低频电磁场对大鼠主动脉平滑肌细胞基质金属蛋白酶的影响

目的 观察不同磁场强度及作用时间的低频电磁场（LFEMF）对培养的大鼠主动脉血管平滑肌细胞（VSMC）表达基质金属蛋白酶-2（MMP-2）的影响。方法 用含10％小牛血清的DMEM培养液体外培养大鼠主动脉VSMC，随机分为对照组、LFEMF不同磁场强度（20，40，60mT）及时间（10，20，30min）干预组，运用MMP-2活性酶图分析法结合光密度扫描分析，观察LFEMF对VSMC的MMP-2表达的影响。结果 不同磁场强度组均明显抑制MMP-2的活性，且具有剂量依赖性，但抑制作用不随时间延长而变化，不具有时间依赖性。结论 适当强度和作用时间的LFEMF抑制VSMC的MMP-2活性的表达，磁场对经皮腔内冠状动脉成形术（PTCA）及支架植入术后的再狭窄（RS）可能具有防治作用。     

低频电磁场对培养的人脐静脉内皮细胞一氧化氮合酶的影响

目的：观察不同磁场强度,不同作用时间的低频电磁场（LFEMF）对培养的人脐静脉内皮细胞（HUVEC）一氧化氮合酶（NOS）活性表达的影响,以探讨LFEMF用于经皮冠脉腔内成形术（PTCA）及支架植入（IVS）术后冠脉再狭窄（RS）防治的意义。方法：分别用不同磁场强度,不同作用时间的LFEMF作用于体外培养人脐静脉内皮细胞系ECV304,行ABC免疫酶染色法,图像分析法分析LFEMF对HUVEC的NOS表达的影响。结果：除60mT20min组,60mT30min组外,其余各实验组内皮细胞结构型NOS（ecNOS)表达均较对照组增强,差异显著（P＜0．05）。所有实验组与对照组诱生型NOS（iNOS)均无表达。结论：LFEMF增强了培养的HUVEC的ecNOS的表达,可能适用于RS的防治。     

大剂量阿托伐他汀对大鼠主动脉平滑肌细胞基质金属蛋白酶-2的影响

目的研究不同剂量阿托伐他汀对培养的大鼠主动脉血管平滑肌细胞(VSMC)基质金属蛋白酶-2(MMP-2)活性的影响。方法用含10%小牛血清的DMEM培养液体外培养大鼠主动脉VSMC,随机分为对照组,不同剂量阿托伐他汀干预组,运用MMP-2活性酶图分析法结合光密度扫描分析,观察不同剂量阿托伐他汀对VSMC的MMP-2表达的影响。结果不同剂量阿托伐他汀组均明显抑制MMP-2的活性,且具有剂量依赖性。结论阿托伐他汀可以显著降低VSMC的MMP-2的表达,大剂量抑制作用更强。     

丹参酮ⅡA对AngⅡ诱导的大鼠血管平滑肌细胞增殖的影响

目的：探讨丹参酮ⅡA（TSN）对血管紧张素Ⅱ（AngⅡ）诱导的大鼠血管平滑肌细胞增殖的影响,以寻找药物作用的靶点。方法：分离大鼠胸主动脉中层平滑肌,贴壁法培养平滑肌细胞,建立AngⅡ诱导的血管平滑肌细胞（VSMC）增殖模型;以四甲基偶氮唑蓝（MTT）法观察丹参酮ⅡA对VSMC增殖的影响;用酶促反应定磷法测定钙调神经磷酸酶（CaN）活性;应用免疫细胞化学法观察原癌基因c-fos和c-myc的表达。结果：成功建立AngⅡ诱导的VSMC增殖模型;随着TSN浓度增加VSMC增殖活性呈明显下降趋势（P〈0.05,0.01）;TSN各组随着浓度的增加,VSMC中CaN活性显著下降（P〈0.01）,VSMC中c-fos和c-myc表达水平也显著下降（P〈0.01）。结论：TSN能抑制血管平滑肌细胞的增殖,并呈浓度依赖性。其机制可能与下调VSMC中CaN活性、c-fos及c-myc表达有关。     

