在高血压动脉重建中microRNA-21对血管平滑肌细胞细胞外基质表达的调控作用

目的探讨在高血压动脉重建中microRNA-21(miR-21)对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)细胞外基质(extracellular matrix,ECM)的调控作用及其机制。方法建立腹主动脉缩窄型大鼠高血压模型,大鼠分为假手术对照组、高血压2周组和高血压4周组;对体外培养的大鼠主动脉VSMCs施加频率为1.25 Hz周期性张应变,加载幅度分别为0%(静态对照组)、5%(正常张应变组)、15%(模拟高血压状态的高张应变组),加载持续时间均为12 h。采用Western blotting和Real time RT-PCR技术,分别检测动脉和细胞样品ECM以及miR-21的表达。用miR-21特异干扰片段抑制培养的VSMCs miR-21表达,然后检测VSMCs的ECM、miR-21和Smad 7表达变化。结果与假手术对照组相比,高血压2周组胸主动脉ECM和miR-21的表达显著上升;高血压4周组胸主动脉的I型胶原、III型胶原和miR-21表达显著上升。与静态对照组和5%张应变组相比,15%张应变组VSMCs的I型胶原表达无显著变化,而III型胶原表达显著升高,Smad 7表达显著下降,周期性张应变增强VSMCs的miR-21表达。干扰miR-21降低周期性张应变状态下VSMCs的miR-21表达以及III型胶原蛋白水平表达,上调VSMCs的Smad 7表达。结论高血压血管重建导致大鼠胸主动脉ECM和miR-21高表达。周期性高张应变可诱导VSMCs的miR-21高表达,再通过其调节Smad 7蛋白,进而调控VSMCs的ECM,尤其是III型胶原的表达,参与高血压血管重建。     

张应变调控的核骨架蛋白在高血压血管平滑肌细胞增殖中的作用

目的:高张应变诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)异常增殖在高血压血管重建发生发展中起重要作用。本研究探讨细胞核骨架(nuclear envelope,NE)在其中的作用及其机制。方法:应用腹主动脉缩窄构建高血压大鼠动物模型;应用FX4000张应变加载系统对体外培养大鼠胸主动脉VSMCs分别施加5%(正常生理状态)和15%(模拟高血压状态)幅度的周期性张应变;Western blot检测NE蛋白emerin和lamin A蛋白表达水平;染色质免疫共沉淀、芯片(CHIP-onchip)结合MOTIF生物信息学分析检测与emerin和lamin A结合的DNA序列及其特性;protein/DNA array检测抑制emerin或lamin A表达后转录因子活性变化。结果:高血压大鼠颈总动脉emerin和lamin A表达水平明显降低,中膜VSMCs增殖明显增加;体外加载15%周期性张应变模拟高血压病理条件下VSMCs受到的张应变力学刺激,VSMCs的emerin和lamin A表达明显降低,细胞增殖明显增加,这一作用可被emerin或lamin A的高表达载体转染所部分逆转。emerin和lamin A能够分别与包含多种转录因子启动子结合位点的DNA片段结合,进而调控多种与VSMCs增殖相关的转录因子活性。结论:NE蛋白emerin和lamin A能够响应力学刺激,并通过调控与特异性转录因子启动子区域的结合调控转录因子活性,参与VSMCs增殖功能调控和高血压血管重建。     

