【作者】 郭瑞威；

【导师】 黄岚；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景动脉粥样硬化性疾病的发病率呈明显的增长趋势,给人类的健康带来严重的威胁。动脉粥样硬化进程的特点是单核细胞和淋巴细胞被招募到血管内膜,随后诱导血管中层血管平滑肌细胞（vascular smooth muscle cells, VSMCs）向内膜迁移并增殖,引起血管腔狭窄导致病变发展。近年来,冠脉介入治疗临床应用给冠心病患者带来了福音,但术后再狭窄也严重影响着冠脉介入治疗的远期效果,研究证实VSMCs过度增殖是导致冠脉介入术后再狭窄的主要原因。由此看出:VSMCs过度增殖在动脉粥样硬化和冠脉介入术后再狭窄中都有着重要的作用,然而起增殖的内在机制目前尚未完全阐明。基础研究表明Ca2+是调节细胞增殖的重要物质基础,去除细胞外钙后细胞增殖被明显抑制。VSMCs胞内Ca2+的调节依赖于Ca2+跨膜转运与细胞内钙库释放和再摄取Ca2+等过程。在VSMCs胞膜上有两种钙内流:1、由电压门控钙通道介导的钙内流,电压门控钙通道介导的钙内流可以引起VSMCs中Ca2+浓度的迅速增加,从而引起VSMCs的收缩;2、由钙库操纵型钙通道（SOC）介导的钙库Ca2+释放激活的Ca2+内流（CRAC）。研究表明在VSMCs在疾病状态下发生表型转换后,电压门控钙通道相关蛋白的表达逐渐下调。那么在这种状态下,是什么Ca2+通道介导细胞的Ca2+内流?近年来研究证明由SOC引发的胞浆内Ca2+浓度持续缓慢上升是细胞增殖的关键因素,在VSMCs上也发现了SOC通道的存在。SOC激活机制是:外来刺激通过IP3和兰尼定受体途径引发细胞内钙库中Ca2+的释放,当胞内钙库中Ca2+的浓度下降到一定程度时,细胞膜上SOC开放产生CRAC,引起细胞内Ca2+浓度持续缓慢上升,从而补充胞浆和钙库中的Ca2+,维持细胞的兴奋性,CRAC是SOC介导的典型Ca2+内流。目前细胞生物学的研究确定STIM1（stromal interaction molecule 1）是侦测胞内钙库中Ca2+下降从而导致细胞膜CRAC形成的分子。STIM1在多种细胞中表达（包括VSMCs）。Putney等用RNAi技术抑制了STIM1表达后,全细胞电位箝制法也无法测得胞膜上CRAC的形成。STIM1基因位于人的11号染色体上,蛋白分子位于内质网。在此,本研究证明STIM1分子可能在血管平滑肌细胞增殖中有着重要的作用,可能是抑制血管平滑肌细胞增殖新的药物靶点。实验方法1、大鼠siRNA靶序列的设计根据文献报道选取2条25nt的序列（rSTIM1, GenBank NO:NM₀01108496）,即STIM11:GCAUGGAAGGCAUCAGAAGUGUAUA;STIM12:GGAUGAGGUGAUACAGUGGCUGAUU,在引物设计时两端分别加入BamHI和HindIII酶切位点。稀释退火片段分别与线性化PEGFP6-1、PEGFP6-4载体的连接:STIM11稀释退火片段与PEGFP6-1载体的连接为STIM11-22;STIM12稀释退火片段与PEGFP6-4载体的连接为STIM12-4。质粒STIM11-22、STIM12-4的SalI+PstI双酶切分别回收大片段和小片段:STIM11-22回收大片段与STIM12-4回收小片段的连接,筛选后鉴定,将其连接到穿梭质粒PGSadeno,包装重组腺病毒,命名为Ad-si/rSTIM1;人源STIM1（hSTIM1, GenBank NO:U52426）重组腺病毒质粒命名为Ad-hSTIM1,由新西兰奥克兰大学分子生物实验室惠赠。纯化病毒滴度在109以上,满足在体转染的要求。2、在体动物实验部分:建立球囊损伤模型,用2%戊巴比妥钠按50mg/kg腹腔注射麻醉。沿颈前正中线切开皮肤,在颈前三角区暴露左颈总动脉及颈内、外动脉,自颈外动脉向近心端插入1.5F球囊导管至颈总动脉起始部,2个大气压使球囊膨胀,阻断血流30秒,后慢速回拉球囊至颈内外动脉分叉处,反复3次,退出导管,注入病毒液约10μl,夹闭动脉约10分钟。结扎颈外动脉,逐层缝合皮下组织与皮肤,常规饲养。术后常规肌注青霉素以预防感染。术后7和14天取颈动脉,中间切片HE染色,图象分析软件下计算内膜与中膜面积比值。定量RT-PCR和western blot检测血管组织中STIM1的表达。免疫荧光检测血管壁STIM1的分布。Western blot来检测血管壁PCNA表达情况。3、体外细胞实验:无菌条件下取大鼠胸主动脉,采用组织贴块法培养于含20%FBS的DMEM培养液中, 37℃、5% CO2培养箱中静置培养,实验用4-6代VSMCs;将病毒液加入细胞培养上清中感染细胞,4h后更换培养基,12h后可以看见绿色荧光。采用3H-TdR掺入法和细胞记数法检测VSMCs增殖情况。激光共聚焦检测细胞内钙变化情况。流式细胞仪分析细胞周期情况,并用western blot检测细胞周期蛋白P21和PRb表达情况。Transwell小室分析细胞迁移能力。4、统计学方法:所得结果用医学图像分析系统测定其灰度值,所得数据用均值±标准差表示。用SPSS13.0对数据进行处理,两组间比较用t检验;多组间进行单因素的方差分析,组间比较用Tukey’s方法进行。以P<0.05为差异有显著性。结果1、成功构建了大鼠STIM1基因干扰的腺病毒表达载体;通过反复扩增,病毒滴度约为3×109 pfu/ml。Ad-GFP在体转染后3、7和14天,GFP蛋白水平在3天达到最高,7和14天仍可检测到表达,这表明构建的病毒质粒达到在体转染要求。对细胞进行病毒转染后,DAPI染色细胞核,记数表明转染效率达到92.4±7.6%。质粒的成功构建魏进一步研究STIM1的生物学作用以及血管平滑肌细胞增殖的内在机制奠定了基础。2、在体动物实验部分:血管壁STIM1 mRNA和蛋白表达在血管损伤后7和14天后逐渐升高,与对照组比较有显著差异（P<0.05）。免疫荧光检测表明血管壁STIM1表达与血管平滑肌细胞a-actin表达有共区域性,表明在增殖的平滑肌细胞中STIM1表达上调。分别用腺病毒阴性对照、Ad-si/rSTIM1及Ad-hSTIM1转染球囊损伤的大鼠颈动脉,在转染后7天和14天处死动物取颈动脉,Western Blotting检测STIM1表达。结果表明Ad-si/rSTIM1转染组的STIM1表达较Ad-empty转染组显著下降（P<0.05）。Ad-hSTIM1和Ad-si/rSTIM1共转染后STIM1恢复至正常水平。转染Ad-si/rSTIM1后内膜与中膜（I/M）比值较转染阴性对照组明显减少（P<0.05）。而Ad-hSTIM1和Ad-si/rSTIM1共转染后I/M值恢复到转染对照组水平。