雷公藤内酯醇抑制血管平滑肌细胞增殖的研究

目的:探讨雷公藤内酯醇对血清诱导的大鼠血管平滑肌细胞(VSMC)增生的影响及其作用机制。方法:体外培养大鼠胸主动脉平滑肌细胞,用细胞计数法观察雷公藤内酯醇对细胞增生的抑制作用,采用流式细胞仪进行细胞周期分析,逆转录-聚合酶链反应(RT-PCR)法检测细胞c-fos的mRNA表达水平。结果:雷公藤内酯醇明显抑制血清诱导的大鼠VSMC增生;阻断细胞周期中细胞由G0/G1期向S期转化;RT-PCR检测显示雷公藤内酯醇能明显抑制原癌基因c-fos的表达,其作用呈剂量依赖性。结论:雷公藤内酯醇可抑制血清诱导的大鼠VSMC增生,下调原癌基因c-fos的表达。     

低频电磁场对培养的人脐静脉内皮细胞一氧化氮合酶的影响

背景目前认为经磁场作用的酶,大多有增强活性的倾向,而关于低频电磁场(1owfrequency electromagneticfield,LFEMF)对培养的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)一氧化氮合酶(nitric oxide synthase,NOS)活性表达的影响研究不多见.目的探讨不同磁场强度,不同作用时间的LFEMF对HUVEC的NOS活性表达的影响,以探讨LFEMF用于经皮冠脉腔内成形术(percutanuoustransluminal coronary angioplasty,PTCA)及支架植入术后冠脉再狭窄防治的意义.设计随机对照的实验研究.地点、材料和干预本研究在解放军第四军医大学西京医院心内科实验室完成.体外培养的人脐静脉内皮细胞系ECV304按LFEMF的不同磁场强度,不同作用时间分为9个实验组和1个对照组进行干预.进行ABC免疫酶染色后图像分析法观察HUVEC的NOS表达.主要观察指标非磁场干预与不同强度磁场干预的体外培养的人脐静脉内皮细胞NOS结果.结果除60mT 20min组,60mT 30min组外,其余各实验组内皮细胞结构型NOS(Endothelium Constructive nitric oxide synthase,ecNOS)表达均较对照组增强,差异有显著性意义(P＜0.05).所有实验组与对照组诱生型NOS(Induced nitric oxide synthase,iNOS)均无表达.结论LFEMF增强了培养的HUVEC的ecNOS的表达,可能适用于再狭窄的防治.     

低频电磁场对培养的人脐静脉内皮细胞一氧化氮合酶的影响

背景：目前认为经磁场作用的酶，大多有增强活性的倾向，而关于低频电磁场(low frequency electromagnetic field，LFEMF)对培养的人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVEC)一氧化氮合酶(nitric oxide synthase，NOS)活性表达的影响研究不多见。目的：探讨不同磁场强度，不同作用时间的LFEMF对HUVEC的NOS活性表达的影响，以探讨LFEMF用于经皮冠脉腔内成形术(percutanuous transluminal coronary angioplasty，FFCA)及支架植入术后冠脉再狭窄防治的意义。设计：随机对照的实验研究。地点、材料和干预：本研究在解放军第四军医大学西京医院心内科实验室完成。体外培养的人脐静脉内皮细胞系ECV304按LFEMF的不同磁场强度，不同作用时间分为9个实验组和1个对照组进行干预：进行ABC免疫酶染色后图像分析法观察HUVEC的NOS表达。主要观察指标：非磁场干预与不同强度磁场干预的体外培养的人脐静脉内皮细胞NOS结果。结果：除60mT20min组，60mT30min组外，其余各实验组内皮细胞结构型NOS(Endothelium Constnmtive nitric oxide synthase，ecNOS)表达均较对照组增强，差异有显著性意义(P&;lt;0．05)。所有实验组与对照组诱生型NOS(Induced nitric oxide synthase，iNOS)均无表达。结论：LFEMF增强了培养的HUVEC的ecNOS的表达，可能适用于再狭窄的防治。     