长链非编码RNA XR007793在病理性高张应变诱导平滑肌细胞增殖中的作用

目的探讨高血压条件下异常升高的周期性张应变刺激对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖活性的影响以及VSMCs中一种长链非编码RNA(long non-coding RNA,lncRNA)XR007793在其中可能的作用。方法应用体外周期性张应变加载系统Flexcell-4000对VSMCs分别施加5%生理性张应变和15%病理性高张应变,加载频率为1.25 Hz,加载时间24 h。荧光实时定量PCR检测XR007793及其共表达基因:信号转导与转录激活因子2(STAT2)、细胞分裂相关蛋白8(CDCA8)、原癌基因LMO2和干扰素调节因子(IRF7)的表达变化,Western bloting方法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达变化,RNA干扰技术抑制VSMCs的XR007793表达,静态条件下流式细胞术检测VSMCs周期变化,牵拉条件下Brdu检测VSMCs增值变化情况。结果与5%生理性张应变组相比,15%病理性高张应变显著下调XR007793表达水平,促进VSMCs增值活性并上调STAT2和CDCA8的表达水平。静态条件下干扰XR-007793,VSMC增殖水平显著上升。高周期性张应变条件下干扰XR-007793,VSMC增殖水平上升,CDCA8表达水平上升。结论病理性高张应变可能通过降低XR007793表达来影响CDCA8表达变化,进而调控VSMCs增殖功能变化过程。研究结果为阐明高血压条件下血管重建机制和药物治疗靶标的研究提供新的实验依据。     

Rho蛋白解离抑制因子α在高血压大鼠胸主动脉的表达

目的 研究高血压大鼠胸主动脉以及周期性张应变刺激下血管平滑肌细胞(VSMCs)的Rho蛋白解离抑制因子(RhoGDIα)的表达变化,探讨血管紧张素Ⅱ(AngⅡ)信号通路对其表达的调控影响. 方法 应用实时PCR和Western blotting技术分别检测4周、12周和18周自发性高血压大鼠(SHR, n =4)和正常血压京都种鼠(WKY, n =4)胸主动脉RhoGDIα mRNA和蛋白的表达;免疫组织化学检测RhoGDIα在SHR和WKY胸主动脉的定位;Western blotting技术检测腹主动脉缩窄性高血压大鼠(ACR, n =6)胸主动脉RhoGDIα蛋白的表达;应用细胞应变加载系统对大鼠胸主动脉VSMCs施加1Hz、10%的周期性张应变,在有或无血管紧张素亚型Ⅰ(AT1)受体拮抗剂 L-158809的条件下观察周期性张应变刺激对VSMCs RhoGDIα蛋白表达的影响. 结果 4周与12周SHR和WKY胸主动脉RhoGDIα表达无显著性差异,而在18周组,SHR胸主动脉RhoGDIα表达显著高于WKY.RhoGDIα主要存在于血管中膜VSMCs.2周和4周ACR胸主动脉RhoGDIα表达较正常对照组显著上调,提示在高血压状态下的主动脉RhoGDIα的表达上调.10%周期性张应变加载抑制了VSMCs的RhoGDIα表达,加入ATI受体拮抗剂后RhoGDIα表达显著低于10%周期性张应变加载组. 结论 大鼠高血压时主动脉RhoGDIα表达上调;Ang Ⅱ信号通路在RhoGDIα表达调控中起重要作用.     

张应变诱导分泌型miR-27a调控GRK6在高血压内皮细胞异常增殖中的机制研究

目的:本研究旨在揭示高血压条件下血管平滑肌细胞(vascular smooth muscle cells,VSMCs)响应高周期性张应变后调控血管内皮细胞(endothelial cells,ECs)异常增殖的可能机制。方法:在体条件下,构建腹主动脉缩窄型高血压大鼠模型;体外条件下,用FX-4000T张应变加载系统对VSMCs施加5%和15%的周期性张应变。结果:与正常组相比,高血压组大鼠胸主动脉ECs中GRK6的表达水平显著降低,ECs增殖水平显著上升;体内和体外条件均存在VSMCs源性MPs(VSMC-MPs);mi R-27a存在于VSMC-MPs中,并可靶向调控GRK6;15%周期张应变条件下产生的VSMC-MPs中mi R-27a的含量显著高于5%组,作用于ECs后,15%组ECs中mi R-27a的水平显著高于5%组,GRK6的表达水平显著低于5%组,ECs的增殖能力显著高于5%组;用从转染生物素连接的mi R-27a(B-mi R-27a)的VSMCs培养液中分离得到的MPs作用于ECs,在ECs中可以检测到B-mi R-27a的存在;mi R-27a正向调控ECs的增殖,GRK6负向调控ECs的增殖。结论:在高血压条件下,高周期性张应变促进VSMCs分泌mi R-27a,其可通过VSMC-MPs转移到ECs中,抑制GRK6表达,并最终诱导ECs异常增殖。     

