ORC1在大鼠血管平滑肌细胞增殖过程中的表达

目的检测不同增殖状态大鼠血管平滑肌细胞(vascu lar smooth musc le cells,VSMCs)中起始识别复合物1(origin recogn ition comp lex 1,ORC1)的表达。方法采用组织块贴壁法培养大鼠胸主动脉原代培养VSMCs;提取细胞总RNA,应用逆转录聚合酶链反应(RT-PCR)技术测定不同增殖状态VSMCs中ORC1 mRNA表达;采用免疫荧光、激光共聚焦显微镜检测不同增殖状态VSMCs中ORC1蛋白表达。结果经光镜、免疫荧光鉴定证实分离培养的细胞为VSMCs;处于静止状态的VSMCs无ORC1 mRNA表达,血清刺激6 h后ORC1 mRNA表达量明显增加,12~24 h达到高峰,继后逐渐减少;处于静止期的VSMCs无ORC1蛋白表达,处于增殖状态的VSMCs中有大量ORC1蛋白表达。结论ORC1可能在VSMCs增殖过程中起重要作用。     

雷帕霉素对大鼠血管平滑肌细胞起始识别复合物1 mRNA表达的影响

目的观察血管平滑肌细胞(VSMC)起始识别复合物1(ORC1)mRNA在雷帕霉素作用下的表达变化。方法采用组织块贴壁法获得VSMC并用免疫细胞化学法鉴定,含体积分数10%血清的培养基培养。血清饥饿法饥饿细胞24 h使其同步化。将雷帕霉素加入含体积分数10%血清的培养基(终浓度为10 nmol.L-1)中继续培养细胞,分别于0 h、3 h、6 h、9 h、12 h、15 h、18 h、21 h、24 h收集细胞,采用反转录-聚合酶链反应技术法检测各时间点ORC1 mRNA的表达(内参为β-actin)。用Quantity One软件计算各时间点ORC1 mRNA与内参的光密度比值,并作统计学分析。结果各时间点ORC1与β-actin光密度比值均有显著性差异(F=410.170,P=0.001)。雷帕霉素作用3～12 h ORC1 mRNA的表达持续升高(与0 h比较,P<0.05),12～18 h逐渐下降(与0 h比较,P依次为0.001,0.001,1.000),18～24 h与0 h比较,无统计学差异(P依次为1.000,0.986,1.000)。结论VSMC的ORC1 mRNA表达随雷帕霉素作用时间呈动态变化。     

西罗莫司对大鼠血管平滑肌细胞增殖、起始识别复合物1表达的影响及机制探讨

目的探讨西罗莫司对体外培养的大鼠血管平滑肌细胞(VSMCs)增殖、起始识别复合物1(ORC1)表达的影响及机制。方法细胞随机分为对照组和实验组,每组3瓶/孔,血清饥饿法培养细胞24h使其同步化。对照组采用含10%胎牛血清的DMEM培养液培养,实验组采用含10μmol/L西罗莫司+10%胎牛血清的DMEM培养液培养。继续培养0～48h,采用免疫细胞化学法检测增殖细胞核抗原(PCNA)表达,电子显微镜观察细胞超微结构,RT-PCR法检测ORC1mRNA的表达,蛋白质印迹法检测P53、cyclin D1、cyclin A和ORC1蛋白的表达。结果与对照组相比,实验组48hPCNA阳性表达率降低[(20±2.1)%vs(80±3.0)%,P<0.05],P53蛋白表达升高(P<0.05);细胞核有固缩的迹象;实验组cyclin D1蛋白表达轻微升高(P>0.05),48hcyclin A蛋白的表达下降(P<0.05)。对照组ORC1mRNA的表达在0～12h升高,24～48h降低,高峰期在12h。实验组细胞ORC1mRNA的表达始终处于高水平。与同时间对照组比较,实验组48hORC1mRNA的表达升高(P<0.05)。蛋白质印迹分析结果与之相似。结论西罗莫司抑制VSMCs增殖,同时促进其凋亡;其作用环节可能是通过阻止细胞由G0/G1期向S期转化,诱导细胞处于静止状态;ORC1的作用环节位于西罗莫司作用点的上游,进一步支持ORC1参与细胞复制起始过程。     

起始识别复合物1对血管平滑肌细胞增殖活性的影响

目的起始识别复合物1(origin recognition complex1,ORC1)与血管平滑肌细胞增殖密切相关[1],文中探讨ORC1对血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖活性的影响。方法用组织块贴片法培养大鼠胸主动脉VSMC;用流式细胞术测定VSMC静止期和增殖期的细胞周期,并计算出不同状态的增殖活性;用免疫荧光法测定ORC1在VSMC中的表达位置,用流式细胞术测定不同细胞周期中ORC1蛋白表达。结果静止期VSMC的增殖活性很低[(10.40±3.52)%],在VSMC中ORC1不表达或低水平表达。血清剌激24 h后,处于增殖期的VSMC的增殖活性明显增加,达(33.91±13.42)%,ORC1蛋白表达也明显增加(P<0.01)。结论 ORC1可能与VSMC增殖活性有关。     