免疫组化检测血管壁PCNA的表达,结果表明与阴性对照转染组相比,Ad-si/rSTIM1转染组PCNA表达明显降低（P<0.05）,而Ad-hSTIM1和Ad-si/rSTIM1共转染后PCNA表达恢复到转染Ad-empty组水平。同时,Ad-hSTIM1转染增加了p21表达,降低了pRb表达（P<0.05）,Ad-hSTIM1和Ad-si/rSTIM1共转染恢复到对照组水平。3、体外细胞实验部分:分别用不同感染复数的腺病毒阴性对照、Ad-si/rSTIM1及Ad-hSTIM1转染VMSC,在转染后48h检测STIM1表达。结果表明感染复数为15和30 MOI的Ad-si/rSTIM1转染组的STIM1表达较腺病毒阴性对照转染组显著下降（P<0.05）。15 MOI的Ad-hSTIM1和15 MOI的Ad-si/rSTIM1共转染后STIM1恢复至正常水平。转染后各组细胞增殖情况:结果显示转染Ad-si/rSTIM1后3H-TdR掺入量较转染腺病毒阴性对照组明显降低,细胞记数也明显减少（P<0.05）。而Ad-hSTIM1和Ad-si/rSTIM1共转染后3H-TdR掺入量和细胞数目恢复到转染腺病毒阴性对照组水平,转染后各组细胞内Ca2+浓度变化的比较:腺病毒转染48h后用1μM TG刺激细胞,使细胞内Ca2+耗竭,加入DEME培养基后,观察细胞内Ca2+浓度的变化,取增加值比较。结果表明与腺病毒阴性对照转染组相比,Ad-si/rSTIM1转染组细胞内Ca2+浓度增加幅度明显减少（P<0.05）,而Ad-hSTIM1和Ad-si/rSTIM1共转染后Ca2+浓度增加幅度恢复到转染腺病毒阴性对照组水平。转染后各组细胞周期分布情况:结果显示转染Ad-si/rSTIM1后细胞分布于G0/G1期的细胞数目较转染腺病毒阴性对照组明显增多,S和G2/M期细胞数目明显减少（P<0.05）。而Ad-hSTIM1和Ad-si/rSTIM1共转染后细胞周期分布与转染腺病毒阴性对照组无明显差异。转染后各组细胞p21和pRb表达变化:腺病毒转染48h后对细胞内p21和pRb表达进行检测。结果表明与腺病毒阴性对照转染组相比,Ad-si/rSTIM1转染组细胞内p21表达上调、pRb表达下调（P<0.05）,而Ad-hSTIM1和Ad-si/rSTIM1共转染后p21和pRb表达恢复到转染腺病毒阴性对照组水平。结论本研究主要结论:1、球囊损伤动脉血管后STIM1表达随着VSMCs增殖逐步升高;2、RNA干扰沉默STIM1表达后显著抑制了血管损伤后内膜增生;3、RNA干扰沉默STIM1表达后可以抑制血清诱导的VSMCs增殖和迁移。这些结果表明STIM1是调控血管平滑肌细胞增殖和再狭窄形成的一个关键因素,其可能成为治疗动脉硬化和再狭窄的药物靶点。更多还原

【Abstract】 BACKGROUNDAtherosclerosis is a common medical problem and has major impact on survival, quality of life, and health services. The atherosclerotic process is characterized by the recruitment of monocytes and lymphocytes to the arterial intima. The subsequent accumulation and proliferation of vascular smooth muscle cells (VSMCs), which immigrate from the medial layer, lead to lesion progression and encroachment on the coronary vascular lumen. Despite the fact that the use of percutaneous coronary intervention (PCI) has improved the results of atherosclerosis significantly, understanding restenosis after PCI remains a challenge. Restenosis refers to the re-occurrence of stenosis on the basis of intimal lesion, and the major contribution to this process is the proliferation and migration of medial VSMCs. Therefore, proliferation of VSMCs is a key event in atherosclerosis and restenosis after vascular injury; however, the underlying mechanism of VSMCs proliferation is unclear.Ca2+ channels are of particular interest in cell proliferation because of the profound anti-proliferative effect of removing extracellular Ca2+, and evidence from studies of many cell types that Ca2+ entry mechanisms have an essential role. Elevation of Ca2+ levels in VSMCs can result from entry of extracellular Ca2+ as well as release of Ca2+ sequestered within organelles such as the sarcoplasmic reticulum (SR). Ca2+ influx across the plasma membrane (PM) is mediated by voltage-dependent Ca2+ channels, and voltage-independent cation channels, including store-operated Ca2+ channels(SOC). It has been recognized that phenotypic modulation of VSMCs is associated with downregulation of voltage-dependent Ca2+ channels, which provide Ca2+ entry for contraction when the cells are in the contractile phenotype of the physiological blood vessel, and are the target of antihypertensive calcium antagonist drugs. What are the Ca2+ channels of the proliferating VSMC? Store-operated Ca2+ entry (SOCE), also known as capacitative Ca2+ entry, is thought to have an essential role in the regulation of contraction, cell proliferation, and apoptosis. Meanwhile, it has been reported that SOCE has been detected in VSMCs.The activation of SOCE is triggered by a reduction in the concentration of SR Ca2+. Transient discharge of SR Ca2+ occurs during the course of signaling events that activate inositol 1,4,5-trisphosphate receptors (IP3R) or ryanodine receptors in the SR membrane. SR Ca2+ stores can be depleted by inhibiting sarcoendoplasmic reticulum Ca2+ ATPases (SERCA) with thapsigargin. Although several biophysically distinct SOCE have been reported, the best characterized are the Ca2+ release-activated Ca2+ (CRAC) channels. Over the years, many genes have been claimed to code for the CRAC channel. Recently, an RNAi-based screening approach revealed a novel membrane-spanning protein named stromal interaction molecule 1 (STIM1) to be required for activation of SOCE. STIM1 is dispersed on the endoplasmic reticulum (ER) membrane under quiescence. Ca2+ store depletion stimulates redistribution of STIM1 to the PM. The redistribution is thought to transmit a store depletion signal to the CRAC channels in the PM. Evidence indicates that STIM1 may function as a Ca2+ sensor in the ER, leading to transduction of this signal to the PM, and opening of store-operated Ca2+ channels located in the PM. Nevertheless, there has been relatively little association of STIM1 with human disease, little direct evidence that STIM1 knockdown could be an effective therapeutic strategy, and no link between STIM1 and organ function. We have focused on the suggestion that STIM1 might have a role in vascular disease. Here, we present evidence from in vivo and in vitro studies that STIM1 does have an essential role in the proliferation of VSMCs, and we consider the relevance to the adaptive injury response of blood vessels.METHODS1. Construction of adenoviral vectors: A mixture of two siRNA duplex sequences exclusively targeting rat STIM1, but not human STIM1 were used. (i) Start nucleotide 935, GCAUGGAAGGCAUCAGAAGUGUAUA; and (ii) start nucleotide 970, GGAUGAGGUGAUACAGUGGCUGAUU. Target sequences for rSTIM1 were chemically synthesized as complementary oligonucleotides. Annealed oligonucleotides encoding sense and antisense strands linked by the loop sequence were subcloned into pGS-1. Recombinant viral genomes were transfected into 293 cells in a 6-well plate. Eight days after transfection, the recombinant virus was collected and subjected to one round of amplification in a T-75 flask with 1.5×106 293 cells, resulting in 2 