丙泊酚、瑞芬太尼对大鼠缺血再灌注损伤肺泡细胞凋亡的影响
摘要
目的:本实验旨在探讨麻醉药丙泊酚(propofol)和瑞芬太尼(remifentanil)对大鼠缺血再灌注损伤(ischemia reperfusion injury,IR)后肺泡细胞凋亡的影响。
方法:清洁级雄性Wistar大鼠126只,体重280—300g。大鼠依随机原则分5组,其中6只进入空白对照组(不对动物作任何处理,直接麻醉后剖胸取肺组织及左房血检测,此组作为各组术前空白对照),假手术组(仅行右开胸和右肺门穿带)、手术组(活结阻断右肺动脉、肺静脉和右主支气管)、丙泊酚组以及瑞芬太尼组各30只,并再随机分为5小组。各小组依照结扎前及缺血30min、再灌注1、2、4hr依实验步骤取材待检。建立大鼠原位热缺血再灌注模型,于结扎前、缺血30分钟、再灌注1小时、2小时、4小时给予药物干预,采取右肺组织及右房血标本,测定①血氧分压,②肺组织湿/干重比,③光镜和透射电镜观察组织细胞形态及超微结构,确定细胞凋亡发生,④采用流式细胞仪测定肺组织中凋亡情况。
结果:缺血再灌注组与假手术组比较,血氧分压降低(P＜0.05),肺组织湿/干重比增高(P＜0.05),肺泡细胞凋亡指数增高(P＜0.05),丙泊酚组、瑞芬太尼组与缺血再灌注组比较,血氧分压增高(P＜0.05),肺组织湿/干重比降低(P＜0.05),肺泡细胞凋亡指数下降(P＜0.05)。
结论:丙泊酚组和瑞芬太尼组血气分析、湿干比值、细胞凋亡率等指标测定结果明显优于IR组,显示丙泊酚和瑞芬太尼在治疗剂量对IR时的细胞凋亡过程有明显的缓解作用,能够减轻肺组织IR及凋亡的程度。
Objective The experiment wi1l research the effect of propofol and remifentanil against the apoptosis of alveolar cells induced by ischemia reperfusion injury.
Methods 126 male Wistar rats weighing 220-250g were randomly divided into 5groups:GroupA:control group(n=6,species were taken directly after anesthesia and thoracic incision.This group served as the pre-operative controls to the other groups),Group B:Sham group(n=30,suture was only placed in right hilus.The other operation progress and analysis of species were same to IR group),Group C: Ischemia-reperfusion group(n=30,according to time,It is further divided into 5 sub-groups stands for before deligation,30min after ischemis,1h、2h、4h after reperfusion),Group D:propofol treatment group(n=30,five sub-groups as groupC), Group E:remifentanil treatment group(n=30,five sub-groups as group C).We use the rat model of warm ischemia and repefusion in situ in single lung.After the establishment of the ischemia reperfusion in situ models,all rats will give pharmaceutical intervention.Before the ligation,after 30min of the ischemia and after 1h,2h,4h repectively of the reperfusion,We get the lung tissue and blood samples of the right atrium,then detect the partial pressure of blood oxygen, the wet weight/dry weight ratio of lung tissue and observe the configuration and ultrastructure of the tissue cells to confirm that the apoptosis happens and detect apoptosis of alveolar cells with the flow cytometer.
Results Compared with shame group,Ischemia-reperfusion group showed decreased PaO_2 and elevated wet/dry ratio(P＜0.05).Apoptotic index of lung was elevated.Compared with Ischemia-reperfusion group,propofol treatment group and remifentanil treatment group showed elevated PaO_2 and decreased wet/dry ratio (P＜0.05).Apoptotic index of lung was decreased(P＜0.05).
Conclusions In conclusion,compared with Ischemia-reperfusion group,propofol treatment group and remifentanil treatment group showed elevated PaO_2 and decreased wet/dry ratio and apoptotic index of lung was decreased.It is said that propofol and remifentanil have suppression to the apoptosis of alveolar cells induced by ischemia reperfusion injury.