ORC1基因RNA干扰对大鼠血管平滑肌细胞表型的影响

目的 以RNA干扰抑制血管平滑肌细胞(vascular smooth muscle cells,VSMCs)ORC1基因,探讨ORCl基因表达抑制后对VSMCs表型的影响.方法 实验设置正常对照组、阴性siRNA组及阳性siRNA组.应用Western blot检测ORC1基因表达的变化;免疫荧光染色后激光共聚焦显微镜观察细胞形态结构;应用流式细胞仪检测细胞表型标志蛋白表达.结果 ①siRNA转染后,阳性siRNA组ORCI基因表达水显著降低,而正常对照组及阴性siRNA组间ORC1基因表达水平无显著差异.②siRNA转染后,阳性转染组细胞呈现收缩型形态特征,空白对照组及阴性对照组细胞呈现合成型形态特征.③siRNA转染使ORCI表达减弱后,VSMCs收缩型标志物α-平滑肌肌动蛋白(α-SM actin)和平滑肌肌动蛋白重链2(SM-2)表达水平明显升高.合成型标志物骨桥蛋白(osteopontin)表达水平明显降低,空白对照组及阴性对照组间3种标志物表达水平无显著差异.结论 RNA干扰介导的ORC1基凶沉寂可促使VSMCs由合成型向收缩型转化.     

大鼠血管平滑肌细胞分离培养的探讨

目的：培养大鼠血管平滑肌细胞。方法：手术显微镜下分离主动脉和肺动脉，再用翻转干涸法进行操作。结果：显微镜下分离组织，岢基本除净动脉纤维脂肪层和外膜，再用刀片轻刮内膜，可保证所贴组织块为地动脉中膜。传至第三代时，用台盼蓝检查细胞传代成活率９５％，光镜、电镜和免疫组化鉴定培养细胞，培养细胞纯度９６％。结论：贴块片简便易行、经济、又可以防止膜损伤，可为血管病理变化研究提供适宜的细胞。     

ORC1在大鼠血管平滑肌细胞G1/S和S期的表达

目的检测大鼠血管平滑肌细胞(VASCULAR SMOOTH MUSCLE CELLS,VSMCS)G1/S、S期中起始识别复合物(ORIGINRECOGNITION COMPLEX1,ORC1)的表达。方法采用组织块贴壁法培养大鼠胸主动脉原代培养VSMCS。利用胸苷双阻断法造成细胞同步,应用逆转录聚合酶链反应(RT-PCR)技术、流式细胞仪检测分别测定G1/S、S期VSMCS的ORC1MRNA及蛋白表达。结果经光镜、免疫组化鉴定证实分离培养的细胞为VSMCS。ORC1MRNA在GO期的VSMCS无明显表达、G1/S期达到高峰,到S、G2/M期明显减少。流式细胞仪检测的VSMCS ORC1蛋白质表达与MRNA变化相似。结论ORC1可能在VSMCS细胞周期调节过程中起重要作用。     

大鼠血管平滑肌细胞的培养与鉴定

目的:改进大鼠血管平滑肌细胞(VSMCs)的培养方法。方法:采用机械刮除、差异贴壁等手段进行组织贴块法原代培养,胰蛋白酶消化传代,并应用相差显微镜和即用型SABC免疫组化染色试剂盒对细胞进行形态学和免疫组化鉴定。结果:镜下培养细胞呈典型的"谷-峰状"生长,免疫组化染色显示胞浆内-αactin阳性表达,锥虫蓝染色检测细胞传代成活率95%。结论:本法可获纯度高、结构和功能良好的VSMCs。     

ORC1基因RNA干扰对大鼠血管平滑肌细胞增殖的影响

目的以RNA干扰抑制血管平滑肌细胞(vascu lar smooth musc le cells,VSMCs)ORC1基因,探讨ORC1基因表达抑制后VSMCs增殖的变化。方法实验设置正常对照组、阴性siRNA组及阳性(ORC1 A、ORC1 B、ORC1 C)siR-NA组。应用W estern b lot检测ORC1基因表达的变化;应用MTT比色试验、3H-TdR掺入试验检测VSMCs增殖的情况。免疫细胞化学染色观察增殖细胞核抗原(proliferating cell nuc lear antigen,PCNA)表达。结果①siRNA转染后,3个阳性siRNA转染组ORC1基因表达水平均降低,尤以第2对阳性siRNA抑制效果最为显著,而空白对照组及阴性对照组间ORC1基因表达水平无显著差异。②siRNA转染使ORC1表达减弱后,VSMCs的MTT吸光度值3、H-TdR掺入量和PCNA表达量均较空白对照组及阴性对照组显著降低。结论RNA干扰介导的ORC1基因沉寂可显著抑制VSMCs增殖。     

