铜蒸气激光照射对兔血管平滑肌细胞凋亡的诱导作用及对增殖抑制作用的研究

【作者】 刘爱琴；

【导师】 唐建民； 顾瑛；

【作者基本信息】 第三军医大学 ， 康复理疗， 2001， 博士

【摘要】 经皮冠状动脉成形术（PTCA）是治疗冠状动脉狭窄的重要手段之一，然而术后再狭窄（RS）的发病率在6个月时可高达30-50％。血管平滑肌细胞（VSMC）增殖、迁移，新生内膜形成、增厚是血管成形术后再狭窄发生的主要原因，增生活跃的VSMC在诱发因子的刺激下易发生凋亡，为再狭窄的防治提供了新思路。激光具有定位准确、剂量易于控制、安全简便、无全身不良反应等其他方法所不能替代的优点，将激光用于再狭窄防治方面的研究，具有深远的理论和实际意义。 本课题研究目的：1、观察激光诱导VSMC凋亡的形态学及生物化学特性的改变；2、筛选出适当的诱导VSMC凋亡的激光照射剂量，了解激光诱导VSMC凋亡的剂量规律；3、观察细胞的增殖代谢状态和细胞周期时相、增殖细胞核抗原（PCNA）、以及c-myc、bcl-2 mRNA表达量对激光诱导VSMC凋亡率的影响；4、观察激光照射对细胞增殖代谢状态的影响，并通过观察激光照射对增殖细胞核抗原（PCNA）及对p53、c-myc及bcl-2 mRNA表达的影响，探讨激光照射诱导VSMC凋亡、抑制其增殖的机制，为进一步的在体研究及建立激光在血管内局部照射防治再狭窄的方法提供理论依据。 一、铜蒸气激光照射诱导VSMC凋亡的形态学、生物化学以及分子生物学研究 组织贴块法培养兔胸主动脉SMC，实验取第四代离体培养之VSMC。1、在光学显微镜及透射电镜下观察到经激光照射后VSMC呈现细胞体积变小、染色质浓缩、边集，胞质、胞核固缩，凋亡小体形成等典型的凋亡细胞形态学改变。2、激光照射后VSMC DNA电泳分析结果表明激光照射组出现特征性的凋亡 DNA条带。3、经功率密度为 300 mwcmZ、能量密度为200J／cm‘的激光照射后 24h，流式细胞仪oCM）及 TUNEL法检测 VSMC的凋亡率分别为O．1土8．9）％和O0．0土2．8％卜 明显高于对照组［凋亡率为O＊土3．7）％］。 二、铜蒸气激光诱导血管儡鹏凋亡的照光剂量的筛选及剂量规律研究 以不同照光剂量的铜蒸气激光照射VSMC，照光后24h采用TUNEL法检测各组细胞凋亡率。结果显示：1、各照射组凋亡率均较对照组明显增高O＜0刀匀，当激光剂量为功率密度300mWCm＼ 能量密度200J／cm‘时VSMC凋亡率达到（ O土2．8）％，高于其他照射组o＜0刀5卜2、能量密度与＊S＊C凋亡诱导率之间的关系：在能量密度 100wt00Jfom’范围内，激光的功率密度为 100thw儿m时，随激光能量密度的增加，细胞的凋亡诱导率无明显变化…＞0刀5）；激光的功率密度为200mwtamZ或300mw／cmZ时，随激光能量密度的增加，凋亡率逐渐升高O＞0刀5卜 当能量密度达到200卫口n‘时凋亡率最高，以后逐渐下降。3、激光功率密度与VSMC凋亡诱导率之间的关系：能量密度为 100J／Cin‘时，100niw尔tti‘及 200ttiWCtti‘照射组 VSMC的凋亡率无显著性差异…＞0D5卜而功率密度 300mwtom‘照射组 VSMC凋亡率明显高于前两者…＜0．05）；能量密度分别为200Mm‘，叨＊Mm‘，400Mm’时，在实验所采用剂量范围内，V＊＊C凋亡率随激光功率密度的增加而升高…功．05X4、从实验所采用的照射剂量范围及温度实验之结果分析，激光诱导VSMC凋亡是细胞发生光化学反应的结果。 三、铜蒸气激光诱导血管平滑肌细胞凋亡的影响因素的研究 VSMC同步化后，随机分为3组：①10％新生牛血清组＊0％NCS卜②PDGF组＊＋20ng／ml PDGF），③40％FBS组。各组分别加入相应的生长因于，16J 后，MTT法检测细胞增殖代谢状态，流式细胞仪检测细胞周期分布情况，兔疫组化法检测 PCNA的表达情况，逆转录FCR（RTICR）法检测细胞内原癌基因c－myc、hcf上 的mRNA表达情况，以功率密度300mw／cm’，能量密度 200J／cm‘激光照射备组 VSMC，照光后 24h TUNEL法检测各组细胞凋亡率，分析各影响因素与VSMC凋亡诱导率之间的相关 XI D ＼，－性，表明细胞的增殖代谢状态、PCNA阳性表达率、S＋GZ／M期细胞百分数、c，myc、bclZ的mRNA表达量均与VSMC激光凋亡诱导率之间呈不同程度的正相关，而G广G；期细胞百分数与VSMC的凋亡诱导率呈明显负相关。 四、激光照射对 VSMC增殖代谢及对 c－myc、bel－2及 p53 mRNA表达的影响 铜蒸更多还原