方法:清洁级雄性Wistar大鼠126只,体重280—300g。大鼠依随机原则分5组,其中6只进入空白对照组(不对动物作任何处理,直接麻醉后剖胸取肺组织及左房血检测,此组作为各组术前空白对照),假手术组(仅行右开胸和右肺门穿带)、手术组(活结阻断右肺动脉、肺静脉和右主支气管)、丙泊酚组以及瑞芬太尼组各30只,并再随机分为5小组。各小组依照结扎前及缺血30min、再灌注1、2、4hr依实验步骤取材待检。建立大鼠原位热缺血再灌注模型,于结扎前、缺血30分钟、再灌注1小时、2小时、4小时给予药物干预,采取右肺组织及右房血标本,测定①血氧分压,②肺组织湿/干重比,③光镜和透射电镜观察组织细胞形态及超微结构,确定细胞凋亡发生,④采用流式细胞仪测定肺组织中凋亡情况。
结果:缺血再灌注组与假手术组比较,血氧分压降低(P＜0.05),肺组织湿/干重比增高(P＜0.05),肺泡细胞凋亡指数增高(P＜0.05),丙泊酚组、瑞芬太尼组与缺血再灌注组比较,血氧分压增高(P＜0.05),肺组织湿/干重比降低(P＜0.05),肺泡细胞凋亡指数下降(P＜0.05)。
结论:丙泊酚组和瑞芬太尼组血气分析、湿干比值、细胞凋亡率等指标测定结果明显优于IR组,显示丙泊酚和瑞芬太尼在治疗剂量对IR时的细胞凋亡过程有明显的缓解作用,能够减轻肺组织IR及凋亡的程度。
Objective The experiment wi1l research the effect of propofol and remifentanil against the apoptosis of alveolar cells induced by ischemia reperfusion injury.
Methods 126 male Wistar rats weighing 220-250g were randomly divided into 5groups:GroupA:control group(n=6,species were taken directly after anesthesia and thoracic incision.This group served as the pre-operative controls to the other groups),Group B:Sham group(n=30,suture was only placed in right hilus.The other operation progress and analysis of species were same to IR group),Group C: Ischemia-reperfusion group(n=30,according to time,It is further divided into 5 sub-groups stands for before deligation,30min after ischemis,1h、2h、4h after reperfusion),Group D:propofol treatment group(n=30,five sub-groups as groupC), Group E:remifentanil treatment group(n=30,five sub-groups as group C).We use the rat model of warm ischemia and repefusion in situ in single lung.After the establishment of the ischemia reperfusion in situ models,all rats will give pharmaceutical intervention.Before the ligation,after 30min of the ischemia and after 1h,2h,4h repectively of the reperfusion,We get the lung tissue and blood samples of the right atrium,then detect the partial pressure of blood oxygen, the wet weight/dry weight ratio of lung tissue and observe the configuration and ultrastructure of the tissue cells to confirm that the apoptosis happens and detect apoptosis of alveolar cells with the flow cytometer.
Results Compared with shame group,Ischemia-reperfusion group showed decreased PaO_2 and elevated wet/dry ratio(P＜0.05).Apoptotic index of lung was elevated.Compared with Ischemia-reperfusion group,propofol treatment group and remifentanil treatment group showed elevated PaO_2 and decreased wet/dry ratio (P＜0.05).Apoptotic index of lung was decreased(P＜0.05).
Conclusions In conclusion,compared with Ischemia-reperfusion group,propofol treatment group and remifentanil treatment group showed elevated PaO_2 and decreased wet/dry ratio and apoptotic index of lung was decreased.It is said that propofol and remifentanil have suppression to the apoptosis of alveolar cells induced by ischemia reperfusion injury.
