海带多糖L01抑制内皮损伤大鼠血栓形成与对内皮细胞超微结构的影响
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摘要
目的:观察海带多糖对两种内皮细胞损伤大鼠模型血栓形成的情况,以及对体外培养内皮细胞超微结构的影响,为研究和开发新型抗血栓药物提供理论和实验室依据。
方法:(1)体内给药对内皮损伤大鼠动静脉环路血栓形成的影响。采用肾上腺素(Adr)法制备内皮细胞损伤的大鼠模型。大鼠随机分为7组,生理盐水对照组,腹腔注射生理盐水,每天2次,连续3d;阿司匹林高、中、低剂量组,分别腹腔注射不同浓度阿司匹林,给药1h后皮下注射肾上腺素,每天2次,连续3d;海带多糖高、低剂量组,腹腔注射海带多糖,给药1h后皮下注射肾上腺素,每天2次,连续3d;肾上腺素模型组,皮下注射肾上腺素,每天3次,连续3d,各组于第四天制备动静脉环路前再次皮下注射肾上腺素。通过动—静脉环路法测定血栓湿重和血栓抑制率。(2)体内给药对内毒素血症大鼠肠系膜微循环变化的影响。大鼠随机分为6组,生理盐水对照组,股静脉注射生理盐水;内毒素组,股静脉注射内毒素;阿司匹林高、低剂量组,股静脉给予内毒素后,注射不同浓度阿司匹林;海带多糖高、低剂量组,股静脉给予内毒素后,注射不同浓度海带多糖。将肠系膜固定于微循环观察盒中,观测微血管内血栓形成的时间,通过图像分析系统记录固定时间点的血流速度。(3)对体外培养内皮细胞电镜下超微结构的影响。体外培养的人脐静脉内皮细胞(HUVEC)在培养瓶中随机分组,空白对照组、模型组、L01对照组、L01高剂量组、L01低剂量组,分别培养48h。取材、固定、脱水、浸透、包埋、切片及染色,制成超薄切片,电镜下观察细胞超微结构,并观察各细胞器是否有损伤。
结果:(1)体内给药对内皮损伤大鼠动静脉环路血栓形成的影响。肾上腺素损伤模型中,与生理盐水组比较,模型组血栓湿重明显增加。与模型组比较,海带多糖高剂量组(50mg/kg)和低剂量组(10mg/kg)均能降低大鼠血栓湿重,高剂量组血栓抑制率达57.5%。(2)体内给药对内毒素血症大鼠肠系膜微循环变化的影响。内毒素损伤模型中,与生理盐水组比较,模型组血栓形成时间加快,1h后流速减缓。而与模型组比较,海带多糖高剂量组(5mg/kg)和低剂量组(2.5mg/kg)均能延长血栓形成时间,在1h流速显著高于模型组。(3)对体外培养内皮细胞电镜下各细胞器超微结构的影响。肾上腺素模型组细胞核不规则,细胞间紧密连接,细胞间微绒毛减少、消失,线粒体(Mi)肿胀,嵴溶解消失,核旁溶酶体增多。海带多糖对照组基本接近正常组,低剂量组损伤程度低于模型组,而高剂量组基本接近正常组。
结论:海带多糖对肾上腺素和内毒素制备的内皮损伤大鼠模型中的血栓形成均有抑制作用,并且从分子超微结构方面对各细胞器的损伤也有改善作用。
Objective:TO investigate the mechanism of laminaran in inhibiting the thrombus formation according to the establishment of two rat models with injured endothelial cells,including the effect on ultrastructural organization of cultured endothelial cells,thus providing an experimental basis for developing a new drug for antithrombosis.