【Abstract】 Effects of Copper Vapor Laser Irradiation onApoptosis and Proliferation Inhibiting of VascularSmooth Muscle CellsAbstractPercutaneous transluminal coronary angioplasty (PTCA) has been widely adopted as a therapeutic modality for coronary atherosclerosis. However, late restenosis (RS) is developed among 30-50% of patients who undergo PTCA. The main reason causing restenosis is vascular injury, which can be induced by the inflated balloon or similar devices. Restenosis is accompanied by platelet thrombus formation and a change of the phenotype of vascular smooth muscle cells (VSMC). VSMC’s change from their resting contractile state to one capable of migratory, proliferative and secretory functions. So, excessive proliferation of VSMC is considered to be the major contributing factor to restenosis. One viable strategy for the provention of restenosis is to inhibit this proliferative response and to induce apoptosis of the proliferative VSMC. Most of the previous studies in this field concentrated on coronary stenting, ionizing radiation, drugs and gene therapy. No effective methods have been found to resolve this problem. Laser therapy is expected to be highly effective for the management of restenosis because of the advantages of laser. Some advantages of laser include accurate direction, exact irradiation dosage, easy manipulation and no systematic toxicity. Till now, the prevention of RS with laser irradiation has not been reported and the mechanism of this alternative treatment is still not understood.The objectives of this study were: 1. Select one or several laser irradiation dosages by which quite high apoptotic rate of VSMC can be induced, and obtain the relation between the laser irradiation dose and the apoptotic rate of VSMC; 2.Observe the morphological and biochemica1 characteristics of aPoptotic VSMC; 3.lnvestigate the factors that influnce the VSMC aPoptotic fate induced by laserirradiation, by analyzing the correlation between VSMC aPoPtotic rate and thecell proliferative statllsi PCNA-positive expression Tate, cell cycIe, c-myc and bc1-2 mRNA leve1s in VSMC; 4. Observe the effects of laser irradiation on theproliferative and matabolic statlls of VSMC, PCNA-positive expression, p53, c-myc and bc1-2 mRNA leve1s in VSMC, so as to propose the mechanism ofinduction aPoptosis and inhibition of the proliferation of VSMC by laserirradiation. This stUdy will provide a foundation for the amer stUdy in vivo andfor designing a nove1 management strategies for local laser irradiation by WhichRS can be prevented.Methods and Results:1. Study the effects of the copper vaPor laser on VSMC aPoPtosis. (l)Morphological characteristics of VSMC were observed under both light andtransmission electron microscope. Morpho1ogical characteristics were observed 4,8, 12, 16, and 24h after irradiated by laser with a PD dose of 300mWcm2 and EDof 200J/cm2. Cell shrinkage, dense chromatin, edging in nuclei, cy’toplasm andnuclei condensation, and aPoptosis bodies fOrmation, which were tyPicalcharacteristic of aPoptosis cells were all found. These changes in VSMC werefound to be time-dependellt. After short time irradiation, early aPoptosisaPpeared While after a long time irradiation, the later stage of aPoptosis occurred.(2) DNA 1addering on agarose demonstrated that a slender small molecule DNAfragment (180~200bp) in VSMC irradiated with low dosage of PDl00 mW/cmand ED 200 mWcm2. More distinCt aPoptotic bands were found When using aPD 200 mW/cm2 and ED 200 mW/cm2 irradiation grouP, and when the laser dosewas PD300 mW/cm’, ED 200 mW/cm2, the bands were the most pronounced. (3)The aPoptotic rate of VSMC studied by propidium iodide staining was measuredby flow cytometry (FCM). Measurements were taken 24h after laser irradiationWth a PD300 mWfom’, ED 200 mWfom2 dose. The data was analyzed withCellQuest software and revealed that aPoptotic rate of the irradiation grouP was(31.1 l 8.9)%, which had no diffeence Wth the value measu更多还原