dry) , morphology of thrombus was observed by HE staining. At last, 3ml blood was sampled from the abdominal aorta and centrifuged 3000 rpm / min for 15 minutes at 4℃. Then the plasma left was used to determine the content of TXB2 and 6-keto-PGF1α(metabolite of TXA2 and PGI2) by radio immunoassay.(2) Rat model of arterial thrombosis induced by ferric chloride: The rats were randomly divided into six groups:①Sham group: with distilled water intragastric infusion;②C ontrol group: with distilled water intragastric infusion;③V RE large dose group (VRE-LD): with VRE intragastric infusion, equivalent to crude drug by 4g/kg dose;④VRE medium dose group (VRE-MD): with VRE intragastric infusion, equivalent to crude drug by 2g/kg dose;⑤V RE small dose group (VRE-SD): with VRE intragastric infusion, equivalent to crude drug by 1g/kg dose;⑥C DDP group: with CDDP solution intragastric infusion by 67.5mg/kg dose. After giving prophylactic intragastric infusion once a day for ten continuous days, rats were anaesthetized by chloral hydrate(1ml/100g) at 15 minutes after the last administration in each group. Ambilateral common carotid artery was separated and the right common carotid artery was used for sampling blood. A piece of plastic film (4cm×1.2cm) was put under the 1.5cm long part of the separated left common carotid artery. A piece of filter paper(1cm×0.6cm) with the solution 10μl FeCl3 was put below the left common carotid artery for 20min in group 2 to 5. After the piece of filter paper removed, blood was sampled from the right common carotid artery and centrifuged 3000 rpm / min for 15 minutes at 4℃. The plasma left was used to determine the content of TXB2、6-keto-PGF1αand ET-1 by radio immunoassay. Then the thrombotic segement of the vessel was taken and weighted, morphology of thrombus was observed by HE staining.(3)Acute blood stasis model: The rats were randomly divided into four groups:①N ormal group: with distilled water intragastric infusion;②Control group: with distilled water intragastric infusion;③VRE medium dose group (VRE-MD): with VRE intragastric infusion, equivalent to crude drug by 2g/kg dose;④C DDP group: with CDDP solution intragastric infusion by 67.5mg/kg dose. After giving prophylactic intragastric infusion once a day for ten continuous days, rats of all groups except the normal group were conducted subcutaneous injections with 0.08ml epinephrine (1mg/ml) per 0.1kg weight at 60 minutes after the last administration. Rats of group 2 to 4 were put into ice-cold water for 5 min after 2 hours behind subcutaneous injections, and were administered the same dose epinephrine after 2 hours interval. Rats were fasted without water deprivation and 5ml blood was sampled from the abdominal aorta the next day. The anticoagulant agent is ethylenediamine tetraacetic acid disodium salt(EDTA-Na2). Then the blood samples viscosity were detected by Multifunction Intelligent Blood Coagulation Analyzer TYXN-96.Results:(1) Effect on thrombosis in arteriovenous shunt model: It was shown that each VRE group had significantly inhibited thrombosis and reduced the thrombotic weight compared with the control group(P<0.05). The effect of VRE-MD group was especially markedly, better than that of CDDP group too. Meanwhile, it could down regulate the level of TXB2 and TXB2/6-keto-PGF1αand up regulate the level of 6-keto-PGF1αin plasma, and also showed the best performance in VRE-MD group.(2) Effect on thrombosis in rat model of arterial thrombosis induced by ferric chloride: Thrombosis in lumen of blood vessel of each VRE group was reduced apparently compared with the control group. Thrombus of CDDP group was smaller than that of control group, but bigger than that of VRE group. The effect of VRE-MD group was especially markedly. Meanwhile, it could down regulate the level of TXB2、ET-1 and up regulate the level of 6-keto-PGF1αin plasma, and also showed the best performance in VRE-MD group.(3) Effect on viscosity of acute blood stasis model: Compared with the control group, the VRE group could significantly down regulate the whole blood viscosity (P<0.05), effect of which was better than that of CDDP group.Conclusions:1. It is proved that VRE had an effect on anti-thrombosis in arteriovenous shunt model and rat model of arterial thrombosis induced by ferric chloride. The effect of VRE group was better than that of CDDP group.2. By detecting the variation of TXB2、6-keto-PGF1α、ET-1 and the blood viscosity, we found VRE had the function to down regulate the level of TXB2、ET-1 and the blood viscosity and up regulate the level of 6-keto-PGF1α. Its main anti-thrombosis mechanism may be associated with this variation.更多还原