改良大鼠血管平滑肌细胞的原代培养

目的探讨提高大鼠血管平滑肌细胞原代培养效率的方法。方法采取组织块贴壁法进行原代培养,高糖DMEM培养基中加入血小板源性生长因子-B(platelet-derived growth factor-b,PDGF-B),用细胞形态学及免疫组织化学法对血管平滑肌细胞鉴定。结果原代培养,4～6天可见细胞长出,2周可以传代;传代培养,1周可以传代1次。镜下可见细胞以梭形为主,呈"峰-谷"状生长,免疫组化染色可见细胞浆内有大量染成棕黄色的肌丝。结论在采用组织块贴壁法进行培养时,加入PDGF-B培养,可获得纯度高、活性好的血管平滑肌细胞,并缩短了培养周期。     

腺病毒介导过表达ANT1基因诱导大鼠血管平滑肌细胞凋亡

目的利用腺病毒载体转染体外培养的血管平滑肌细胞(vascular smooth muscle cells,VSMCs),研究过表达腺嘌呤核苷酸转位酶1(adenine nucleotide translocase 1,ANT1)对大鼠VSMCs凋亡的影响。方法用携带ANT1的腺病毒载体(Ad-ANT1)或空载体腺病毒载体(Ad-GFP)感染VSMCs,采用激光共聚焦观察Hoechst33258染色后细胞凋亡情况,流式细胞术检测Annexin V标记细胞凋亡率。Western blot检测Bax和Bcl-2蛋白表达。结果激光共聚焦观察转染Ad-ANT1后48 h细胞出现凋亡,且凋亡率[(13.40±1.14)%]与转染空载体对照组[(1.80±0.84)%]相比较差异有统计学意义(P<0.01)。流式细胞术检测转染Ad-ANT1后48 h细胞凋亡率[(10.66±1.75)%]明显高于转染空载体组细胞[(3.13±0.37)%](P<0.05)。Western blot结果显示转染Ad-ANT1后Bax蛋白表达明显高于转染空载体组,Bcl-2蛋白表达2组比较无明显差异。结论腺病毒介导过表达ANT1可诱导大鼠VSMC的凋亡,其机制可能通过上调Bax实现。     

Artesunate Reduces Proliferation, Interferes DNA Replication and Cell Cycle and Enhances Apoptosis in Vascular Smooth Muscle Cells

This study examined the effect of artesunate (Art) on the proliferation, DNA replication, cell cycles and apoptosis of vascular smooth muscle cells (VSMCs). Primary cultures of VSMCs were established from aortas of mice and artesunate of different concentrations was added into the medium. The number of VSMCs was counted and the curve of cell growth was recorded. The activity of VSMCs was assessed by using MTT method and inhibitory rate was calculated. DNA replication was evaluated by [^3H]-TdR method and apoptosis by DNA laddering and HE staining. Flowmetry was used for simultaneous analysis of cell apoptosis and cell cycles. Compared with the control group. VSMCs proliferation in Art interfering groups were inhibited and [^3H]-TdR incorprating rate were decreased as well as cell apoptosis was induced. The progress of cell cycle was blocked in G0/G1 by Art in a dose-dependent manner. It is concluded that Art inhibits VSMCs proliferation by disturbing DNA replication, inducing cell apoptosis and blocking cell cycle in G0/G1 phase.     

Different responses of cell cycle between rat vascular smooth muscle cells and vascular endothelial cells to paclitaxel

Summary： Although previous reports showed dmg-eluting stent （DES） could effectively inhibit neointima formation, in-stent restenosis （ISR） remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells （VSMCs） and vascular endothelial cells （VECs） of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, prolif- eration of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0-G1 and G2-M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1 S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.     

胰岛素对大鼠血管平滑肌细胞表型转化的影响

目的:探讨胰岛素对体外培养的血管平滑肌细胞(vascular smooth muscle cell,VSMC)表型转化的影响.方法:用酶消化法分离大鼠VSMC,采用免疫组织化学法检测胰岛素作用后VSMC肌动蛋白(α-SM actin),RT-PCR 检测胰岛素作用后VSMC中bFGF、TGF-β、PDGF、matrix Gla和OPN各目的基因mRNA相对表达水平.结果:胰岛素组VSMC的3H-TdR掺入值比对照组升高47％(P<0.01), VSMC α- SM actin免疫组化结果显示对照组的α-SM actin比胰岛素组染色深.而matrix Gla和OPN在培养的VSMC中mRNA的表达量,胰岛素组明显高于对照组(P<0.05),同时bFGF、TGF-β、PDGF的表达胰岛素组也明显高于对照组(P<0.05).并可明显见到细胞骨架F-actin、G-actin重新分布.结论:在合成表型的matrix Gla和OPN表达量明显高于收缩表型,而合成表型的α- SM actin表达量明显低于收缩表型.提示胰岛素对VSMC的表型转化起了一定作用.胰岛素在促进了VSMC增殖的同时,伴有VSMC由收缩型转变为合成型及细胞骨架F-actin、G-actin重新分布.