ml of viral stock. The Ad-si/rSTIM1 and Ad-hSTIM1 viruses were titrated using the standard plaque assay. The titer for Ad-si/STIM1 was 3×109 pfu/ml. All adenoviruses expressed GFP under a separate promoter, allowing verification of infection. A nonsilencing control (NSC) sequence was generated in the same manner. Ad-hSTIM1 was gifted from department of biochemistry and molecular biology, university of Auckland.2. The section of animal experiments in vivo: Angioplasty of the rat left carotid artery was performed by using a balloon embolectomy catheter. Some animals were subjected to anesthesia and surgical procedure without balloon injury (sham-operated rats). After balloon injury, solutions of (20μL) Ad-si/rSTIM1, Ad-hSTIM1 or NSC were infused into the ligated segment of the common carotid artery for 20 minutes. At 7 days and 14 days after angioplasty, the carotid arteries were removed, and 6 cross-sections were cut from the approximate middle of the artery. The lumen loss of each group was measured by staining with hematoxylin and eosin. The expression of STIM1 in vessel wall was assessed using quantity RT-PCR, western blot and double-immunofluorescence staining. The proliferation of VSMCs was evaluated by measuring the PCNA expression in vessev wall.3. The section of in vitro experiments: Rat aortic VSMCs were isolated and subcultured, and VSMCs between 3 and 7 passages were used for the in vitro experiments. VSMCs were transduced with Ad-si/rSTIM1, Ad-hSTIM1, or NSC, and STIM1 protein levels were evaluated by western blot. The proliferation of VSMCs was measured by [3H] thymidine incorporation and cell counting. Changes in [Ca2+]i in individual cells were measured using an Aquacosmos system. Cell-cycle distribution was analyzed using flow cytometry. The expression of p21 and pRb were assessed using western blot. Cell migration was analyzed using transwell migration assay.4. Statistical analysis: Results are expressed as mean±SEM of n rats for in vivo experiments and mean±SEM of multiple experiments for molecular biology. SPSS13.0 software was used for statistical analysis. Student t tests were used to compare 2 groups, or ANOVA was used with the Tukey’s multiple comparison tests for multiple groups. Values of P <0.05 were regarded as statistically significant.RESULTS1. Successful transduction of Ad-GFP in balloon injured rat carotid artery was documented by Western blot analysis at 3, 7, and 14 days after injury. The level of GFP expression reached a maximum at day 3, and remained high until days 7 and 14 after adenoviral infection. These results suggest that the adenovirus-mediated delivery system was effective in rat carotid artery. The efficiency of adenovirus transfection in VSMCs was examined. Cell numbers were obtained by counting nuclei stained with 4’-6’-diamidino-2-phenylindole (DAPI). The transfection efficiency of adenovirus GFP