引文
1、张赛,陈如坤,林敏,何雪明.大鼠肺IR中肺泡细胞凋亡的动态观察.中华医学杂志2004年10月2日第84卷第19期 1597-1600。
2、Harkin DW,BarrosD,sa AA,McCallionK,HoPerM,Halliday Ml,CamPbell FC.Circulating neutroPhil Priming and systemic inflammation in limb ischaemia -reperfusion injury Int Angiol,2001,20(1):78-89
3、Fischer S,Cassivi SD,Xavier AM,Cardella JA,Cutz E.Edwards V,Liu M.Keshavjee S.Cell death in human lung transplantation:apoptosis induction in human lungs during ischemia and after transplantation.[Journal Article]Annals of Surgery.231(3):424-31,2000 Mar.
4、Ozatamer,Tasoz,Kilinc,Unal,N.Propofol attenuates myocardial lipid peroxidation during coronary artery bypass grafting surgery.BJA:British Journal of Anaesthesia.89(2):242-246,August 2002.
5、郭英,张宏,张晓霞.雷米芬太尼与苏芬太尼对失血性休克鼠肺内中性粒细胞聚集激活的对比研究 100521139(2004)0420278203 军医进修学院学报 2004 Aug,25(4)
6、郭英,张宏.雷米芬太尼对肺损伤大鼠粘附因子表达影响的实验研究100853 北京 解放军总医院 解放军医学杂志2003年6月第28卷第6期526。
7、Frass M,Gmeiner B.Sandor,Schumann R,Wagner O,Kaye A.D.Effects of remifentanil on neutrophil adhesion.Acta Anaesthesiologica Scandinavica.44(10):1232-1237,November 2000.
8、Lam A,Bhat MB,Nunez G,et al.Regulation of bcl-xl channel activity by calcium.Biol Chem 1998;273(28):17307-17310
9、Gulbins E,Dreschers S,Bock J.Role of mitochondria in apoptosis.Exp Physiol.2003Jan;88(1):85-90.
10、Harris MH,Thompson CB.The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability.Cell Death Differ.2000 Dec;7(12):1182-91.
1.Kerr JFR,Wyllie AH,Currie AR.Apoptosis:A basic biological phenomenon with wide ranging implication in tissue kinetics[J].Br J Cancer,1972,26(4):2392257.
2.Li,Yunping MD;Cohen,Robert MD International Anesthesiology Clinics.New Therapeutic Targets and Modalities in Cardiovascular Medicine.43(2):77-89,Spring 2005。
3.Nhan,Thomas Q.;Liles,W Conrad;Schwartz,Stephen M.Role of Caspases in Death and Survival of the Plaque Macrophage.Thrombosis & Vascular Biology.25(5):895-903,May 2005.
4.Li,Yunping MD;Cohen,Robert MD International Anesthesiology Clinics.New Therapeutic Targets and Modalities in Cardiovascular Medicine.43(2):77-89,Spring 2005.