Methods:(1)L01 effect on Thrombus formation in the artery-vein circuit rat models in vivo.The experiment adopts the model of endothelial cells injured by adrenaline.Rats were divided into 7 groups randomly.NS control rats were received normal saline 1d,2d,3d,two times a day.H-L01 treatment rats and L-L01 treatment rats were injected L01 50mg/kg and 10mg/kg respectively, and treated with adrenalin in an hour.Correspondingly,Asp treatment rats were instead of H-Asp,M-Asp and L-Asp.Model rats were injected adrenalin 1d,2d,3d,three times a day.Every group were injected adrenalin before experiment in the fourth day.the weight and the inhibition ratio of thrombus were measured in the artery-vein circuit rat models.(2)Effect on Thrombus formation in the mesentery minicirculation models in vivo.Rats were divided into 6 groups randomly.NS control rats were received normal saline.Model rats were injected endotoxin.L01 treatment rats were injected L01 after being injected endotoxin.Asp treatment rats is the same as L01 treatment rats. Thrombus formation time and blood flow rate in veins were measured in the mesentery minicirculation models.(3)Effect on ultrastructural organization of endothelia cells in vitro.H-L01 treatment,L-L01 treatment,model control,L01 control and normal control were administered in vitro for 48 hours.To observe ultrastructural organization of endothelia cells and damage of the cell organ through TEM.
Results:(1)L01 effect on Thrombus formation in the artery-vein circuit rat models in vivo.In the models by injected adrenaline,compared with control group,the weight of thrombus increased significantly.But compared with model group,H-L01(50mg/kg)group and L-L01(10mg/kg)group can decrease the weight of thrombus,and the inhibition ratio in H-L01 group can reach to 57.5%. (2)Effect on Thrombus formation in the mesentery minicirculation models in vivo.In the models by injected endotoxin,compared with control group,thrombus formation time increased.And compared with model group,H-L01(5mg/kg)group and L-L01(2.5mg/kg)group can prolong the thrombus formation time,and on the 1h,the blood flow rate in veins is much higher than model group's.(3)Effect on ultrastructural organization of endothelia cells in vitro.In the models by injected adrenaline,irregularity cellular nucleus,cello-tight junction,microvilli is to decrease and to disaapear. Engorgement of mitochondrium,cristae is to dissolve and disaapear, cytolysosome is to increase.L01 control group and H-L01group is almost the same as the nomal control group;the damage in L-L01group is not as severe as the model group.
Conclusions:Laminaria-polysaccharide can inhibit the thrombus formation in two rat models with injured endothelial cells.And improve the damage from the ultrastructural organization of endothelia cells
方法:(1)体内给药对内皮损伤大鼠动静脉环路血栓形成的影响。采用肾上腺素(Adr)法制备内皮细胞损伤的大鼠模型。大鼠随机分为7组,生理盐水对照组,腹腔注射生理盐水,每天2次,连续3d;阿司匹林高、中、低剂量组,分别腹腔注射不同浓度阿司匹林,给药1h后皮下注射肾上腺素,每天2次,连续3d;海带多糖高、低剂量组,腹腔注射海带多糖,给药1h后皮下注射肾上腺素,每天2次,连续3d;肾上腺素模型组,皮下注射肾上腺素,每天3次,连续3d,各组于第四天制备动静脉环路前再次皮下注射肾上腺素。通过动—静脉环路法测定血栓湿重和血栓抑制率。(2)体内给药对内毒素血症大鼠肠系膜微循环变化的影响。大鼠随机分为6组,生理盐水对照组,股静脉注射生理盐水;内毒素组,股静脉注射内毒素;阿司匹林高、低剂量组,股静脉给予内毒素后,注射不同浓度阿司匹林;海带多糖高、低剂量组,股静脉给予内毒素后,注射不同浓度海带多糖。将肠系膜固定于微循环观察盒中,观测微血管内血栓形成的时间,通过图像分析系统记录固定时间点的血流速度。(3)对体外培养内皮细胞电镜下超微结构的影响。体外培养的人脐静脉内皮细胞(HUVEC)在培养瓶中随机分组,空白对照组、模型组、L01对照组、L01高剂量组、L01低剂量组,分别培养48h。取材、固定、脱水、浸透、包埋、切片及染色,制成超薄切片,电镜下观察细胞超微结构,并观察各细胞器是否有损伤。
