【作者】 陈晓平；

【导师】 祝之明；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 硕士

【摘要】 背景和目的血管舒缩活动(vasomotion)指的是血管组织张力及内径的周期性、节律性收缩与舒张现象,具有一定的频率与振幅,小阻力血管及大弹力血管均可在体内、体外自发产生或是在管腔内压改变、受机械力牵张、血管收缩激动剂等刺激下发生。部分研究认为不同血管组织节律性舒缩活动的产生与电压依赖性钙通道、胞膜电位及由IP3受体介导的胞内钙离子振荡性变化、磷脂酶C及磷脂酶A2通路等密切相关,而在高血压病理状态下,如自发性高血压大鼠肠系膜动脉及先兆子痫患者网膜动脉等阻力血管的节律性舒缩活动振幅均较血压正常者显著升高,研究显示可能与高血压状态下血管平滑肌细胞间缝隙连接增多、内皮一氧化氮(NO)功能不足等相关,但具体机制尚待进一步明确,此外,高血压时血管平滑肌细胞钙内流显著增加也可能与节律性舒缩幅度的增强有关。近年研究发现瞬时型感受器电位通道(transient receptor potential channels, TRPC)家族可介导血管平滑肌细胞钙离子内流,该非选择性阳离子通道可分为TRPC1、2、3、4、5、6、7等几个亚型,在高血压状态下,大鼠单核细胞TRPC3及动脉平滑肌细胞TRPC6蛋白表达和介导的钙离子内流显著增加。钙池操纵钙离子通道(store-operated calcium entry channels, SOCE)是一类由胞内内质网储存钙耗竭而激活的钙离子内流通道,有研究显示SOC的分子组成即是TRPC中的1、3和5亚型,而现已明确节律性舒缩的形成与肌浆网储存钙的周期性释放、再摄取形成的胞内钙离子振荡性变化密切相关,另有研究表明,TRPC通道在ATP诱导的人类脐带平滑肌细胞钙离子振荡中起作用,故TRPC可能在肌浆网的周期性耗竭、再充盈过程中被激活及失活关闭,由此介导的钙离子周期性内流也可能在节律性舒缩中起部分作用。血管紧张素Ⅱ1型受体拮抗剂(ARB)及钙离子拮抗剂(CCB)作为临床上广泛应用的降压药,不仅可有效降低血压,还可改善心血管内皮功能、逆转高血压所致心血管重构损害,有研究显示,长期应用ARB对SHR大鼠进行干预后其主动脉TRPC3通道蛋白表达下降,采用RNA干扰的方法抑制TRPC3在血管平滑肌细胞上的表达后AngⅡ介导的钙离子内流显著减少,但该二种降压药对高血压病理状态下异常的阻力血管节律性舒缩是否有影响尚不清楚,本实验将重点研究应用坎地沙坦、替米沙坦及氨氯地平对SHR大鼠长期干预后对其阻力血管节律性舒缩的影响,并对发生影响的可能机制进行初步探讨。对象与方法一、动物分组:体重250-260g健康3月龄雄性SHR大鼠40只,随机分为4组,每组10只,分别为SHR对照组、替米沙坦干预组(5mg/kg/d)、坎地沙坦干预组(4mg/kg/day)及氨氯地平干预组(10mg/kg/day);健康3月龄雄性WKY大鼠10只为血压正常对照组,干预组均将药物混于少量蒸馏水中灌胃给药,共干预16周,对照组给予等量蒸馏水灌胃。二、血压测量:测定各组大鼠清醒状态下尾动脉收缩压,分别在干预开始时和干预后每4周测定,动态观察各组血压变化。三、血管环实验:干预结束后采用小血管张力测量仪测定各组大鼠肠系膜动脉节律性舒缩振幅,并观察TRPC通道阻断剂对节律性舒缩的影响。四、免疫荧光染色:将SHR大鼠肠系膜动脉外周脂肪结缔组织仔细分离后免疫荧光染色证实TRPC通道蛋白在平滑肌细胞上的表达。五、蛋白表达检测:提取各组大鼠肠系膜动脉蛋白,应用Western blot方法分析各组大鼠肠系膜动脉TRPC1、TRPC3、TRPC4、TRPC5及TRPC6的蛋白表达情况。结果1.与血压正常WKY大鼠相比,SHR大鼠肠系膜动脉节律性舒缩幅度显著增强(65.5±7.1% vs. 2.2±0.5%; n=6, p<0.01);2.与血压正常WKY大鼠相比,SHR大鼠肠系膜动脉TRPC1、TRPC3及TRPC5的蛋白表达显著增加(TRPC1表达:WKY 1.00±0.13 vs. SHR 1.60±0.10; p<0.01; TRPC3表达:WKY 1.00±0.18 vs. SHR 1.70±0.26; p<0.05; TRPC5表达:WKY 1.00±0.13 vs. SHR 1.41±0.10; p<0.05; each n=6);3.TRPC通道阻断剂可部分抑制SHR大鼠肠系膜动脉显著增强的节律性舒缩振幅,使其舒缩节律发生紊乱(Gd3+:从107.3±10.7%降至63.1±10.6%;2-APB:从105.2±13.9%降至30.3±2.5%;SKF96365:从109.9±15%降至23.5±9.6%,n=6,p<0.05);4.坎地沙坦、替米沙坦及氨氯地平干预组SHR大鼠血压均显著下降(SHR对照组:222±7mmHg;坎地沙坦组:123±7mmHg;替米沙坦组:115±6mmHg;氨氯地平组:122±5 mmHg;p<0.01;n=10),但坎地沙坦及替米沙坦组SHR大鼠肠系膜动脉节律性舒缩幅度显著减小,而氨氯地平干预组无明显改变(SHR对照组:66±7%;坎地沙坦组:14±3%;替米沙坦组:8±2%;氨氯地平组:57±7%,p<0.01;n=10);同时,坎地沙坦、替米沙坦干预组肠系膜动脉TRPC1、TRPC3及TRPC5蛋白表达显著下降(p<0.05;n=5),而氨氯地平干预组无明显改变(p>0.05;n=5)。结论1.SHR大鼠肠系膜动脉TRPC1、TRPC3及TRPC5通道蛋白表达显著增强,可能与其节律性舒缩幅度的显著增加有关;2.坎地沙坦、替米沙坦及氨氯地平均可显著降低SHR大鼠血压,但只有坎地沙坦、替米沙坦可降低SHR大鼠肠系膜动脉TRPC1、TRPC3及TRPC5通道蛋白表达及节律性舒缩幅度;更多还原

【Abstract】 Background and Objective: Vasomotion refers to the phenomenon of cyclical, rhythmical change of diameter and contractions with a certain frequency and amplitude in vascular tissue. Vasomotion occurs in small resistance vessels as well as in larger arteries both in vivo and in vitro either spontaneously or in response to pressure, stretch or application of vasoconstrictor agonists. Part of the studies have described a critical role for the voltage-dependent calcium channels, membrane potential, the IP3 receptor-mediated oscillatory change of intracellular calcium, phospholipase C and phospholipase A2 pathway on the vasomotion in different