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
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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．05X
4、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
Effects of Copper Vapor Laser Irradiation on
Apoptosis and Proliferation Inhibiting of Vascular
Smooth Muscle Cells
Abstract
Percutaneous 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 laser
irradiation, by analyzing the correlation between VSMC aPoPtotic rate and the
cell 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 the
proliferative and matabolic statlls of VSMC, PCNA-positive expression, p53, c-
myc and bc1-2 mRNA leve1s in VSMC, so as to propose the mechanism of
induction aPoptosis and inhibition of the proliferation of VSMC by laser
irradiation. This stUdy will provide a foundation for the amer stUdy in vivo and
for designing a nove1 management strategies for local laser irradiation by Which
RS 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 and
transmission electron microscope. Morpho1ogical characteristics were observed 4,
8, 12, 16, and 24h after irradiated by laser with a PD dose of 300mWcm2 and ED
of 200J/cm2. Cell shrinkage, dense chromatin, edging in nuclei, cy'toplasm and
nuclei condensation, and aPoptosis bodies fOrmation, which were tyPical
characteristic of aPoptosis cells were all found. These changes in VSMC were
found to be time-dependellt. After short time irradiation, early aPoptosis
aPpeared While after a long time irradiation, the later stage of aPoptosis occurred.
(2) DNA 1addering on agarose demonstrated that a slender small molecule DNA
fragment (180~200bp) in VSMC irradiated with low dosage of PDl00 mW/cm
and ED 200 mWcm2. More distinCt aPoptotic bands were found When using a
PD 200 mW/cm2 and ED 200 mW/cm2 irradiation grouP, and when the laser dose
was 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 measured
by flow cytometry (FCM). Measurements were taken 24h after laser irradiation
Wth a PD300 mWfom', ED 200 mWfom2 dose. The data was analyzed with
CellQuest software and revealed that aPoptotic rate of the irradiation grouP was
(31.1 l 8.9)%, which had no diffeence Wth the value measu
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．05X
4、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
Effects of Copper Vapor Laser Irradiation on
Apoptosis and Proliferation Inhibiting of Vascular
Smooth Muscle Cells
Abstract
Percutaneous 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 laser
irradiation, by analyzing the correlation between VSMC aPoPtotic rate and the
cell 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 the
proliferative and matabolic statlls of VSMC, PCNA-positive expression, p53, c-
myc and bc1-2 mRNA leve1s in VSMC, so as to propose the mechanism of
induction aPoptosis and inhibition of the proliferation of VSMC by laser
irradiation. This stUdy will provide a foundation for the amer stUdy in vivo and
for designing a nove1 management strategies for local laser irradiation by Which
RS 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 and
transmission electron microscope. Morpho1ogical characteristics were observed 4,
8, 12, 16, and 24h after irradiated by laser with a PD dose of 300mWcm2 and ED
of 200J/cm2. Cell shrinkage, dense chromatin, edging in nuclei, cy'toplasm and
nuclei condensation, and aPoptosis bodies fOrmation, which were tyPical
characteristic of aPoptosis cells were all found. These changes in VSMC were
found to be time-dependellt. After short time irradiation, early aPoptosis
aPpeared While after a long time irradiation, the later stage of aPoptosis occurred.
