干细胞分化为平滑肌细胞参与血管病变

动脉粥样硬化,冠状动脉介入术后再狭窄以及器官移植相关的血管病变,以增生内膜平滑肌细胞增殖,分泌细胞外基质为主要特点。传统观点认为增生内膜的平滑肌细胞系血管中层平滑肌细胞向内膜迁移,增殖的结果。现代认为干细胞能分化为血管平滑肌细胞,参与上述疾病的发生、发展。     

干扰SET8调节血管平滑肌细胞增殖、凋亡促进血管钙化

背景与目的血管平滑肌细胞(VSMC)凋亡在许多血管钙化疾病的病理过程中起关键作用。近期研究表明,赖氨酸甲基转移酶SET8参与调节细胞增殖、凋亡过程。文章旨在探寻SET8是否通过调节VSMC增殖、凋亡而影响血管钙化。方法体外培养原代大鼠VSMC,将VSMC随机分为正常对照组、空质粒组和SET8-shRNA组。以脂质体Lipofectamine~(TM)2000为载体,转染VSMC。采用茜素红染色法和钙含量测定法判断细胞钙化程度;采用MTT法检测三组细胞的增殖能力;实时荧光定量PCR、Western blot法检测三组细胞中增殖基因Survivin和凋亡基因Caspase-3的表达水平,分析干扰SET8对VSMC增殖、凋亡及其相关基因和蛋白表达的影响。结果 (1)干扰SET8可成功抑制VSMC中SET8蛋白的表达(P0.05)。(2)转染VSMC后,与正常对照组和空质粒组比较,SET8-shRNA组钙含量明显升高(P0.05)。(3)MTT结果显示,在第12 h、24 h、36 h、48 h,SET8-shRNA组VSMC的增殖能力较正常对照组和空质粒组明显降低(P0.05)。(4)实时荧光定量PCR和Western blot结果显示SET8-shRNA组Survivin的mRNA和蛋白表达较正常对照组和空质粒组明显下降,而Caspase-3的mRNA和蛋白表达则相反(P0.05)。结论干扰SET8能够增加促凋亡基因表达,抑制增殖基因表达,SET8有可能参与调节大鼠血管钙化。     

miR-181a-5p对人主动脉平滑肌细胞成骨样分化的影响

目的 观察miR-181a-5p对动脉钙化细胞模型的影响,探讨其可能的作用机制.方法 培养人主动脉平滑肌细胞(HASMC),经成骨样分化诱导后使用茜红素染色检测细胞成骨样分化的程度,采用real-time PCR检测成骨样分化标志基因骨钙素(OCN)、Runt相关转录因子2(RUNX2)和碱性磷酸酶(ALP)的mRNA...     

葛根素对血管平滑肌细胞增殖及增殖细胞核抗原和凋亡抑制蛋白表达的影响

目的:通过观察葛根素对培养的脐动脉平滑肌细胞增殖细胞核抗原(PCNA)和凋亡抑制蛋白(Survivin)表达的影响,探讨葛根素抑制平滑肌细胞增殖的机制。方法:分离培养人脐动脉平滑肌细胞,加入不同浓度葛根素与细胞孵育,采用免疫组化法检测血管平滑肌细胞的PCNA表达;提取RNA,采用Rt-PCR检测Survivin表达水平,并经核苷酸序列分析验证,灰度扫描测定Survivin/GAPDH(s/G)比率。结果:葛根素抑制血管平滑肌细胞的PCNA表达(P=0.0000);葛根素组Survivin/磷酸甘油醛脱氢酶(GAPDH)(s/G)比率较对照组低。结论:葛根素具有抑制血管平滑肌细胞增殖的作用。     

老年大鼠血管平滑肌细胞对氧化应激反应的研究

目的观察老年大鼠血管平滑肌细胞（VSMC）对氧化应激损伤的反应。方法贴块法培养雄性Wistar大鼠胸主动脉VSMC，采用50umol／L H2O2处理细胞6h形成氧化应激，比较老年组和成年组VSMC对氧化应激反应的差异。RT—PCR法检测单核细胞趋化因子-1（MCP-1）mRNA的表达，比色法测定细胞丙二醛（MDA）含量。结果给予H2O2氧化应激前，老年组VSMC的MCP-1mRNA表达及MDA含量无显著差异（P〉0．05）；给予H2O2氧化应激后，老年组VSMC的MCP-1mRNA表达及细胞MDA含量的显著高于成年组VSMC。结论老年大鼠VSMC对氧化应激有着更强烈的反应，这可能是老年期易患动脉粥样硬化性血管疾病的重要机制之一。     

