自发性高血压大鼠肠系膜微淋巴管自律运动的改变及基质金属蛋白酶抑制剂减轻血管钙化的机制研究

发布时间：2018-01-26 23:41

  本文关键词： 高血压 微循环 微淋巴管 自律运动 自发性高血压 血管钙化 血管平滑肌细胞 基质金属蛋白酶-2 凋亡　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：第一部分 自发性高血压大鼠肠系膜微淋巴管自律运动的改变目的动态监测并定量分析不同周龄自发性高血压大鼠(SHR)肠系膜微淋巴管自律运动的特点。方法取3周、8周和13周的雄性Wistar大鼠和SHR,麻醉、固定并暴露肠系膜后,在活体显微镜下动态监测肠系膜微淋巴管的自律运动并录像,运用VasTrack自动测量系统对录像进行定量和分析。分析的指标包括微淋巴管自律运动的相对振幅、频率、收缩期及舒张期的幅度、时长和速度,并根据频率、最大舒张管径和最大收缩管径计算出肠系膜微淋巴管收缩功能的两个指标：收缩分数和总收缩活性指数。结果(1)SHR与Wistar大鼠肠系膜微淋巴管自律运动在不同周龄的差别：3周龄SHR与3周龄Wistar大鼠微淋巴管自律运动在各个指标方面均无显著差异(均为P0.05)。到8周龄时,SHR微淋巴管自律运动的相对振幅、收缩幅度、收缩时长、收缩速度、舒张幅度、舒张速度及总收缩活性指数均显著低于Wistar大鼠(分别为P＜0.001,P＜0.01, P＜0.05,P0.01, P＜0.001,P0.01,P0.05), SHR微淋巴管自律运动的频率、舒张时长和收缩分数与Wistar大鼠无显著差异(均为P0.05)。到13周龄时,SHR微淋巴管自律运动的相对振幅、收缩幅度、收缩时长、舒张幅度、舒张时长和收缩分数显著低于Wistar大鼠(均为P0.001),频率和舒张速度显著高于Wistar大鼠(均为P0.05),收缩速度和总收缩活性指数与Wistar大鼠无显著差异(均为P0.05)。(2)SHR肠系膜微淋巴管自律运动在不同周龄的差别：与3周龄SHR相比,8周龄SHR淋巴管自律运动的相对振幅、频率、舒张幅度、舒张速度和总收缩活性指数显著降低(分别为P＜0.05, P0.05, P0.05, P0.01,P＜0.01)。8周龄SHR的收缩幅度、收缩时长、收缩速度、舒张时长和收缩分数与3周龄SHR相比,均无显著差异(均为P0.05)。13周龄SHR的收缩频率、收缩速度、舒张速度和总收缩活性指数均显著高于8周龄SHR(分别为P0.05,P0.05,P0.01,P0.01)；13周龄SHR的收缩时长、舒张时长和收缩分数均显著低于8周龄SHR(分别为P0.05,P0.01,P0.05)；13周龄SHR的相对振幅、收缩幅度和舒张幅度与8周龄SHR相比,均无显著差异(均为P0.05)。结论(1)SHR与Wistar大鼠的肠系膜微淋巴管均存在自律运动。(2)高血压可能是引起SHR肠系膜微淋巴管自律运动障碍的一个重要因素。(3)高血压发生后,SHR肠系膜微淋巴管自律运动的舒张期活动减弱,收缩功能显著下降。(4)高血压进展过程中,SHR微淋巴管自律运动的紊乱体现在收缩功能增强,而收缩功能增强更有利于机体内环境的稳态。第二部分 基质金属蛋白酶抑制剂通过下调BMP-2抑制大鼠主动脉血管平滑肌细胞钙化目的探讨基质金属蛋白酶(MMPs)在血管钙化中的作用及其机制。方法取8周龄雄性VVistar大鼠,贴块法培养主动脉血管平滑肌细胞(VSMCs),形态学和免疫荧光法鉴定VSMCs。对照组的VSMCs采用DMEM培养基,钙化组用p-甘油磷酸(p-GP)诱导VSMCs发生钙化,MMPs抑制剂组用β-GP+MMPs抑制剂多西环素(doxycycline, Doxy)。培养12天后,对VSMCs进行von kossa染色和钙含量分析,以检测VSMCs钙化。用试剂盒检测碱性磷酸酶(ALP)活性,用、Westernblot检测a-SMA, Desmin及ALP的蛋白表达,以观察VSMCs表型改变。、Western blot检测MMP-2、MMP-9蛋白表达水平,明胶酶谱法检测MMP-2、MMP-9的酶活性,以观察MMPs抑制剂多西环素的抑制效果。Western blot检测骨形态发生蛋白-2(BMP-2)及其受体、RUNX2. Msx-2的表达水平,以探讨MMPs印制后BMP-2通路的变化。用TUNEL法检测VSMCs凋亡状况,Western blot检测凋亡相关蛋白caspase-3、cleaved-caspase-3、Bax和Bcl-2的表达,caspase-3活性试剂盒检测caspase-3活性。结果(1)培养的细胞镜下呈梭形、纺锤形或菱形,细胞有多个突起,胞浆丰富,12天后,细胞出现“峰和谷”样的生长模式。平滑肌a-actin (smooth muscle a-actin, SM a-actin)抗体免疫荧光染色后,可见胞浆呈绿色荧光,胞浆内见大量与细胞长轴平行的纤维细丝,表明培养的细胞为VSMCs.(2)钙化组的钙含量和钙化面积显著高于对照组(P＜0.001,P＜0.001); MMPs抑制剂组的钙含量和钙化面积显著低于钙化组(P0.001,P0.001)。钙化组可见VSMCs表型改变,而MMPs抑制导致VSMCs表型改变减轻。(3)Western blot检测结果发现,与钙化组相比,MMPs抑制导致MMP-2和MMP-9蛋白表达降低(P0.01,P0.01)；明胶酶谱检测结果表明,与钙化组相比,MMPs抑制导致MMP-2和MMP-9酶活性降低(P0.01,P＜0.01)。(4) MMPs抑制导致BMP-2、RUNX2和Msx-2蛋白表达降低(P＜0.01,P＜0.001, P0.01)。 (5)BMP-2可促进VSMCs表达RUNX2和Msx-2(P0.01, P0.01),而noggin可减少两者的表达(P0.05, P0.05)。(6) TUNEL检测结果表明,钙化组的凋亡细胞比例显著高于对照组(P0.001)；与钙化组相比,MMPs抑制剂组的凋亡细胞比例显著降低(P0.001)。钙化组的caspase-3活性及cleaved-caspase-3蛋白表达均显著关于对照组(P0.001,P0.001)。与对照组相比,钙化组的Bax表达显著升高、Bcl-2表达显著降低(P0.01,P0.05)。结论(1)成功培养了大鼠VSMC并诱导钙化。(2)抑制MMPs可减轻VSMCs钙化,并减轻钙化过程中VSMCs的表型改变。(3)抑制MMPs可通过下调BMP-2.进而降低RUNX2口Msx-2的表达,减轻血管钙化。(4)抑制MMPs可能通过减轻VSMCs凋亡来减轻血管钙化。