(2) DNA 1addering on agarose demonstrated that a slender small molecule DNA
fragment (180~200bp) in VSMC irradiated with low dosage of PDl00 mW/cm
and ED 200 mWcm2. More distinCt aPoptotic bands were found When using a
PD 200 mW/cm2 and ED 200 mW/cm2 irradiation grouP, and when the laser dose
was 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 measured
by flow cytometry (FCM). Measurements were taken 24h after laser irradiation
Wth a PD300 mWfom', ED 200 mWfom2 dose. The data was analyzed with
CellQuest software and revealed that aPoptotic rate of the irradiation grouP was
(31.1 l 8.9)%, which had no diffeence Wth the value measu
引文
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2．李琴 等，猪冠状动脉平滑肌细胞原代培养．上海医科大学学报，1994；21(3)：31
3．鄂征 主编，《组织培养和分子细胞学技术》1995 北京出版社
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18． Pop M, Payette Y. Results of bilateral photorefractive keratectomy. Ophthalmology 2000;107(3) :472-9
19． Douki T, Cadet J. Individual determination of the yield of the main uv-induced dimeric pyrimidine photoproducts in dna suggests a high mutagenicity of cc photolesions. Biochemistry 2001;40(8) :2495-501
20． van Steeg H. The role of nucleotide excision repair and loss of p53 in mutagenesis and carcinogenesis. Toxicol Lett 2001;120(1-3) :209-19
21． Liu CD, Kwan D, Saxton RE, McFadden DW. Hypericin and photodynamic therapy decreases human pancreatic cancer in vitro and in vivo. J Surg Res 2000;93(l):137-43
22． Lee YJ, Lee KH, Kim HR, Jessup JM, et al. Sodium nitroprusside enhances TRAIL-induced apoptosis via a mitochondria-dependent pathway in human colorectal carcinoma CX-1 cells. Oncogene 2001 ;20(12) : 1476-85
23． Almeida A, Bolanos JP. A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical
neurons. J Neurochem 2001;77(2):676-90
24. Perez MJ, Cederbaum AI. Spin trapping agents (tempol and POBN) protect HepG2 cells overexpressing CYP2E1 against arachidonic acid toxicity. Free Radio Biol Med 2001;30(7):734-46
25. Liu Z, Martin LJ. Motor neurons rapidly accumulate DNA single-strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy. J Comp Neurol 2001;432(1):35-60
26. Debbasch C, Brignole F, Pisella PJ, Warnet JM, et al. Quaternary ammoniums and other preservatives' contribution in oxidative stress and apoptosis on Chang conjunctival cells. Invest Ophthalmol Vis Sci 2001;42(3):642-52
27. Penkowa M, Molinero A, Carrasco J, Hidalgo J. Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures. Neuroscience 2001;102(4):805-18
28. Kaufmann JA, Bickford PC, Taglialatela G. Oxidative-stress-dependent upregulation of Bcl-2 expression in the central nervous system of aged Fisher344 rats. J Neurochem 2001 ;76(4): 1099-108
29. Igase M, Okura T, Nakamura M, Takata Y, et al. Role of GADD153 (growth arrest-and DNA damage-inducible gene 153) in vascular smooth muscle cell apoptosis. Clin Sci (Colch) 2001;100(3):275-81
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32．彭黎明，Annexin 法定量分析淋巴瘤细胞凋亡与坏死，中华医学检验杂志，1999；22(1)：49
33．彭黎明，六种细胞凋亡检测方法的比较，中华病理学杂志，1999，28(1)：
55-57
34． Maulik N, Kagan VE, Tyurin VA, et al. Redistribution of phosphatidylethanolamine and phosphatidylserine precedes reperfusion-induced apoptosis. Am J Physiol, 1998;274(1Pt2) :H242-8
35． Alcocer F, Whitley D, Salazar J, Jordan W, et al. Mutual exclusion of apoptosis and hsp70 in human vein intimal hyperplasia in vitro. J Surg Res 2001;96(1) :75-80
36． Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear Dna fragmentaion. J Cell Biol, 1992; 119:493-501
37． Freimer G, Gao F, Mo QZ, Christopher TA, et al. Anti-apoptotic effect of gik in myocardial ischemia-reperfus ion injury. Acad Emerg Med 2001;8(5) :549
38． Kokawa K, Shikone T, Otani T, Nishiyama R, et al. Apoptosis and the expression of bcl-2 and bax in patients with endometrioid, clear cell, and serous carcinomas of the uterine endometrium. Gynecol Oncol 2001 May;81(2) : 178-83
39． Wang G, Xia Z, Yu B, Xiao S, et al. Cardiac apoptosis in burned rats with delayed fluid resuscitation.Burns 2001 May;27(3) :250-3
