【作者】 李丽琦；

【导师】 肖洪彬；

【作者基本信息】 黑龙江中医药大学 ， 方剂学， 2012， 博士

【摘要】 目的：从红细胞膜分子层次探讨大鼠寒冷刺激血瘀模型、饥饿加力竭游泳血瘀模型、声光电复合刺激加钳尾激惹血瘀模型在血液流变学方面的生物学差异；应用少腹逐瘀汤、补阳还五汤、丹参饮反证寒冷刺激血瘀模型红细胞膜生物学特征方法：以大鼠为研究对象。(1)用冰水浸泡模拟寒冷刺激而致血瘀模型、以饥饿加力竭游泳模拟气虚血瘀模型、以不可预期的声光电复合刺激加钳尾激惹模拟惊恐、愤怒过度而致气滞血瘀模型。持续刺激直至2周后各组大鼠分别出现寒凝血瘀、气虚血瘀、气滞血瘀表征,颈动脉取血测血液黏度、红细胞变形性、血沉K值、凝血四项、血浆胆固醇及甘油三酯含量；取血制备红细胞膜,检测红细胞膜Na+-K+-ATP酶活力、唾液酸含量、巯基含量、胆固醇含量、SOD活性、MDA含量。(2)寒冷刺激血瘀模型大鼠分别灌胃给予高、低剂量少腹逐瘀汤,高、低剂量补阳还五汤及高、低剂量丹参饮,连续2周,颈动脉取血,除检测上述指标外,以激光共聚焦显微镜观察各组大鼠红细胞膜骨架结构,并检测红细胞膜蛋白组成及百分含量。结果：(1)与空白对照比较,寒冷刺激血瘀模型组全血黏度、血沉K值、FIB含量均显著增高,红细胞变形能力明显降低,APTT明显缩短,红细胞膜Na+-K+-ATP酶活力、SOD活性显著下降,唾液酸含量显著降低(P<0.05或P<0.01)；饥饿加力竭游泳血瘀模型高切变率全血黏度、红细胞变形性降低,APTT及PT明显缩短,血浆胆固醇含量下降,红细胞膜红细胞膜Na+-K+-ATP酶活力及SOD活性显著降低(P<0.05或P<0.01)；不可预期声光电刺激加钳尾激惹血瘀大鼠APTT明显缩短,红细胞膜Na+-K+-ATP酶活力及SOD活性显著下降、唾液酸含量显著降低,MDA含量增加(P<0.05或P<0.01)。(2)与寒冷刺激血瘀模型组比较,少腹逐瘀汤、补阳还五汤、丹参饮均可显著降低低切变率全血黏度,在高切变率下全血黏度少腹逐瘀汤降低效果最好；三方均能显著升高红细胞变形指数显著降低FIB含量,增加红细胞膜上唾液酸的含量及SOD酶活力(P<0.05或P<0.01)；丹参饮可显著降低K值(P<0.05),升高红细胞膜带1蛋白百分含量；少腹逐瘀汤可显著纠正APTT缩短及降低MDA含量,提高Na+-K+-ATP酶活力,明显改善F-actin解聚状态,升高红细胞膜上带1蛋白、带2蛋白、带3蛋白百分含量(P<0.05或P<0.01)。结论：(1)宏观血液流变学方面：寒冷刺激血瘀模型血液流变学呈高流变性特征；饥饿加力竭游泳血瘀模型血液流变学呈现低流变特点；声光电复合刺激加钳尾激惹血瘀模型除APTT明显缩短,其余宏观血液流变学指标变化不明显；(2)三种血瘀模型均表现为红细胞膜Na+-K+-ATP酶活性、SOD活性降低；寒冷刺激和声光电复合刺激血瘀模型红细胞膜唾液酸含量减少；(3)补阳还五汤、少腹逐瘀汤、丹参饮三方可纠正寒冷刺激血瘀模型全血黏度增高、红细胞变形性降低、聚集性增强、纤维蛋白原含量增加及膜骨架构象异常；纠正APTT缩短、提高膜Na+-K+-ATP酶活性、减少膜MDA含量方面,少腹逐瘀汤优于其他两方；少腹逐瘀汤可纠正寒冷刺激血瘀模型红细胞膜带1蛋白、带2蛋白、带3蛋白百分含量异常,丹参饮作用于膜带1蛋白、带7蛋白。更多还原