恒磁场对大鼠主动脉平滑肌细胞骨桥蛋白基因表达的影响

背景:适当强度的恒磁场可抑制血管平滑肌细胞增殖,可能用于抑制经皮冠状动脉介入治疗术后再狭窄。目的:观察不同磁感应强度恒磁场对培养的大鼠主动脉血管平滑肌细胞骨桥蛋白基因表达的影响,以探讨磁场是否能用于经皮冠状动脉介入治疗术后再狭窄的防治。设计:随机分组,对照观察。单位:解放军第四军医大学西京医院心内科。材料:实验于2006-02/12在解放军第四军医大学西京医院心内科实验室(全军心血管病研究所)完成。纯种雄性SD大鼠,体质量200～250g。方法:用含体积分数为0.1小牛血清的DMEM培养液体外培养大鼠主动脉血管平滑肌细胞,实验随机分为对照组,1Gs恒磁场组、5Gs恒磁场组、10Gs恒磁场组、50Gs恒磁场组,其中对照组不予磁场干预,其他各组分别给予磁场干预继续培养48h。应用反转录-聚合酶链反应及蛋白免疫印迹技术结合吸光度扫描分析,观察恒磁场对血管平滑肌细胞骨桥蛋白及骨桥蛋白mRNA表达的影响。主要观察指标:各组血管平滑肌细胞骨桥蛋白及骨桥蛋白mRNA的表达。结果:对照组细胞表达一定量的骨桥蛋白及骨桥蛋白基因,各恒磁场组均较对照组降低,差异显著(P<0.05)。不同磁场强度组间分析显示,恒磁场的刺激作用具有磁场强度依赖性,随磁场强度加大,抑制骨桥蛋白及骨桥蛋白mRNA表达作用增强。结论:适当强度的恒磁场能在基因水平上抑制血管平滑肌细胞骨桥蛋白的表达,磁场对经皮冠状动脉介入治疗术后的再狭窄可能具有防治作用。     

bFGF和地塞米松对骨髓基质干细胞增殖与分化的影响

目的：探讨碱性成纤维生长因子（bFGF）和地塞米松（Dex）在体外培养条件下对SD大鼠骨髓基质干细胞（MSC）增殖与分化的影响。方法：用DMEM冲洗股骨、胫骨骨髓腔，收集骨髓细胞悬液，接种在塑料培养瓶内进行体外培养及扩增，并分别加入不同浓度bFGF和Dex，通过MTT法检测2者对MSC增殖的影响，通过RT-PCR技术测定碱性磷酸酶（ALP）、骨钙素（OCN）和骨桥蛋白（OPN）的表达，了解2者对MSC分化的影响。结果：bFGF对MSC增殖呈剂量依赖性，10ng／ml时促增殖最明显；Dex对骨髓基质细胞的增殖起抑制作用，并随Dex浓度的升高而增强。bFGF降低MSC的ALP活性及OCN、OPN的表达；而Dex以及2者联合应用则增加ALP活性，促进MSC的ALP、OCN及OPN表达增加。结论：bFGF促进MSC增殖、抑制其分化；Dex抑制MSC增殖，但可促进其分化，bFGF（10ng／m1）和Dex（10^-8mol／L）联合应用能有效的促进MSC的增殖和成骨分化。     

超声造影剂对血管平滑肌细胞增殖、迁移和凋亡的影响

目的探讨超声波联合超声造影剂对平滑肌细胞增殖、迁移和凋亡的影响.方法体外培养大鼠胸主动脉血管平滑肌细胞（VSMC）.采用MIT法、Millicell小室、Annexin V-FITC/PI双标染色和流式细胞仪,检测VSMC的增殖、迁移能力和凋亡,观察血小板衍生生长因子--BB（PDGF-BB）对VSMC增殖、迁移和凋亡的影响,频率1 MHz、声强0.3 W/cm^2的连续波超声联合声学造影剂辐照VSMC后上述指标的变化.结果PDGF-BB对VSMC的凋亡无影响,但可促进VSMC增殖和迁移.超声联合造影剂辐照VSMC 30 s即可显著抑制PDGF-BB所致的VSMC增殖（P〈0.05）,同时细胞凋亡比例显著增高（P〈0.01）,辐照60 s可显著抑制PDGF-BB所致的VSMC迁移（P〈0.05）,随着辐照时间延长,以上作用增强,辐照60 s时对VSMC增殖的抑制程度最强.结论低强度超声波辐照联合超声造影剂可抑制VSMC增殖、迁移,并促进细胞凋亡.     