40． Hishita T, Tada-Oikawa S, Tohyama K, Miura Y, et al. Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39． Cancer Res 2001 Apr l;61(7) :2878-84
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16． Rosen ES. LASIK mania. J Cataract Refract Surg 2000;26(3) :303-4
17． Nogradi A, Hopp B, Revesz K, Szabo G, et al. Atomic force microscopic study of the human cornea following excimer laser keratectomy. Exp Eye Res 2000 Mar;70(3) :363-8
18． Pop M, Payette Y. Results of bilateral photorefractive keratectomy. Ophthalmology 2000;107(3) :472-9
19． Douki T, Cadet J. Individual determination of the yield of the main uv-induced dimeric pyrimidine photoproducts in dna suggests a high mutagenicity of cc photolesions. Biochemistry 2001;40(8) :2495-501
20． van Steeg H. The role of nucleotide excision repair and loss of p53 in mutagenesis and carcinogenesis. Toxicol Lett 2001;120(1-3) :209-19
21． Liu CD, Kwan D, Saxton RE, McFadden DW. Hypericin and photodynamic therapy decreases human pancreatic cancer in vitro and in vivo. J Surg Res 2000;93(l):137-43
22． Lee YJ, Lee KH, Kim HR, Jessup JM, et al. Sodium nitroprusside enhances TRAIL-induced apoptosis via a mitochondria-dependent pathway in human colorectal carcinoma CX-1 cells. Oncogene 2001 ;20(12) : 1476-85
23． Almeida A, Bolanos JP. A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical
neurons. J Neurochem 2001;77(2):676-90
24. Perez MJ, Cederbaum AI. Spin trapping agents (tempol and POBN) protect HepG2 cells overexpressing CYP2E1 against arachidonic acid toxicity. Free Radio Biol Med 2001;30(7):734-46
25. Liu Z, Martin LJ. Motor neurons rapidly accumulate DNA single-strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy. J Comp Neurol 2001;432(1):35-60
26. Debbasch C, Brignole F, Pisella PJ, Warnet JM, et al. Quaternary ammoniums and other preservatives' contribution in oxidative stress and apoptosis on Chang conjunctival cells. Invest Ophthalmol Vis Sci 2001;42(3):642-52
27. Penkowa M, Molinero A, Carrasco J, Hidalgo J. Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures. Neuroscience 2001;102(4):805-18
28. Kaufmann JA, Bickford PC, Taglialatela G. Oxidative-stress-dependent upregulation of Bcl-2 expression in the central nervous system of aged Fisher344 rats. J Neurochem 2001 ;76(4): 1099-108
29. Igase M, Okura T, Nakamura M, Takata Y, et al. Role of GADD153 (growth arrest-and DNA damage-inducible gene 153) in vascular smooth muscle cell apoptosis. Clin Sci (Colch) 2001;100(3):275-81
30. Darzyrkiewicz Z, Juan G, Gorczyca, W, et al. Cytometry in cell necrobiology:analysis of apoptosis and accidental cell death(necrosis). Cytometry,1997;27:1-20
31. Ormerod MG, The study of apoptotic cells by flow cytometry. Leukemia,1998;12:1013-25
32．彭黎明，Annexin 法定量分析淋巴瘤细胞凋亡与坏死，中华医学检验杂志，1999；22(1)：49
33．彭黎明，六种细胞凋亡检测方法的比较，中华病理学杂志，1999，28(1)：
55-57
34． Maulik N, Kagan VE, Tyurin VA, et al. Redistribution of phosphatidylethanolamine and phosphatidylserine precedes reperfusion-induced apoptosis. Am J Physiol, 1998;274(1Pt2) :H242-8
35． Alcocer F, Whitley D, Salazar J, Jordan W, et al. Mutual exclusion of apoptosis and hsp70 in human vein intimal hyperplasia in vitro. J Surg Res 2001;96(1) :75-80
36． Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear Dna fragmentaion. J Cell Biol, 1992; 119:493-501
37． Freimer G, Gao F, Mo QZ, Christopher TA, et al. Anti-apoptotic effect of gik in myocardial ischemia-reperfus ion injury. Acad Emerg Med 2001;8(5) :549
38． Kokawa K, Shikone T, Otani T, Nishiyama R, et al. Apoptosis and the expression of bcl-2 and bax in patients with endometrioid, clear cell, and serous carcinomas of the uterine endometrium. Gynecol Oncol 2001 May;81(2) : 178-83
39． Wang G, Xia Z, Yu B, Xiao S, et al. Cardiac apoptosis in burned rats with delayed fluid resuscitation.Burns 2001 May;27(3) :250-3
40． Hishita T, Tada-Oikawa S, Tohyama K, Miura Y, et al. Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39． Cancer Res 2001 Apr l;61(7) :2878-84