雌激素对血管平滑肌细胞凋亡及c-myc基因表达的影响

目的探讨17β雌二醇(E2)对血管平滑肌细胞(VSMC)凋亡的影响及其可能机制.方法应用流式细胞仪检测低、高浓度17βE2(10、100 nmol/L)对传代的VSMC凋亡率的影响,并通过细胞免疫组化方法观察对凋亡相关基因c-myc表达的影响.结果17β E2作用下VSMC凋亡率显著高于对照组(P＜0.01),伴随着VSMC凋亡率的增加,c-myc蛋白的表达亦增强,且表现明显的浓度依赖性.结论17β E2具有诱导VSMC凋亡的作用,其部分机制可能是通过上调促凋亡基因c-myc的表达有关.     

同型半胱氨酸对人血管平滑肌细胞凋亡及死亡受体表达的研究

目的探讨同型半胱氨酸（Hcy）对体外培养的人血管平滑肌细胞（VSMC）凋亡的影响，以及Hcy诱导人VSMC凋亡的机制。方法体外培养人VSMC，给予Hcy干预，然后用流式细胞仪检测细胞的凋亡率；同时做免疫组化，检测细胞死亡受体（Fas和TNFR-1）及其相关蛋白（Caspase-8和NF—KB）的表达。结果给予Hcy干预后，其凋亡率均高于对照组（P〈0．01）；凋亡率与Hcy干预浓度和干预时间之间直线相关关系存在（P〈0．01），且相关性良好（r分别为0．998、0．999）；其死亡受体和相关蛋白与对照组相比均有不同程度的表达。结论Hcy可诱导人VSMC的凋亡，而且这一效应呈浓度和时间依赖性，其致人VSMC凋亡的机制之-可能是通过死亡受体通路（TNFR-1和Fas）激活胞浆内的Caspase-8和NF—kB介导的。     

HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro

Objectives

Vascular calcification is a major clinical problem and elucidating the underlying mechanism is important to improve the prognosis of patients with cardiovascular disease. We aimed to elucidate the role and mechanism of action of Hepatocyte Growth Factor (HGF)/c-Met signalling in vascular calcification and establish whether blocking this pathway could prevent mineralisation of vascular smooth muscle cells (VSMCs) in vitro.

Methods and results

We demonstrate increased HGF secretion and c-Met up-regulation and phosphorylation during VSMC osteogenic differentiation. Adenoviral-mediated over-expression of HGF (AdHGF) in VSMCs accelerated mineralisation, shown by alizarin red staining, and significantly increased ⁴⁵Calcium incorporation (1.96 ± 0.54-fold [P < 0.05]) and alkaline phosphatase (ALP) activity (3.01 ± 0.8-fold [P < 0.05]) compared to controls. AdHGF also significantly elevated mRNA expression of bone-related proteins, Runx2, osteocalcin, BMP2 and osterix in VSMCs. AdHGF-accelerated mineralisation correlated with increased Akt phosphorylation, nuclear translocation of Notch3 intracellular domain (N3IC) and up-regulation of the Notch3 target protein, HES1. In contrast, adenoviral-mediated over-expression of the HGF antagonist, NK4, markedly attenuated VSMC mineralisation, and reduced c-Met phosphorylation, Akt activation and HES1 protein expression compared to AdHGF-treated cells. Furthermore, the Notch inhibitor, DAPT, attenuated N3IC nuclear translocation and AdHGF-induced mineralisation.

Conclusion

We demonstrate HGF induces VSMC osteogenic differentiation via c-Met/Akt/Notch3 signalling, highlighting these pathways as potential targets for intervention of vascular calcification.     