expression in cultured VSMCs was 92.4±7.6%,2. The section of animal experiments in vivo: STIM1 mRNA and protein were detected in carotid arteries subjected to vascular injury, whereas their expression was low in uninjured right carotid arteries at 7 days and 14 days after balloon angioplasty. Meanwhile, when compared to the sham groups, STIM1 mRNA and protein in the left injured carotid had increased significantly at 7 days after balloon angioplasty, and further increased at 14 days after balloon angioplasty. In addition, immunoreactivity for STIM1 and SMαA was observed mostly in neointima at days 7 and 14. These results suggest that STIM1 is expressed in proliferating SMCs, contributing greatly to neointimal formation.We delivered adenovirus constructs expressing nonsilencing control (NSC), Ad-hSTIM1, and Ad-si/rSTIM1 to rat carotid arteries. Expression of STIM1 protein was confirmed by Western blotting. Compared with the NSC group, incubation with Ad-si/rSTIM1 attenuated injury-induced STIM1 upregulation significantly. Cotransfection with Ad-hSTIM1 reversed the downregulation of STIM1 by RNAi. Interestingly, the neointimal formation area and lumen loss ratio at day 14 were reduced significantly by transfection with Ad-si/rSTIM1 compared with transfection with NSC. When Ad-hSTIM1 was transfected with Ad-si/rSTIM1, the neointimal formation area and lumen loss ratio were restored to near the control level. Concomitantly, the expression of PCNA in rat carotid arteries at 14 days after injury was much lower in the Ad-si/rSTIM1-treated group than in the NSC group. The transfection of Ad-hSTIM1 with Ad-si/rSTIM1 restored the expression of PCNA in rat carotid arteries to near the level of that in the NSC group. In addition, STIM1 knockdown caused an increase in expression of p21, and a significant reduction in phosphorylation of Rb (pRb) in vivo at 14 days after injury. No significantly differences were found between sham group and Ad-si/rSTIM1-treated group on the expression of PCNA, p21 and pRb. 3. The section of in vitro experiments: Transduction of VSMCs with Ad-si/rSTIM1(MOI 15 and 30 pfu/cell) effectively decreased STIM1 protein expression at 48 hours post transduction. The cotransfection of Ad-hSTIM1 (MOI 15 pfu/cell) with Ad-si/rSTIM1 (MOI 15 pfu/cell) restored the expression of STIM1 protein. Interestingly, transfection of VSMCs with Ad-si/rSTIM1 decreased the uptake of [3H]thymidine by VSMCs significantly at 48 hours after infection. The cotransfection of Ad-hSTIM1 reversed the effect of STIM1 knockdown on [3H]thymidine uptake. Concomitantly, transfection of VSMCs with Ad-si/rSTIM1 suppressed proliferation of VSMCs significantly (MOI 15 pfu/cell), and hSTIM1 re-expression reversed the effect of STIM1 knockdown on VSMC proliferation. In addition, transfection of VSMCs with Ad-si/rSTIM1 decreased the number of migrating cells significantly