[Abstract]:Different week old spontaneously hypertensive rats to dynamic monitoring and quantitative analysis of changes in the first part of spontaneously hypertensive rat mesentery micro lymphatic vasomotion (SHR) characteristics of mesentery micro lymphatic vasomotion. Methods 3 weeks, 8 weeks and 13 weeks of male Wistar rats and SHR, anesthesia, fixed and mesentery after exposure in vivo microscopy, dynamic monitoring of mesentery micro lymphatic self motion and video, using the VasTrack automatic measurement system for quantitative analysis of video analysis. And the indexes including relative amplitude, microlymphatics vasomotion frequency, systolic and diastolic amplitude, duration and speed, and according to the frequency, maximum diastolic diameter and maximum the contraction diameter to calculate two indicators of mesentery micro lymphatic systolic function: the systolic fraction and total systolic activity index. Results (1) SHR and Wistar in rat mesentery micro lymphatic self-discipline The difference in motion in different age: 3 week old SHR and Wistar rats aged 3 weeks microlymphatics vasomotion were no significant differences in all indexes (P0.05). By 8 weeks of age, relative amplitude, SHR microlymphatics vasomotion contraction amplitude, contraction velocity, shrinkage, diastolic amplitude, diastolic velocity and total systolic activity index were significantly lower than that of Wistar rats (P < 0.001, P < 0.01, P < 0.05, P0.01 < 0.001, P, P0.01, P0.05, SHR) microlymphatics vasomotion frequency, duration and diastolic systolic fraction and Wistar rats no significant the difference (P0.05). By 13 weeks of age, relative amplitude, SHR microlymphatics vasomotion contraction, contraction duration, diastolic amplitude, duration and diastolic systolic fraction was significantly lower than that of Wistar rats (P0.001), diastolic velocity and frequency was significantly higher than that of Wistar rats (P0.05) the systolic velocity and total shrinkage, active index 鏁颁笌Wistar澶ч紶鏃犳樉钁楀樊寮，

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