生理性周期性张应变通过激活AMPK磷酸化抑制血管平滑肌细胞迁移

目的 探讨细胞能量代谢的关键调节因子 AMP激活的蛋白激酶AMPK在血管平滑肌细胞（vascular smooth muscle cells, VSMCs）响应生理性周期性张应变力学刺激后对VSMCs迁移的影响。方法 采用 Flexcell-5000T体外细胞张应变加载系统,对大鼠原代培养的 VSMCs 施加10%幅度、1.25 Hz 频率的周期性张应变,模拟VSMCs在体内的生理性力学环境;以未加载周期性张应变的静态细胞为对照组,Western blotting 检测 VSMCs的 p-AMPK蛋白表达;划痕实验检测 VSMCs 迁移功能。结果 与静态组的细胞相比,生理性周期性张应变加载24 h后显著减少划痕愈合面积,提示生理性周期性张应变抑制VSMCs迁移;生理性周期性张应变加载3 h后,VSMCs的p-AMPK蛋白表达显著升高,而加载24 h后p-AMPK蛋白表达显著降低。在生理性周期性张应变加载条件下,孵育AMPK抑制剂可以在张应变加载3 h后显著降低 p-AMPK蛋白表达,而在张应变加载24 h后显著促进VSMCs迁移;在静态条件下孵育AMPK激活剂 AICAR 3 h后显著诱导p-AMPK蛋白表达,孵育24 h后显著抑制VSMCs迁移;提示p-AMPK蛋白表达参与调控VSMCs迁移。结论 生理性周期性张应变能通过激活p-AMPK蛋白表达,进而抑制VSMCs迁移,提示生理性周期性张应变调控VSMCs迁移对维持血管稳态具有重要意义。     

ROCK1及其相关信号分子参与张应变调控的血管平滑肌细胞增殖

目的探讨Rho相关卷曲蛋白激酶1(Rho-associated coiled-coil containing protein kinase 1,ROCK1)及其相关信号分子在感受张应变机械刺激、调控血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖功能中的作用。方法应用张应变加载系统对体外培养VSMCs施加牵张幅度10%、频率1.25 Hz生理性周向张应变;Brdu检测VSMCs增殖水平;Western blotting检测力学加载后VSMCs的ROCK1表达水平以及蛋白激酶C(protein kinase C,PKC)α/βII、蛋白激酶D(protein kinase D,PKD)、胞外信号调节激酶(extracellular regulated protein kinase,ERK)磷酸化水平;采用RNA干扰技术(RNA interference,RNAi)检测ROCK1对VSMC增殖和PKCα/βII、PKD、ERK磷酸化的调控作用。结果 10%生理性张应变加载12、24 h显著抑制VSMCs的ROCK1表达,并显著抑制PKD和ERK的磷酸化;10%生理性张应变加载12 h显著抑制PKCα/βⅡ的磷酸化,但加载24 h PKCα/βⅡ的磷酸化与静止对照组相比无显著差异。RNAi抑制VSMCs的ROCK1表达后,VSMCs增殖水平显著降低,同时PKCα/βⅡ和PKD磷酸化水平显著降低,但ERK磷酸化无明显变化。结论 10%生理性张应变可能通过抑制ROCK1表达调控PKCα/βⅡ和PKD的磷酸化水平,从而影响VSMCs增殖,维持血管稳定性。探讨张应变力学刺激调控血管细胞功能的细胞内信号转导网络,对心血管生理和疾病病理机制研究具有一定意义。     