【Abstract】 Objectives:To investigate biological difference of hemorheology of three kinds of blood stasis model from molecular level of red cell membrane. There were cold stimulation blood stasis model, starvation plus exhausted swimming blood stasis model and acoustics, optics, electricity complex stimulus plus clamp tail blood stasis model. Shaofu Zhuyu decoction, Buyang Huanwu decoction and Danshen decoction were applied to reverse approve the biological characteristics of red cell membrane of cold stimulation blood stasis model.Methods:The rats were observed in this article.(1) Dipping in ice water to simulate cold stimulation blood stasis model. Starvation plus exhausted swimming to simulate qi deficiency blood stasis model. Unpredictable acoustics, optics, electricity complex stimulus plus clamp tail irritation to simulate qi depression to blood stasis. After stimulation continual for two weeks, the token of cold stagnation blood stasis, qi deficiency blood stasis and qi depression blood stasis were appeared. The whole blood viscosity, erythrocyte deformability, blood sedimentation K value, four kinds of thrombin, plasma cholesterol and triglyceride were determined after taking blood from carotid. Then erythrocyte membrane were prepared to determine Na+-K+-ATP enzyme activity, the content of sialic acid, hydrosulphonyl, cholesterol, MDA and activity of SOD.(2) The cold stagnation blood stasis rats were intragastric administrated high and low dose Shaofu Zhuyu decoction, Buyang Huanwu decoction and Danshen decoction for two weeks. Except the above-mentioned index were determined, the structure of membrane skeleton were observed under laser scanning confocal fluorescence microscope. The constituent of membrane protein and percentage composition were determined.Results:(1) Compare with the blank group, the whole blood viscosity, blood sedimentation K value and the content of FIB of cold stagnation blood stasis group is increasing significantly. The erythrocyte deformability is decreasing and APTT is curtailing significantly. SOD and Na+-K+-ATP enzyme activity of erythrocyte membrane are decreasing and the content of sialic acid is decreasing (P<0.05or P<0.01); Under high shear the whole blood viscosity and erythrocyte deformability of Starvation plus exhausted swimming group is decreasing. APTT and PT is curtailing significantly. The content of plasma cholesterol is decreasing distinctly. SOD and Na+-K+-ATP enzyme activity of erythrocyte membrane are decreasing significantly (P<0.05or P<0.01); Compare with the blank group, APTT of unpredictable acoustics, optics, electricity complex stimulus plus clamp tail irritation group is curtailing significantly. SOD and Na+-K+-ATP enzyme activity of erythrocyte membrane are decreasing. The content of sialic acid is decreasing and MDA is increasing (P<0.05or P<0.01).(2) Compare with the cold stagnation blood stasis group, under low shear the whole blood viscosity is decreased significantly after i.g. Shaofu Zhuyu decoction, Buyang Huanwu decoction and Danshen decoction. Under high shear the whole blood viscosity is decreased distinctly by Shaofu Zhuyu decoction. After i.g. three pieces of prescription to stagnation blood stasis model rats, the erythrocyte deformability and the content of sialic acid of erythrocyte membrane is increased, SOD activity of erythrocyte membrane are increasing, the content of FIB is decreased significantly (P<0.05or P<0.01). The blood sedimentation K value could be decreased by Danshen decoction; Shaofu Zhuyu decoction not only could curtail APTT and decrease the content of MDA, but also could increase Na+-K+-ATP enzyme activity, improve depolymerization state of F-actin and increase the percentage composition of band1protein, band2protein and band3protein significantly.Conclusions:(1) The difference of three kinds of blood stasis model in macro-hemorheology registered as the following points:The cold stagnation blood stasis group demonstrated high rheological behavior; Starvation plus exhausted swimming group demonstrated low rheological behavior; Acoustics, optics, electricity complex stimulus plus clamp tail irritation group showed the curtailing time of APTT. In molecular rheology, SOD and Na+-K+-ATP enzyme activity of the three models were reduced, the content of sialic acid was decreased in the cold stagnation blood stasis group and the complex stimulus group.(2) The three prescriptions could improve the following performance in the cold stagnation blood stasis model rats such as whole blood viscosity and erythrocyte aggregation was increased, erythrocyte deformability was decreased and the content of FIB was increased; But Shaofu Zhuyu decoction was more effective than the other two in curtail APTT, increase Na+-K+-ATP enzyme activity and decrease the content of MDA; Most of all, Shaofu Zhuyu decoction had particularity in improve the abnormal phenomena of red cell membrane skeleton and band1protein, band2protein, band3protein.Danshen Yin influenced the band1and band7protein.更多还原