果蝇样受体4介导游离脂肪酸调节人血管平滑肌细胞单核细胞趋化蛋白-1的表达

目的探讨软脂酸对人主动脉血管平滑肌细胞（HA—VSMC）单核细胞趋化蛋白-1（MCP-1）基因表达的调节及果蝇样受体4（TLR4）信号通路的作用。方法采用不同剂量的软脂酸（100、200、400μmol/L）处理HA—VSMC6、12、24h,采用实时荧光定量聚合酶链反应（PCR）检测细胞MCP．1mRNA表达,酶联免疫吸附实验（ELISA）检测MCP-1蛋白表达,观察软脂酸调节MCP-1表达的剂量依赖关系和时间效应;采用蛋白激酶C（PKC）抑制剂白屈菜红碱、磷脂酰肌醇3激酶（P13K）抑制剂wortmannin、神经酰胺抑制剂myriocin和核因子KB（NF—KB）抑制剂parthenolide分别处理细胞30min,再加入软脂酸（400μmol/L）处理细胞6h后,实时荧光定量PCR检测MCP-1mRNA的表达水平,ELISA检测MCP-1的蛋白表达水平,研究软脂酸调节MCP-1表达依赖的信号通路;构建TLR4短发夹RNA（shRNA）腺病毒pGSadeno-TLR4并感染HA—VSMC沉默TLR4基因表达,实验同时设空白对照组（PBS缓冲液）和对照病毒（pGSadeno-GFP）组。细胞感染96h后更换培养基,再加入软脂酸（400μmoL／L）处理细胞6h,采用实时荧光定量PCR检测MCP-1mRNA表达水平,ELISA检测MCP-1的蛋白表达水平。提取细胞核蛋白,采用ELISA检测NF—KBp65亚基活性,观察TLR4／NF-KB通路在软脂酸调节HA-VSMCMCP-1基因表达中的作用。组间均数比较采用单因素方差分析。结果采用软脂酸处理细胞6h后,对照组及100、200和400μmoVL软脂酸组MCP-1mRNA表达分别为1．00±0．02、3．30±2．84、8．21±4．31和11．25±2．73（F=7．57,P〈0．05）;MCP-1蛋白表达分别为（127±10）、（147±10）、（163±18）和（194±14）ng／L（F=7．81,P〈0．05）。软脂酸可促进HA-VSMCMCP-1mRNA和蛋白表达且具有明显的剂量依赖关系。细胞培养12h和24h后,随着软脂酸浓度的增加,MCP-1mRNA和蛋白表达水平呈逐渐增加趋势,但时间效应则不明显。采用不同的信号通路抑制剂和软脂酸处理细胞6h后,对照组、软脂酸组、软脂酸＋parthenolide组、软脂酸＋白屈菜红碱组、软脂酸＋wortmannin组和软脂酸＋myriocin组MCP-1mRNA表达分别为1．00±0．02、10．80±1．23、3．49±0．28、10．84±0．24、11．24±0．27和10．62±0．36（F=1313．07,P〈0．05）;MCP-1蛋白表达分别为（132±8）、（218±12）、（152±4）、（213±12）、（225±7）和（226±9）ng／L（F=106．83,P〈0．05）。成功地构建并获得TLR4shRNA腺病毒pGSadeno—TLR4,采用pGSadeno-TLR4感染HA—VSMC阻断TLR4信号后,软脂酸＋pGSadeno．TLR4组的NF—KB065结合活性、MCP-1mRNA和蛋白表达均显著低于软脂酸组[分别为0．48±0．12比1．24±0．16、1．88±0．33比10．80±1．23、（154±10）比（218±12）ng／L;F=591．86、659．16、118．37,均P〈0．01]。而对照病毒pGSadeno．GFP对软脂酸诱导的NF—KB065结合活性和MCP：I表达均无明显影响。结论TLR4／NF—KB信号通路介导了软脂酸诱导的人主动脉血管平滑肌细胞MCP-1基因表达。     

雌激素抑制血管平滑肌细胞增殖和胶原合成的实验研究

目的　探讨雌激素对于胎儿血管平滑肌细胞 (HVSMC)增殖和胶原合成的影响。方法　分别以不同浓度 (0 .1nmol/ L～ 10 0 nm ol/ L)的雌二醇作用于体外培养的 HVSMC,利用 3H-脯氨酸掺入法测定培养平滑肌细胞胶原合成及 3H- Td R掺入法测定细胞增殖率。结果　 1.0 nm ol/ L～ 10 0 nmol/ L 雌激素以剂量依赖性方式抑制 10 %胎牛血清诱导 HVSMC增殖和胶原合成。结论　小剂量雌二醇具有抗动脉粥样硬化作用 ,而超生理剂量雌二醇可能诱发血管损伤     