生理性张应变通过增加核骨架蛋白Emerin表达抑制血管平滑肌细胞凋亡

目的探讨细胞核骨架蛋白Emerin及其调控的转录因子在感受张应变力学刺激影响血管平滑肌细胞(vascular smooth muscle cells,VSMCs)凋亡中的作用。方法应用FX-5000T张应变加载系统,对体外培养的VSMCs施加5%幅度、1.25 Hz频率生理性张应变,以静止组为对照;应用cleaved-caspase3 ELISA试剂盒检测VSMCs凋亡水平,Western blotting检测VSMCs细胞核骨架蛋白Emerin蛋白表达水平。静态条件下,RNA干扰抑制VSMCs的Emerin表达,Protein/DNA芯片检测345种转录因子活性;将特异性干扰Emerin后活性发生明显变化(上调或下调超过2倍)的转录因子进行IPA(Ingenurity Pathway Analysis)信息学分析,筛选与凋亡功能相关的转录因子;染色质免疫共沉淀(chromatin immunoprecipitation,CHIP)结合q PCR检测特异性干扰Emerin对其与两种转录因子motif区域结合能力的影响。结果与静止组相比,5%生理性张应变加载24 h后,VSMCs凋亡水平显著降低,提示生理性张应变对细胞具有保护作用;5%张应变作用6、12和24 h均显著增加VSMCs的Emerin表达水平。静态条件下RNA干扰抑制Emerin表达,VSMCs凋亡水平显著增加,且10种参与细胞凋亡功能调控的转录因子活性显著上调(2倍以上),包括CREB-BP1、p300、p55、MAX、NRF-1、STAT1、STAT3、TEF1、TR和BZP;CHIP-q PCR结果显示,Emerin特异性干扰可以显著降低Emerin与STAT家族的两个成员STAT1和STAT3的motif区域结合能力。结论生理性张应变可能通过增加核骨架蛋白Emerin表达而调控Emerin与STAT1、STAT3等多种凋亡相关转录因子motif区域结合,进而调控转录因子活性影响VSMCs凋亡。探讨张应变力学刺激调控VSMCs功能的力学生物学分子机制,对揭示血管生理稳态维持和血管病理重建的分子机制具有一定意义。     

周期性高张应变通过下调 PGC1α 表达抑制血管平滑肌细胞线粒体生物发生

目的 探讨高血压背景下病理性高张应变对血管平滑肌细胞(vascular smooth muscle cells, VSMCs)线粒体生物发生的影响,以及PGC1α蛋白在这一过程中的作用。方法 采用Flexcell-5000T体外细胞张应变加载系统对VSMCs施加频率为1.25 Hz、幅度分别为5%和15%的周期性张应变,模拟正常生理情况和高血压病理情况下的力学环境;通过蛋白免疫印迹(Western blotting)和荧光定量PCR(qPCR)方法检测正常生理和高血压病理力学条件下VSMCs的PGC1α蛋白表达,以及柠檬酸合酶和线粒体DNA(mitochondrial DNA,mtDNA)拷贝数变化情况;应用PGC1α特异性激活剂ZLN005和有效干扰片段siRNA检测PGC1α表达上调或下调对柠檬酸合酶和mtDNA拷贝数的影响。结果 与5%生理性周期性张应变相比,15%病理性高张应变显著抑制VSMCs的PGC1α和柠檬酸合酶的表达,mtDNA拷贝数显著降低。与对照组相比,对VSMCs转染PGC1α干扰片段siRNA或孵育PGC1α特异性激活剂ZLN005,分别下调和上调PGC1α蛋白...     

钙离子参与周期性高张应变和血小板源性微囊协同诱导的血管平滑肌细胞迁移

目的 探究周期性高张应变和血小板源性微囊(platelet-derived microvesicles,PMVs)对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)迁移功能的影响,以及Ca2+在其中的作用.方法 应用FX-5000T应变加载系统对体外培养VSMCs施加5％、15％幅...     