5.龚菲力 医学免疫学 科学出版社 2004.6 251-264。
6.张赛 陈如坤 林敏 何雪明 大鼠肺缺血再灌注损伤中肺泡细胞凋亡的动态观察 中华医学杂志2004年10月2日第84卷第19期 1597-1600。
7.谢东甫,秦雄,徐志飞 大鼠肺缺血再灌注损伤引起肺细胞的凋亡 第二军医大学学报2004Mar;25(3)295-297。
8.Harkin DW,BarrosD,sa AA,McCallionK,HoPerM,Halliday Ml,CamPbell FC.Circulating neutroPhil Priming and systemic inflammation in limb ischaemia-reperfusion injury Int Angiol 2001.20(1):78-89。
8.王雨 缺血再灌注损伤与细胞凋亡 中国普外基础与临床杂志2002年1月第9卷第1期 61-64。
9.Ilan Y,Roy Chowdhury N,Prakash R,et al.Massive repopulation of rat liver by transplantation of hepatocytes into specific lobes of liver and ligation of portal vein branches to other lobes[J].Transplantation,1997;64(1):8。
10.Hayashi A,Ito H,Shomori K,et al.Frequent occurrence of hepatocytic apoptosis in acute rejection of the grafted rat liver[J].Pathol Int,1997;47(8):518。
11.Yokomise H.Ueno T.Yamazaki F.Keshavjee S.Slutsky A.Patterson G The effect and optimal time of administration of veraPamil on lung Preservation Transplantation.49(6):1039-43,1990 Jun。
12.Pickford MA.Gower JD.Dore C.Fryer PR.Green CJ.Lipid peroxidation and ultrastructural changes in rat lung isografts after single-passage organ flush and 48-hour cold storage with and without one-hour reperfusion in vivo Transplantation.50(2):210-8,1990 Aug。
13.Feng Zhang,MD,PhD,Eric C.Hu,MD,Jacob Gerzenshtein,MD,Man-Ping Lei,MD,PhD,and William C.Lineaweaver,MD The Expression of Proinflammatory Cytokines in the Rat Muscle Flap With Ischemia-Reperfusion Injury Ann Plast Surg 2005;54:313-317。
14.Cooke,David Tom;Hoyt,E Grantl;Robbins,Robert C.Overexpression of Human Bcl-2 in Syngeneic Rat Donor Lungs Preserves Posttransplant Function and Reduces Intragraft Caspase Activity and Interleukin-1[beta]Production(?)2005 Lippincott Williams & Wilkins,Inc.Volume 79(7),15 April 2005,pp 762-767。
15.Yoshinaga,Keiko,Arimura,Shin-Ichi 2,Niwa,Yasuo 3,Tsutsumi,Nobuhiro,Uchimiya,Hirofumi 1,Kawai-Yamada,Maki 1,~* Mitochondrial Behaviour in the Early Stages of ROS Stress Leading to Cell Death in Arabidopsis thaliana.Annals of Botany.96(2):337-342,August 2005。
16.沈方,沈倩 细胞凋亡与线粒体通透性转变孔的研究进展浙江大学学报(医学版)第34卷 第2期 2005年。
17.Imahashi,Kenichi;Schneider,Michael D.;Steenbergen,Charles;Murphy,Elizabeth Transgenic Expression of Bcl-2 Modulates Energy Metabolism,Prevents Cytosolic Acidification During Ischemia,and Reduces Ischemia/Reperfusion Injury.Circulation Research.95(7):734-741,October 1,2004.
18.Mano,Akiko MD;Tatsumi,Tetsuya MD,PhD;Shiraishi,Jun MD,PhD;Keira,Natsuya MD,PhD;Nomura,Tetsuya MD;Takeda,Mitsuo MD;Nishikawa,Susumu MD;Yamanaka,Satoshi MD,PhD;Matoba,Satoaki MD,PhD;Kobara,Miyuki MD,PhD;Tanaka,Hideo MD,PhD;Shirayama,Takeshi MD,PhD;Takamatsu,Tetsuo MD,PhD;Nozawa,Yoshihisa PhD;Matsubara,Hiroaki MD,PhD Aldosterone Directly Induces Myocyte Apoptosis Through Calcineurin-Dependent Pathways.Circulation.110(3):317-323,July 20,2004。
19.Cooke,David Tom;Hoyt,E Grantl;Robbins,Robert C.Overexpression of Human Bcl-2 in Syngeneic Rat Donor Lungs Preserves Posttransplant Function and Reduces Intragraft Caspase Activity and Interleukin-1[beta]Production(?)2005 Lippincott Williams & Wilkins,Inc.Volume 79(7),15 April 2005,pp 762-767。