50 Hz电磁场对小鼠骨髓间充质干细胞增殖的影响

目的　探讨小鼠骨髓间充质干细胞经脉冲电磁场辐射不同时间后其细胞增殖水平的变化。方法　用密度梯度离心法分离小鼠骨髓间充质干细胞 ,再经贴壁筛选法筛选 ,对生长良好的第 3代小鼠骨髓间充质干细胞进行不同时间的脉冲电磁场辐射 (频率 50Hz、正弦波形、不同强度 ) ,用MTT法、流式细胞仪法测定细胞生长增殖水平及细胞周期变化。结果　小鼠骨髓间充质干细胞经脉冲电磁场辐射 3d(每天 2次 ,每次 3 0min)后 ,各实验组细胞增殖水平均有明显提高 ,差异有显著性意义 (P <0 .0 5)。采用流式细胞仪测定细胞周期 ,发现各实验组 (S G2 /M )期细胞数量较对照组均有不同程度的增长 ,差异有显著性意义 (P <0 .0 5)。各组均未发现DNA倍体异常细胞。结论　小鼠骨髓间充质干细胞经频率为 50Hz、正弦波形、强度分别为 4mT ,3mT ,2mT ,1mT的脉冲电磁场辐射 3d(每天 2次 ,每次 3 0min ,间隔 12h)后 ,能明显促进该细胞体外培养的增殖水平     

Short-term vascular smooth muscle cells with platelet-derived growth factor (PDGF)-BB and angiotensin II induces stimulation of [3H]thymidine incorporation without mitosis

Growth factors such as the platelet-derived growth factor (PDGF)-BB and angiotensin II (Ang II) have been shown to induce vascular smooth muscle cell (VSMC) proliferation after long stimulation periods. Little is known though, about the effects of PDGF-BB and Ang II on VSMC proliferation after short stimulation periods. The purpose of our study was to examine whether a short term (3-60 min) stimulation of VSMC with PDGF-BB or Ang II is sufficient to induce cell proliferation. Incubation of VSMC with Ang II (100 nM) or PDGFBB (50 ng/ml) caused a significant increase in [3H]thymidine incorporation starting after a 3-min stimulation, while the cell counts required 32 and 8 h of stimulation, respectively. Mitogen-activated protein kinase activation reached a maximum at 5-10 min of PDGF-BB or Ang II stimulation. This study demonstrates that the growth-promoting effects of PDGF-BB and Ang II are strongly dependent on the length of the stimulation period and that while prolonged stimulation periods (&gt;8-32 h) result in VSMC proliferation, short ones (3-60 min) result only in [3H]thymidine incorporation without an increase in cell count, a fact of considerable pathophysiological significance, considering that the time kinetics of growth factors in the VSMC microenvironment have not as yet been clarified.     

Short-term stimulation of vascular smooth muscle cells with platelet-derived growth factor (PDGF)-BB and angiotensin II induces

Growth factors such as the platelet-derived growth factor (PDGF)-BB and angiotensin II (Ang II) have been shown to induce vascular smooth muscle cell (VSMC) proliferation after long stimulation periods. Little is known though, about the effects of PDGF-BB and Ang II on VSMC proliferation after short stimulation periods. The purpose of our study was to examine whether a short term (3-60 min) stimulation of VSMC with PDGF-BB or Ang II is sufficient to induce cell proliferation. Incubation of VSMC with Ang II (100 nM) or PDGF-BB (50 ng/ml) caused a significant increase in [3H]thymidine incorporation starting after a 3-min stimulation, while the cell counts required 32 and 8 h of stimulation, respectively. Mitogen-activated protein kinase activation reached a maximum at 5-10 min of PDGF-BB or Ang II stimulation. This study demonstrates that the growth-promoting effects of PDGF-BB and Ang II are strongly dependent on the length of the stimulation period and that while prolonged stimulation periods (>8-32 h) result in VSMC proliferation, short ones (3-60 min) result only in [3H]thymidine incorporation without an increase in cell count, a fact of considerable pathophysiological significance, considering that the time kinetics of growth factors in the VSMC microenvironment have not as yet been clarified.     

Autocrine and paracrine effects of atrial natriuretic peptide gene transfer on vascular smooth muscle and endothelial cellular growth.