vessels. In pathological conditions of hypertension, such as in mesenteric artery from spontaneously hypertensive rat or from patients with pre-eclampsia, the amplitude of vasomotion in resistance artery increased significantly as compared with those of normal blood pressure. According to some studies, such phenomenon may be related to the increased gap junctions among vascular smooth muscle cells or the nitric oxide (NO) deficiency in endothelium, but the exact mechanism remains to be further defined. In addition, the significantly increased calcium influx in the vascular smooth muscle cells may also contribute to the enhanced vasomotion under the condition of hypertension.Recent studies found that the calcium influx in vascular smooth muscle cell can be mediated by transient receptor potential channels (TRPC) family, which can be divided into TRPC1, 2, 3, 4, 5, 6, 7 subtypes. In the hypertensive state, the expressions of TRPC3 channel in mononuclear cells and TRPC6 channel in artery smooth muscle cells as well as TRPC channel mediated calcium influx were increased significantly as compared with those of normal blood pressure. Studies have showed that the molecular composition of the store-operated calcium entry channels (SOCE), in which the emptying of intracellular calcium stores activates calcium influx, are TRPC1, TRPC3 and TRPC5. Now it is clear that the oscillatory change of intracellular calcium which result from the cyclical release and refilling of the intracellular calcium stores is associated with the formation of vasomotion, so it is possible that such a process can activate the TRPC channels rhythmically, which mediated calcium influx may be take part in the forming of enhanced vasomotion and recent study has showed that TRPC channels play an important role in the forming of calcium oscillations induced by ATP in human umbilical cord smooth muscle cells. This study will investigate the relationship between the enhanced vasomotion and the TRPC channels in resistance artery.At present, angiotensinⅡreceptor (type 1) blocker (ARB) and calcium channel blocker (CCB) are two widely used antihypertensive agents in clinic, not only can effectively lower the blood pressure but also can improve the vascular endothelial function and reverse the cardiovascular remodeling induced by hypertension. Studies have shown that long-term application of ARB on the SHR, the expression of TRPC3 was significantly reduced in aorta and the inhibition of TRPC3 channel by using RNA interference in vascular smooth muscle cells could significantly reduce the AngⅡ-mediated calcium influx. But, it is still largely unknown about the impact of ARB and CCB on the abnormal enhanced vasomotion in resistance artery from SHR. One of the important aims of the current study is to identify the impact of long-term application of candesartan, telmisartan and amlodipine on the abnormal vasomotion in resistance artery from SHR, and make a preliminary study on the possible mechanism.Methods:1. Animal grouping and treatment.In this study, three-month-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats weighing 250-260g