20.Chinnaiyan,Arul M.;O'Rourke,Karen;Lane,Brian R.;Dixit,Vishva M.Interaction of CED-4 with CED-3 and CED-9:A Molecular Framework for Cell Death.[Report]Science.275(5303):1122-1126,February 21,1997。
21.Chanan-Khan,Asher Bcl-2 antisense therapy in hematologic malignancies(?)2004 Lippincott Williams & Wilkins,Inc.Volume 16(6),November 2004,pp 581-585。
22.RICCIO,VERONICA DEL;VAN TUYL,MINKE;POST,MARTIN Apoptosis in Lung Development and Neonatal Lung Injury.Pediatric Research.55(2):183-189,February 20040
23.RICCIO,VERONICA DEL;VAN TUYL,MINKE;POST,MARTIN Apoptosis in Lung Development and Neonatal Lung Injury.[Review]Pediatric Research.55(2):183-189,February 2004。
24.Bank,Micha I MD ~*;Gudbrand,Charlotte MD ~*;Rengtved,Pia ~*;Carstensen,Henrik MD +;Fadeel,Bengt MD,PhD ++[S];Henter,Jan-Inge MD,PhD[S];Petersen,Bodil Laub MD,
PhD * Immunohistochemical Detection of the Apoptosis-Related Proteins FADD, FLICE, and FLIP in Langerhans Cell Histiocytosis. [Article] Journal of Pediatric Hematology/Oncology.27(6):301-306,June 2005。
2、Harkin DW,BarrosD,sa AA,McCallionK,HoPerM,Halliday Ml,CamPbell FC.Circulating neutroPhil Priming and systemic inflammation in limb ischaemia -reperfusion injury Int Angiol,2001,20(1):78-89
3、Fischer S,Cassivi SD,Xavier AM,Cardella JA,Cutz E.Edwards V,Liu M.Keshavjee S.Cell death in human lung transplantation:apoptosis induction in human lungs during ischemia and after transplantation.[Journal Article]Annals of Surgery.231(3):424-31,2000 Mar.
4、Ozatamer,Tasoz,Kilinc,Unal,N.Propofol attenuates myocardial lipid peroxidation during coronary artery bypass grafting surgery.BJA:British Journal of Anaesthesia.89(2):242-246,August 2002.
5、郭英,张宏,张晓霞.雷米芬太尼与苏芬太尼对失血性休克鼠肺内中性粒细胞聚集激活的对比研究 100521139(2004)0420278203 军医进修学院学报 2004 Aug,25(4)
6、郭英,张宏.雷米芬太尼对肺损伤大鼠粘附因子表达影响的实验研究100853 北京 解放军总医院 解放军医学杂志2003年6月第28卷第6期526。
7、Frass M,Gmeiner B.Sandor,Schumann R,Wagner O,Kaye A.D.Effects of remifentanil on neutrophil adhesion.Acta Anaesthesiologica Scandinavica.44(10):1232-1237,November 2000.
8、Lam A,Bhat MB,Nunez G,et al.Regulation of bcl-xl channel activity by calcium.Biol Chem 1998;273(28):17307-17310
9、Gulbins E,Dreschers S,Bock J.Role of mitochondria in apoptosis.Exp Physiol.2003Jan;88(1):85-90.
10、Harris MH,Thompson CB.The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability.Cell Death Differ.2000 Dec;7(12):1182-91.
1.Kerr JFR,Wyllie AH,Currie AR.Apoptosis:A basic biological phenomenon with wide ranging implication in tissue kinetics[J].Br J Cancer,1972,26(4):2392257.
2.Li,Yunping MD;Cohen,Robert MD International Anesthesiology Clinics.New Therapeutic Targets and Modalities in Cardiovascular Medicine.43(2):77-89,Spring 2005。
3.Nhan,Thomas Q.;Liles,W Conrad;Schwartz,Stephen M.Role of Caspases in Death and Survival of the Plaque Macrophage.Thrombosis & Vascular Biology.25(5):895-903,May 2005.
4.Li,Yunping MD;Cohen,Robert MD International Anesthesiology Clinics.New Therapeutic Targets and Modalities in Cardiovascular Medicine.43(2):77-89,Spring 2005.