大鼠血管平滑肌细胞中以COX-2/JAK2/STAT3为靶向的信号转导分子调控机制的研究

目的:研究转录信号转导子和激活子3(STAT3)信号转导通路与选择性环氧化酶2(COX-2)抑制剂调控血管平滑肌细胞(VSMC)增殖凋亡的关系,明确COX-2抑制剂作用的细胞内信号转导机制。方法:将选择性COX-2抑制剂NS-398,作用于鼠VSMCs,运用MTT法检测细胞增殖状态;流式细胞仪观察NS-398对细胞凋亡的影响,进一步用RT-PCR检测药物作用前后VSMCs中COX-2mRNA表达;Westernblot检测药物作用前后STAT3通路相关蛋白JAK2、STAT3、CyclinD1、Bcl-2的表达及磷酸化活性。结果:鼠VSMCs中COX-2mRNA呈高表达,NS-398呈时间、剂量依赖性方式抑制VSMCs细胞增殖,促进其凋亡。加入NS-398的VSMCs中COX-2mRNA表达水平显著下降(P<0·01)。同时P-JAK2、P-STAT3、CyclinD1、Bcl-2表达水平随作用时间延长而下降(P<0·01)。结论:选择性COX-2抑制剂NS-398对VSMCs作用与COX-2mRNA水平相关,癌基因Stat3信号转导通路调控了NS-398对VSMCs作用的细胞内信号转导机制,最终通过其下游靶基因CyclinD1、Bcl-2影响VSMCs的增殖与凋亡。     

Gas6 inhibits apoptosis in vascular smooth muscle: role of Axl kinase and Akt

Axl is a receptor tyrosine kinase originally identified as a transforming gene product in human myeloid leukemia cells. Previously, we showed that Axl expression correlated with neointima formation in balloon-injured rat carotid, and that Axl expression was highly regulated by angiotensin II. In the present study we tested the mechanisms by which Axl regulates vascular smooth muscle cell (VSMC) growth focusing on its ability to inhibit apoptosis. Treatment of cultured rat aortic VSMC for 24 h with 0% serum resulted in 19.8 +/- 1.4% apoptotic cells. Treatment of VSMC with 100 ng/ml Gas6 (the putative ligand for Axl) decreased apoptosis to 8.9 +/- 0.7% (P = 0.002, N = 17) as compared to a decrease with 10% serum to 3.0 +/- 0.2% (P = 0.001, N = 17). The ability of Gas6 to prevent apoptosis required both Gas6 binding to Axl and Axl kinase activity since treatment with a soluble, competitive Axl extracellular domain protein or transfection of a kinase inactive mutant (Axl-K567R) completely prevented the anti-apoptotic effect. Prevention of apoptosis by Gas6-Axl required activation of phosphatidyl inositol 3-kinase (PI3K) as shown by treatment with LY294002 or transfection of an Axl deletion mutant that does not bind PI3K (Axl- triangle up PI3K). There was no significant role for ERK1/2 in the anti-apoptotic effects of Gas6-Axl since ERK1/2 activity was maintained in cells transfected with Axl- triangle up PI3K and Axl-K567R. These findings establish the Gas6-Axl-PI3K-Akt pathway as an anti-apoptotic mechanism for VSMC that may be important in the response to vascular injury.     

槲皮素及异鼠李素对人血管平滑肌细胞增殖的抑制作用

目的观察槲皮素(QUE)及异鼠李素(ISOR)对培养人血管平滑肌细胞增殖的影响。方法本实验利用培养的人主动脉平滑肌细胞(VSMC),采用细胞计数、3H-胸腺嘧啶核苷酸(3H-TdR)掺入法,观察QUE、ISOR、去甲肾上腺素(NE)对VSMC增殖、DNA合成的影响,以及QUE、ISOR、酚托拉明(Ph)对NE促VSMC增殖、DNA合成的的影响。结果①QUE有明显抑制VSMC增殖、DNA合成的作用,而ISOR作用较弱。在所观察的1～200(滋mol/L的浓度范围内,QUE和ISOR均在200滋mol/L浓度发挥了最大的抑制作用,其抑制作用有剂量依赖关系。②NE有促进VSMC增殖、DNA合成的作用,而这些促进作用能被Ph所阻滞。③QUE和ISOR均剂量依赖地抑制NE促VSMC增殖和DNA合成的作用,且QUE和ISOR有明显的协同作用。④QUE和ISOR对NE刺激作用的抑制明显强于Ph的作用。⑤QUE和ISOR对VSMC无细胞毒作用。结论QUE和ISOR是细胞毒性很低,对VSMC的增殖、DNA合成尤其是对NE刺激的VSMC增殖、DNA合成有很强的抑制作用的天然黄酮类物质。     

p53 negatively regulates the osteogenic differentiation of vascular smooth muscle cells in mice with chronic kidney disease

Aim

To investigate the osteogenic differentiation of vascular smooth muscle cells (VSMCs) in mice with chronic kidney disease (CKD) and to evaluate the effects of p53 on the osteogenic differentiation of the VSMCs.

