【作者】 郭雪峰；

【导师】 张继东；

【作者基本信息】 山东大学 ， 中西医结合临床， 2011， 硕士

【摘要】 目的1.观察芩丹胶囊对自发性高血压大鼠血压的影响。2.观察芩丹胶囊对血管外膜Ⅰ、Ⅲ胶原表达的影响,并探讨芩丹胶囊逆转高血压血管外膜重构的机制。方法1.研究对象：32只40周龄雄性SHR大鼠随机分为4组,模型组(SHR组)、芩丹胶囊大剂量组(SHR+QDH组)、芩丹胶囊小剂量组(SHR+QDL组)、氯沙坦组(SHR+Los组),另设40周龄雄性WKY大鼠16只分为空白对照组(WKY空白组)及正常药物对照组(WKY+QD组)。SHR+QDH组给予芩丹胶囊750mg/(kg·d)；SHR+QDL组给予芩丹胶囊150mg/(kg·d)；SHR+Los组给予氯沙坦30mg/(kg·d)；WKY+QD组给予芩丹胶囊750mg/(kg·d)；WKY空白组和SHR组给予相等剂量生理盐水,1次/d,连续给药12周。各组大鼠在末次给药后禁食24 h,不禁水。10%水合氯醛腹腔注射麻醉后进行后续实验。2.研究内容：(1)大鼠尾动脉收缩压测定。(2)免疫组化法观察胸主动脉外膜ⅠⅢ胶原表达。(3)实时定量PCR检测胸主动脉外膜Ⅰ、Ⅲ胶原表达。(4)用western blotting检测Ⅰ胶原蛋白表达。结果1.各组大鼠治疗前后收缩压比较见图1,从给药第4周起,SHR+QDH组、SHR+QDL组、SHR+Los组分别与SHR组比较,血压下降明显,其差异均具有统计学意义(P<0.05)。给药6周到12周间,SHR+QDH组比SHR+QDL组血压下降更为显著,差异具有统计学意义(P<0.05)。2.胸主动脉组织免疫组织化学测定结果显示,见表2,图2、3,SHR组与WKY空白组比较,Ⅰ、Ⅲ胶原表达明显增多,差异具有统计学意义(P<0.05)；SHR+QDH组、SHR+QDL组及SHR+Los组表达较SHR组均减少(P<0.05)。WKY空白组与WKY+QD组Ⅰ、Ⅲ胶原表达量比较,其差异无统计学意义(P>0.05),SHR+QDH组与SHR+QDL组比较差异有统计学意义(P<0.05),但与SHR+Los组比较,差异无统计学意义(P>0.05)。3.主动脉壁Ⅰ、Ⅲ胶原mRNA表达水平比较采用2-ΔΔCt方法,将Ⅰ、Ⅲ型胶原基因表达Ct值转换为相对于WKY空白组的变化倍数,可以直接得到的目的基因相对于参照基因的定量。如图6和7所示分别为Ⅰ、Ⅲ胶原的扩增、溶解曲线和溶解峰,说明Ⅰ、Ⅲ胶原是特异性基因表达,无引物二聚体及非特异性扩增。研究结果显示：见图4,WKY空白组和WKY+QD组间差异无统计学意义(P>0.05),SHR+QDH组和SHR+QDL组间差异有统计学意义(P<0.05)。SHR组大鼠胸主动脉壁Ⅰ、Ⅲ型胶原mRNA表达量与WKY组之间的差异有统计学意义(P<0.05)。SHR+QDH组、SHR+QDL组及SHR+Los组大鼠胸主动脉壁Ⅰ、Ⅲ型胶原表达与WKY组大鼠相比明显增多,与SHR组相比有所减少,其差异有统计学意义(P<0.05)。4.Ⅰ胶原在免疫印迹中的蛋白表达WKY空白组,WKY+QD组,SHR+Los组,SHR+QDH组,SHR+QDL组,SHR组蛋白表达依次增强,Ⅰ型胶原分子量大小在120KD左右,参照actin,分子量为43KD,可看出蛋白表达依次增强的趋势。见图5,用Ⅰ型胶原与actin的比值做统计分析结果显示：WKY空白组和WKY+QD组间差异无统计学意义(P>0.05),SHR+QDH组和SHR+QDL组间差异有统计学意义(P<0.05)。SHR模型组和正常WKY相比,差异有统计学意义(P<0.05)；SHR+QDH组.SHR+QDL组.SHR+Los组分别与SHR组比较,差异均有统计学意义(P<0.05)；SHR+QDH组与SHR+Los组比较差异无统计学意义(P>0.05)。结论芩丹胶囊对SHR大鼠有显著的降压疗效,其降压机制与芩丹胶囊降低SHR大鼠Ⅰ、Ⅲ胶原表达水平有关,同时与逆转血管外膜重构,抑制外膜纤维化,减少Ⅰ、Ⅲ胶原在外膜的合成与沉积有关。更多还原