结果:(1)体内给药对内皮损伤大鼠动静脉环路血栓形成的影响。肾上腺素损伤模型中,与生理盐水组比较,模型组血栓湿重明显增加。与模型组比较,海带多糖高剂量组(50mg/kg)和低剂量组(10mg/kg)均能降低大鼠血栓湿重,高剂量组血栓抑制率达57.5%。(2)体内给药对内毒素血症大鼠肠系膜微循环变化的影响。内毒素损伤模型中,与生理盐水组比较,模型组血栓形成时间加快,1h后流速减缓。而与模型组比较,海带多糖高剂量组(5mg/kg)和低剂量组(2.5mg/kg)均能延长血栓形成时间,在1h流速显著高于模型组。(3)对体外培养内皮细胞电镜下各细胞器超微结构的影响。肾上腺素模型组细胞核不规则,细胞间紧密连接,细胞间微绒毛减少、消失,线粒体(Mi)肿胀,嵴溶解消失,核旁溶酶体增多。海带多糖对照组基本接近正常组,低剂量组损伤程度低于模型组,而高剂量组基本接近正常组。
结论:海带多糖对肾上腺素和内毒素制备的内皮损伤大鼠模型中的血栓形成均有抑制作用,并且从分子超微结构方面对各细胞器的损伤也有改善作用。
Objective:TO investigate the mechanism of laminaran in inhibiting the thrombus formation according to the establishment of two rat models with injured endothelial cells,including the effect on ultrastructural organization of cultured endothelial cells,thus providing an experimental basis for developing a new drug for antithrombosis.
Methods:(1)L01 effect on Thrombus formation in the artery-vein circuit rat models in vivo.The experiment adopts the model of endothelial cells injured by adrenaline.Rats were divided into 7 groups randomly.NS control rats were received normal saline 1d,2d,3d,two times a day.H-L01 treatment rats and L-L01 treatment rats were injected L01 50mg/kg and 10mg/kg respectively, and treated with adrenalin in an hour.Correspondingly,Asp treatment rats were instead of H-Asp,M-Asp and L-Asp.Model rats were injected adrenalin 1d,2d,3d,three times a day.Every group were injected adrenalin before experiment in the fourth day.the weight and the inhibition ratio of thrombus were measured in the artery-vein circuit rat models.(2)Effect on Thrombus formation in the mesentery minicirculation models in vivo.Rats were divided into 6 groups randomly.NS control rats were received normal saline.Model rats were injected endotoxin.L01 treatment rats were injected L01 after being injected endotoxin.Asp treatment rats is the same as L01 treatment rats. Thrombus formation time and blood flow rate in veins were measured in the mesentery minicirculation models.(3)Effect on ultrastructural organization of endothelia cells in vitro.H-L01 treatment,L-L01 treatment,model control,L01 control and normal control were administered in vitro for 48 hours.To observe ultrastructural organization of endothelia cells and damage of the cell organ through TEM.
Results:(1)L01 effect on Thrombus formation in the artery-vein circuit rat models in vivo.In the models by injected adrenaline,compared with control group,the weight of thrombus increased significantly.But compared with model group,H-L01(50mg/kg)group and L-L01(10mg/kg)group can decrease the weight of thrombus,and the inhibition ratio in H-L01 group can reach to 57.5%. (2)Effect on Thrombus formation in the mesentery minicirculation models in vivo.In the models by injected endotoxin,compared with control group,thrombus formation time increased.And compared with model group,H-L01(5mg/kg)group and L-L01(2.5mg/kg)group can prolong the thrombus formation time,and on the 1h,the blood flow rate in veins is much higher than model group's.(3)Effect on ultrastructural organization of endothelia cells in vitro.In the models by injected adrenaline,irregularity cellular nucleus,cello-tight junction,microvilli is to decrease and to disaapear. Engorgement of mitochondrium,cristae is to dissolve and disaapear, cytolysosome is to increase.L01 control group and H-L01group is almost the same as the nomal control group;the damage in L-L01group is not as severe as the model group.
Conclusions:Laminaria-polysaccharide can inhibit the thrombus formation in two rat models with injured endothelial cells.And improve the damage from the ultrastructural organization of endothelia cells
引文
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