周期性张应变调控血管平滑肌细胞黏附血小板微体及其在自噬中的作用

目的探讨周期性张应变力学刺激对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)与血小板微体(platelet-derived microparticles,PMPs)黏附能力的影响,以及黏附的PMPs对VSMCs自噬的调控作用。方法应用FX-5000T张应变加载系统,对体外培养VSMCs施加5%幅度的生理性张应变和15%幅度的高张应变;应用流式细胞术检测不同张应变作用的VSMCs与PMPs的黏附;免疫荧光检测PMPs刺激24 h后自噬标志分子微管相关蛋白轻链3(autophagy microtubule associated protein light chain 3,LC3)的表达水平; Western blotting检测PMPs刺激24 h后VSMCs自噬相关蛋白(autophagy related protein,Atg)的表达水平。结果与5%生理性张应变加载相比,15%高张应变加载24 h能显著增强VSMCs与PMPs的黏附水平,提示高张应变促进PMPs与VSMCs的黏附。免疫荧光和Western blotting结果显示,PMPs刺激可显著上升VSMCs中自噬标志蛋白LC3表达,同时Western blotting检测到PMPs刺激后Atg5、Atg7、Atg12蛋白表达水平显著上升。结论高张应变可以促进VSMCs黏附PMPs,黏附的PMPs可能通过增加Atg5、Atg7、Atg12、LC3表达,从而增强VSMCs自噬。     

活化激酶C受体1在切应力调控血管平滑肌细胞增殖中的作用

目的 探讨活化激酶C受体1（receptor for actived C kinase 1, RACK1）在内皮细胞（endothelial cells, ECs）感受切应力刺激调控血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖中的作用及其机制。方法 应用平行平板流动腔系统,对联合培养的大鼠ECs和VSMCs施加1.5 Pa正常切应力（normal shear stress, NSS）和0.5 Pa低切应力（low shear stress,LowSS）,应用BrdU ELISA方法检测VSMCs增殖水平,对蛋白质组学研究发现的力学响应分子RACK1表达以及Akt磷酸化,应用Western blot技术进行检测。静态条件下,应用RNA干扰技术特异性抑制VSMCs的RACK1表达,检测其对细胞增殖和Akt磷酸化的作用。应用ECs与VSMCs隔开培养和联合培养模型,检测ECs对VSMCs的RACK1表达和Akt磷酸化水平的影响。结果 血管差异蛋白质组学的结果发现,与NSS组相比,RACK1在LowSS组血管组织的表达水平明显升高。细胞实验结果显示,LowSS诱导了与ECs联合培养的VSMCs增殖,上调VSMCs的RACK1表达和Akt磷酸化。静态条件下,特异性抑制VSMCs的RACK1表达后,VSMCs的增殖水平和Akt磷酸化水平均显著下降。与ECs联合培养VSMCs,其RACK1表达和Akt磷酸化水平较隔开培养组均上调。结论 VSMCs的RACK1表达受细胞接触与切应力的影响,并可能通过PI3K/Akt信号通路参与LowSS诱导的VSMCs增殖的调控。探讨VSMCs增殖功能变化及其力学生物学机制对于认识动脉粥样硬化等疾病发病机理和疾病防治有重要意义。     

Vascular damages in rats immunized by alpha1-adrenoceptor peptides

Autoantibodies against the α1-adrenoceptor which had agonist activity as norepinephrine might play roles in the progression of hypertension, but whether the autoantibodies could induce vascular remodeling as norepinephrine is not clear. In this paper, the models with antibodies against the α1-adrenoceptor were made by immunizing Wistar rats with the synthesized the second extracellular loop of α1-adrenoceptor peptides. The homo-age male Wistar rats received BSA in the same immunizing manner and male spontaneous hypertensive rats （SHR） were used as control. All the rats were raised for one year. The blood pressure and morphological changes of arteries were measured. In the end, despite the systolic blood pressure of immunized rats had no difference with normal control, the media thickness of aortas and ratio of media to lumen in the third-order arteries of mesenteric vasculature were increased in immunized rats. The observation with electron microscope showed that the mitochondria of vascular smooth muscle cells （VSMCs） had notable hyperplasia, and the interstitial collagen fibril was increased too. The effects of purified antibodies against α1-adrenoceptor on the proliferation of cultured VSMCs, and the expressions of c-jun, c-fos and α1-adrenoceptor were detected. The results showed that the antibodies could promote the proliferation of cultured VSMCs, and enhance the expression of c-jun both in vitro and in vivo. So we concluded that antibodies against the α1-adrenoceptor could contribute to vascular damages in rats by stimulating the growth of VSMCs which might be caused by the increased c-jun expression, and might play particular roles in the pathological changes of hypertension.     