were used. SHR were randomly divided into four groups: control group received distilled water (n=10), 5mg/kg/day telmisartan (n=10), 4mg/kg/day candesartan (n=10) and 10mg/kg/day amlodipine (n=10) by oral garage for 16 weeks. WKY were used as normotensive control group (n=10).2. Blood pressure measurement:Systolic blood pressure was measured monthly in conscious and restrained rats by the tail-cuff method.3. Vascular ring experiments: At the end of treatment, the magnitude of vasomotion in mesenteric artery from each group was measured and the effect of TRPC channel blocker on the vasomotion was also observed.4. Immunofluorescent staining:In order to show the expression of TRPC channel in mesenteric artery from SHR, the immunofluorescent staining was performed.5. Detection of protein expression of mesenteric artery from each group:Proteins of mesenteric artery from each group were extracted and western blotting was taken out to detect protein expression of TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6.Results:1. Compared with WKY normotensive rat, the magnitude of vasomotion was significantly higher in mesenteric arteries from SHR (65.5±7.0% vs. 2.2±0.5%; each n=6, p<0.01);2. Compared with WKY normotensive rat, the expression of TRPC1, TRPC3 and TRPC5 channels were significantly higher in mesenteric arteries from SHR (TRPC1 expression for WKY 1.00±0.13 vs. 1.60±0.10 for SHR; p<0.01; TRPC3 expression for WKY 1.00±0.18 vs. 1.70±0.26 for SHR; p<0.05; TRPC5 expression for WKY 1.00±0.13 vs. 1.41±0.10 for SHR; p<0.05; each n=6);3. The blockers of TRPC channel can partially inhibit the markedly enhanced vasomotion in mesenteric artery from SHR and make the rhythm became derangement (gadolinium, from 107.3±10.7% to 63.1±10.6%; 2-APB, from 105.2±13.9% to 30.3±2.5%; SKF96365, from 109.9±15% to 23.5±9.6%; each n=6; p<0.05);4. Both candesartan, telmisartan and amlodipine can significantly reduce the blood pressure of SHR (placebo, 222±7mmHg; candesartan, 123±7mmHg; telmisartan 115±6mmHg; amlodipine, 122±5 mmHg; p<0.01; each n=10;), but only candesartan and telmisartan can significantly improve the abnormal enhanced vasomotion and amlodipine showed no effect on vasomotion (placebo control group, 66±7%; candesartan treated group, 14±3%; telmisartan treated group, 8±2%; amlodipine treated group, 57±7%; p<0.01, each n=10). At the same time, the expression of TRPC1, TRPC3 and TRPC5 were significantly reduced in mesenteric arteries from candesartan and telmisartan treated group compared to placebo-treated SHR as control group (p<0.05; n=5), while the expression of TRPC1, TRPC3 and TRPC5 showed no significantly change in mesenteric arteries from amlodipine treated group (p>0.05; n=5);Conclusions:1. The markedly increased expression of TRPC1, TRPC3 and TRPC5 in mesenteric artery from SHR may be related to the abnormal enhanced vasomotion;2. Candesartan, telmisartan and amlodipine can significantly reduce blood pressure in SHR, but only candesartan and telmisartan can reduce the expression of TRPC1, TRPC3 and TRPC5 in mesenteric artery from SHR and reduce the abnormal enhanced vasomotion;更多还原