at 48 h after infection. In this rSTIM1 knockdown background, hSTIM1 re-expression reversed the effect of STIM1 knockdown on the number of migrating cells.Cultured VSMCs were first synchronized and stimulated with serum to initiate cell-cycle progression, then infected with NSC, Ad-hSTIM1, and Ad-si/rSTIM1 for 48 hours. Fluorescence-activated cell sorting (FACS) was used to examine cell-cycle distribution. Approximately 13% of VSMCs infected by NSC (MOI 30 pfu/cell) or 16% of uninfected VMSCs progressed into S phase. VSMCs infected by Ad-si/rSTIM1 (MOI 30 pfu/cell) were distributed mainly in the G0/G1 phase, and only 1.6% of cells progressed into S phase. After VSMCs were transfected with Ad-hSTIM1 (MOI 15 pfu/cell), approximately 16% of cells progressed into S phase (Figure 5B). The effect of STIM1 knockdown on cell-cycle arrest was also manifest by alterations in key components of the cell-cycle regulatory machinery. STIM1 knockdown caused an increase in expression of the CDK inhibitor p21, and a significant reduction in pRb, thus permitting accumulation of the hypo-phosphorylated, growth-suppressive form of Rb.We evaluated the effect of small interfering RNA (siRNA) targeted against rSTIM1 on SOCE, which was activated by the depletion of intracellular Ca2+ stores using 1μM TG in the absence of extracellular Ca2+, followed by the addition of extracellular Ca2+ to 2 mM. The TG-mediated SOCE may be attributed to the release of Ca2+ from the SR. The infection of Ad-si/rSTIM1 (MOI 15 pfu/cell at 48 hours after infection) resulted in a marked decrease in SOCE. However, the cotransfection of cells with Ad-hSTIM1 (MOI 15 pfu/cell) reversed the effect of STIM1 knockdown on intracellular Ca2+ of VSMCs.CONCLUSIONSThe present results identify a critical role for STIM1 in neointimal formation in a rat model of vascular injury, and suggest a potential role for STIM1 knockdown in the reduction of neointimal development. Meanwhile, we have demonstrated that STIM1 is a powerful regulator of cell proliferation both in vivo and in vitro. This conclusion is based on several independent lines of evidence.First, an overt upregulation of STIM1 expression in vivo was associated with balloon injury-induced VSMC hyper-proliferation in rat carotid arteries. Second, knockdown of endogenous STIM1 by adenoviral delivery of siRNA significantly suppressed neointimal hyperplasia in vivo, which was reversed by STIM1 replenishment. Third, STIM1 knockdown inhibited SOCE in cultured VSMCs, and blocked serum-induced VSMCs proliferation in vitro, which was also reversed by STIM1 replenishment. In addition, suppression of STIM1 also inhibits VSMCs migration in Vitro.In summary, we show that STIM1 is a critical regulator of VSMC proliferation and neointimal hyperplasia. STIM1 may represent a novel therapeutic target in the prevention of restenosis after vascular intervention.更多还原