降钙素基因相关肽对血管平滑肌表型转化和增殖的影响

目的 探讨降钙素基因相关肽(CGRP)对大鼠血管平滑肌细胞(VSMCs)表型转化和增殖的影响以及它们之间的关系.方法 取大鼠主动脉先体外培养8d后再贴块培养(VSMCs);对照组直接用贴块法培养细胞.实验分为CGRP作用组和无CGRP作用组.用5-BrdU标记平滑肌细胞增殖变化;RT-PCR检测HRG-1和SM22α表达变化.结果 血管经体外培养8d后再贴壁培养的平滑肌细胞可见大量棕黄色标记增殖的细胞核,HRG-1和SM22α mRNA表达明显减少;而CGRP作用组标记的血管平滑肌增殖细胞明显减少,HRG-1和SM22α mRNA表达明显上调.结论 CGRP对VSMCs增殖有抑制作用并同时可使VSMCs从合成型向收缩型转化.     

A novel cultured tissue model of rat aorta: VSMC proliferation mechanism in relationship to atherosclerosis

Development of a cultured tissue experimental model of rat aorta was explored in order to study mechanism of vascular smooth muscle (VSMC) proliferation. This particular model has potential with regard to amelioration of atherosclerosis and other vascular diseases in comparison to whole animal and cell culture models. The aorta segments of rats were divided into 4 experimental groups: the injured endothelium, injured endothelium plus BQ123, without injured endothelium and without injured endothelium plus BQ123. Each of group was subdivided into a further 2 subgroups and cultured with 20% serum and with serum-free DMEM. Each group cultured in vitro for 5, 8 and 13 days respectively. The control group was not cultured in vitro. Bromodeoxyuridine (BrDU 8x10(-4) mol/l) was added into the cultured medium of all groups, 24 h prior to harvesting. These segments were fixed in 4% paraformaldehyde for paraffin slice used to HE and immunocytochemical staining and other aorta segments were used to detect the expressions of hypertension-related gene-1 (HRG-1) and smooth muscle 22 alpha (SM22alpha) by RT-PCR. ET-1 content in the supernatant was detected with radioimmunology. Proliferous VSMC can be observed on artery segments cultured in vitro, and conspicuous plaques were developed on model vascular wall cultured for 13 days. Labeled cells increased with an increase in culture time but were not seen in the control group. A greater number of labeled cells were observed in injured endothelium group cultured in 20% serum DMEM. Hyperplasia was inhibited after BQ123 was added into the medium, suggesting that serum and ET-1 are important factors that lead to VSMC proliferation. Expressions of HRG-1 and SM22alpha were decreased while the aorta segments were cultured in vitro, minimum or even absent mRNA expressions of HRG-1 and SM22alpha were detected in injured endothelium cultured in 20% serum DMEM and increased in injured endothelium plus BQ123 group cultured. ET-1 content in the supernatant increased in injured endothelium cultured in 20% serum DMEM. These results show that the phenotypic transform and VSMC proliferation on cultured artery segments were related not only to serum culture, but also to ET-1 secreting. ET-1 and serum may be the main factors of contributing to the proliferation and phenotypic transform. This model provides a favorable experimental platform for research into the mechanism of vascular proliferous diseases as well as its prevention and treatment.