Methods

Experimental models of CKD-associated vascular calcification generated by five-sixth (5/6) nephrectomy (Nx) and a high-phosphate (HP) diet were used in p53+/+ and p53–/– mice. Following 5/6 Nx, aortic calcification, markers of osteogenic differentiation, VSMCs and p53 protein in aortic tissues were studied.

Results

Aortic calcification was observed after eight weeks following 5/6 Nx in mice of both genotypes, and expression of the markers of osteogenic differentiation in the VSMCs was increased. These changes were continuously observed up to 12 weeks after 5/6 Nx, and particularly after 5/6 Nx + HP. Compared with p53+/+ mice, aortic calcification in p53–/– mice was more severe (p < 0.001). Expression of the markers of osteogenic differentiation was noticeably increased (p < 0.001), while expression of the marker of VSMCs had decreased (p < 0.001). Statistical analysis demonstrated that the markers of osteogenic differentiation were negatively correlated with p53, and the marker of VSMCs was positively correlated with p53 (p < 0.001).

Conclusion

p53 has the potential to negatively regulate the osteogenic differentiation of VSMCs in CKD mice.     

胰岛素对猪主动脉平滑肌细胞的增殖和前列环素合成的影响

以体外培养的猪主动脉平滑肌细胞为实验模型，研究了不同浓度胰岛素对其细胞增殖、前列环素合成的影响。结果显示胰岛素抑制平滑肌细胞的前列环素合成、促进细胞增殖都呈现剂量依赖性，提示高胰岛素血症可能是致动脉粥样样硬化的重要因素之一。     

增龄和高血压经PI3K/Akt和MAPK信号通路间平衡介导调控血管平滑肌细胞的表型转换

目的探究增龄和高血压对胸主动脉血管平滑肌细胞(VSMC)表型转换的影响及磷脂酰肌醇激酶(PI3K/Akt)和丝裂原活化蛋白激酶(MAPK)信号通路对VSMC表型的调控。方法选取1、3、9、16月龄的雄性Wistar Kyoto大鼠(WKY)和自发性高血压大鼠(SHR)各12只,尾动脉无创测定血压,取各组大鼠的胸主动脉进行实验。HE染色测量胸主动脉的管壁厚度;免疫组织化学染色检测VSMC表型标志蛋白α平滑肌肌动蛋白(α-SMactin)、调宁蛋白、骨桥蛋白(OPN)的表达和分布;Western blot检测VSMC表型标志蛋白α-SM-actin、调宁蛋白、OPN及信号蛋白p-Akt、内皮型一氧化氮合酶(eNOS)、p-42/44ERK、p-p38MAPK的表达量。结果 HE染色结果显示,增龄导致管壁厚度增加,且在9月龄时WKY和SHR出现显著差异(P0.01)。免疫组织化学染色和Western blot结果表明,3月龄后,WKY和SHR的α-SM-actin、调宁蛋白表达量随月龄增加出现下调,而OPN出现上调;p-Akt、eNOS的蛋白表达量随月龄增加逐渐下调,p-42/44ERK、p-p38MAPK蛋白表达量随月龄增长逐渐上调,且3月龄时两组已有显著差异(P0.01)。结论增龄和高血压均导致大鼠胸主动脉VSMC收缩表型标志蛋白α-SM-actin、调宁蛋白的表达下调,合成表型标志蛋白OPN的表达上调,二者的交互作用更显著。VSMC的表型转换可能是通过PI3K/Akt和MAPK信号通路间的平衡作用进行调控的。     

Endothelin-induced changes in intracellular calcium concentration in rat vascular smooth muscle cells: Effects of extracellular calcium and atrial natriuretic factor

Summary The purpose of this study was to examine whether different mechanisms might underlie the changes in intracellular calcium concentration ([Ca²⁺]_i) stimulated by high and low concentrations of endothelin, and whether atrial natriuretic factor (ANF) has an inhibitory effect on endothelin-induced [Ca²⁺]_i changes in cultured rat vascular smooth muscle cells (VSMCs). In calcium-replete buffer, cultured monolayers of rat VSMCs superfused with endothelin at a high concentration (10 nM) exhibited a marked transient rise in [Ca²⁺]_i, followed by a sustained elevation, whereas a low concentration of endothelin (0.1 nM) induced a sustained monophasic elevation. When calcium-free buffer was used, 10 nM endothelin induced a transient rise in [Ca²⁺]_i of lesser amplitude, whereas 0.1 nM endothelin did not produce a significant rise. Pretreatment of VSMCs with ANF and cosuperfusion with endothelin failed to inhibit either transient or sustained endothelin-induced changes in [Ca²⁺]_i in calcium-replete buffer.