In addition to the atria, recent evidence suggests that atrial natriuretic peptide (ANP) is also synthesized in other tissues. Of particular interest is the location of ANP mRNA in the vessel wall. We and others have shown that exogenously added ANP inhibited the growth of endothelial cells and vascular smooth muscle cells (VSMC) in culture. However, it is not known if the locally synthesized ANP would act similarly. Because cultured endothelial cells and VSMC have lost the ability to express the endogenous ANP gene, we have transfected cells in culture with an expression vector expressing rat ANP and have examined the effects on growth. Cultured endothelial cells transfected with an ANP expression vector synthesized and secreted high levels of ANP. These cells also showed significantly lower rates of DNA synthesis under basal and fibroblast growth factor (FGF)-stimulated conditions. Addition of conditioned medium from endothelial cells transfected with ANP vector to nontransfected endothelial cells resulted in the significant increase in cyclic GMP. Similarly, conditioned media collected from endothelial cells transfected with ANP vector also decreased DNA synthesis in VSMC. Coculture of ANP-transfected endothelial cells with quiescent VSMC showed that released ANP from endothelial cells inhibited DNA synthesis in VSMC. Finally, we examined the autocrine effect of direct transfection of ANP vector into VSMC. Transfection of the ANP vector decreased DNA synthesis in VSMC under basal and angiotensin II-stimulated conditions. Similarly, transfection of the ANP vector resulted in a decrease in the PDGF and serum (5%)-stimulated DNA synthesis of VSMC. These results demonstrate that endogenously produced ANP can exert autocrine and paracrine inhibitory effects on endothelial cell and VSMC growth. In vivo gene transfer of ANP may provide us with the opportunity of gene therapy for vascular diseases in which the abnormalities are vasoconstriction and pathological growth.     

miR-20a过表达促进VEGF表达及血管平滑肌细胞活性和迁移

目的探讨miR-20a过表达对血管内皮生长因子(VEGF)表达及血管平滑肌细胞(VSMC)活性和迁移的影响,并初步探索其可能的机制。方法将miR-20a mimics转染至VSMC,VSMC分为3组:空白对照(Con)组、阴性对照(miR-NC)组和转染(miR-20a)组。Real-time PCR检测miR-20a的表达变化,Real-time PCR和Western blot检测各组VSMC中VEGF的表达,细胞计数试剂盒(CCK-8)法检测各组VSMC活性变化,Transwell实验检测各组VSMC迁移能力,生物信息学和荧光素酶报告基因实验分析miR-20a对CNN1的靶向关系。结果与miR-NC组比较,转染miR-20a mimics后miR-NC组VSMC中miR-20a的表达明显升高,VEGF m RNA和蛋白表达明显上调,VSMC活性升高,VSMC迁移能力增强,差异均具有统计学意义(P<0.05)。与miR-NC组比较,Con组以上各指标差异均无统计学意义(P>0.05)。荧光素酶报告基因实验结果显示,miR-20a可显著抑制野生型钙调蛋白1(CNN1)相对荧光素酶活性(P<0.05),但对突变型CNN1相对荧光素酶活性无明显抑制作用(P>0.05)。结论过表达miR-20a能够促进VSMC活性和迁移以及VEGF的表达,其作用机制可能与靶向调控CNN1的表达有关。     

Adenovirus-mediated transfer of the atrial natriuretic peptide gene in rat pulmonary vascular smooth muscle cells leads to apoptosis

Atrial natriuretic peptide (ANP) exhibits relaxant and growth-inhibiting effects on vascular smooth muscle cells (VSMCs). To obtain ANP gene expression in VSMCs, we built a recombinant adenovirus containing the ANP cDNA controlled by the adenovirus major late promotor (AdMLP-ANP). After pulmonary VSMC treatment with AdMLP-ANP at a multiplicity of infection ranging from 5 to 100 TCID(50)/cell, immunoreactive ANP was detectable in the cell culture medium at a level that reached 101 +/- 27 pmol/well after 2 days. The newly expressed ANP was biologically active, as evidenced by its ability to induce cyclic guanosine monophosphate accumulation in target cells and to mimic the effect of exogenous ANP (10(-8) to 10(-7) mol/L). Cell growth and survival of AdMLP-ANP-infected cells were decreased and were associated with the promotion of VSMC apoptosis. These effects, which occurred at a multiplicity of infection of 10 to 100 TCID(50)/cell, were observed neither in cells infected with the control adenoviral constructs (AdMLP-betaGAL and AdMLP-gD) nor in cells treated with exogenous ANP (10(-7) to 10(-6) mol/L). These results showing VSMC apoptosis in response to ANP gene expression may have important implications for the prevention of vascular remodeling by gene therapy.