【Abstract】 Aim1. Observe Qindan capsule on blood pressure in spontaneously hypertensive rats.2. Observe the Qindan capsule on vascular adventitiaⅠ,Ⅲcollagen expression, and to explore the reversal of hypertensive vascular adventitia remodeling mechanism.Methods1. Study:32 SHR rats of 40 weeks old were randomly divided into 4 groups, the model group (SHR group), Qindan high dosage group (SHR+QDH group), Qindan low dosage group (SHR+QDL group), the Losartan group (SHR+Los group), Besides, 16 Wistar—Kyoto (WKY) rats of the same weeks were taken as the normal control (WKY control group) and Qindan group (WKY+QD group). SHR+QDH group was treated with high dosage 750mg/(kg·d); SHR+QDL group was treated with low dosage 150mg/(kg·d); SHR+Los group was given losartan 30mg/(kg·d); WKY+QD group were treated with high dosage 750mg/(kg·d); WKY and SHR control group were given an equal dosage of saline, once a day, continuous administration for 12 weeks. Rats in each group after the last administration in the fasted 24h, can not help but water. All the rats were anesthetized with intraperitoneal injection of 10% chloral hydrate follow-up experiments. 2. Research:(1) Determination of rat tail artery systolic pressures.(2)Observed aortic adventitiaⅠ,Ⅲcollagen by immunohistochemistry.(3)Real-time quantitative PCR detection of thoracic aortic adventitiaⅠ,Ⅲcollagen.(4)Western blotting detection of expression of I collagen.Results1. Comparison of systolic blood pressure of ratsFig 1,after 4 weeks from the administration, Qindan high dosage, low dosage group, and losartan group compared with the model group blood pressure decreased, the difference was statistically significant (P<0.05). Between 6 weeks to 12 weeks of administration, the rat blood pressure decreased even more significant in the high dosage group than in the low dosage group, the difference was significant (P<0.05).2. Thoracic aorta by immunohistochemistry determination(Table 2, Fig 2, Fig 3)The results showed that the model group than in the control groupⅠ,Ⅲcollagen expression was significantly increased, the difference was statistically significant (P<0.05); Qindan low dosage group and losartan group were lower expression compared with the model group (P<0.05). Qindan control group and normal control groupⅠ,Ⅲcollagen expression was no significant difference (P>0.05), Qindan high dosage group compared with the low dosage group was statistically significant (P<0.05), but compared with the losartan group, the difference was not statistically significant (P>0.05).3. Aortic wallⅠ,Ⅲcollagen mRNA expression comparedUsing 2-ΔΔCt method, typeⅠ,Ⅲcollagen gene expression relative to the Ct is converted to changes in multiples of WKY control group, the target gene can be obtained directly relative to the reference gene quantification. As shown in Figure 6 and 7, respectivelyⅠ,Ⅲcollagen in the amplification curve and dissolution peaks, indicatingⅠ,Ⅲcollagen is a specific gene expression, no primer-dimers and nonspecific amplification. As shown (Fig 4):WKY control group and WKY+QD group was no significant difference (P>0.05), Differences between the SHR+QDH group and SHR+QDL group were statistically significant (P<0.05). There were significant differences between SHR groups and WKY groups in thoracic aorta adventitiaⅠ,Ⅲcollagen mRNA expression (P<0.05). SHR+QDH group, SHR+QDL group and SHR+Los group of rat thoracic aortic adventitiaⅠ,Ⅲcollagen expression compared with the WKY rats increased significantly, reduced compared with the SHR group, the differences were statistical significance (P<0.05).4. I collagen protein expression by Western blottingThe order in expression of collagenⅠfrom low to high:WKY control group, WKY+QD group, SHR+Los group, SHR+QDH group, SHR+QDL group, SHR group.Ⅰcollagen in 120KD molecular weight around, with reference to actin, a molecular weight of 43KD, can be seen followed the trend of increased protein expression. TypeⅠcollagen and actin by the ratio of statistical analysis were shown in Figure 5:WKY control group and WKY+QD have no significant difference (P>0.05), SHR+QDH group and SHR+QDL differences between the groups have statistical significance (P<0.05). SHR group compared with WKY group, the difference was statistically significant (P<0.05); SHR+QDH group, SHR+QDL group, SHR+Los group were compared with SHR group, the difference was statistically significant (P<0.05); SHR+QDH group and the SHR+Los group difference was not statistically significant (P>0.05).ConclusionQindan capsule on SHR rats had significant antihypertensive effects, and its step-down mechanism was possibly associated with reducing SHR ratsⅠ,Ⅲcollagen expression level, and also with the reversal of vascular adventitial remodeling, inhibition of membrane fibrosis, and reducingⅠ,Ⅲcollagen synthesis and deposition of the vascular adventitia.更多还原