5.龚菲力 医学免疫学 科学出版社 2004.6 251-264。
6.张赛 陈如坤 林敏 何雪明 大鼠肺缺血再灌注损伤中肺泡细胞凋亡的动态观察 中华医学杂志2004年10月2日第84卷第19期 1597-1600。
7.谢东甫,秦雄,徐志飞 大鼠肺缺血再灌注损伤引起肺细胞的凋亡 第二军医大学学报2004Mar;25(3)295-297。
8.Harkin DW,BarrosD,sa AA,McCallionK,HoPerM,Halliday Ml,CamPbell FC.Circulating neutroPhil Priming and systemic inflammation in limb ischaemia-reperfusion injury Int Angiol 2001.20(1):78-89。
8.王雨 缺血再灌注损伤与细胞凋亡 中国普外基础与临床杂志2002年1月第9卷第1期 61-64。
9.Ilan Y,Roy Chowdhury N,Prakash R,et al.Massive repopulation of rat liver by transplantation of hepatocytes into specific lobes of liver and ligation of portal vein branches to other lobes[J].Transplantation,1997;64(1):8。
10.Hayashi A,Ito H,Shomori K,et al.Frequent occurrence of hepatocytic apoptosis in acute rejection of the grafted rat liver[J].Pathol Int,1997;47(8):518。
11.Yokomise H.Ueno T.Yamazaki F.Keshavjee S.Slutsky A.Patterson G The effect and optimal time of administration of veraPamil on lung Preservation Transplantation.49(6):1039-43,1990 Jun。
12.Pickford MA.Gower JD.Dore C.Fryer PR.Green CJ.Lipid peroxidation and ultrastructural changes in rat lung isografts after single-passage organ flush and 48-hour cold storage with and without one-hour reperfusion in vivo Transplantation.50(2):210-8,1990 Aug。
13.Feng Zhang,MD,PhD,Eric C.Hu,MD,Jacob Gerzenshtein,MD,Man-Ping Lei,MD,PhD,and William C.Lineaweaver,MD The Expression of Proinflammatory Cytokines in the Rat Muscle Flap With Ischemia-Reperfusion Injury Ann Plast Surg 2005;54:313-317。
14.Cooke,David Tom;Hoyt,E Grantl;Robbins,Robert C.Overexpression of Human Bcl-2 in Syngeneic Rat Donor Lungs Preserves Posttransplant Function and Reduces Intragraft Caspase Activity and Interleukin-1[beta]Production(?)2005 Lippincott Williams & Wilkins,Inc.Volume 79(7),15 April 2005,pp 762-767。
15.Yoshinaga,Keiko,Arimura,Shin-Ichi 2,Niwa,Yasuo 3,Tsutsumi,Nobuhiro,Uchimiya,Hirofumi 1,Kawai-Yamada,Maki 1,~* Mitochondrial Behaviour in the Early Stages of ROS Stress Leading to Cell Death in Arabidopsis thaliana.Annals of Botany.96(2):337-342,August 2005。
16.沈方,沈倩 细胞凋亡与线粒体通透性转变孔的研究进展浙江大学学报(医学版)第34卷 第2期 2005年。
17.Imahashi,Kenichi;Schneider,Michael D.;Steenbergen,Charles;Murphy,Elizabeth Transgenic Expression of Bcl-2 Modulates Energy Metabolism,Prevents Cytosolic Acidification During Ischemia,and Reduces Ischemia/Reperfusion Injury.Circulation Research.95(7):734-741,October 1,2004.
18.Mano,Akiko MD;Tatsumi,Tetsuya MD,PhD;Shiraishi,Jun MD,PhD;Keira,Natsuya MD,PhD;Nomura,Tetsuya MD;Takeda,Mitsuo MD;Nishikawa,Susumu MD;Yamanaka,Satoshi MD,PhD;Matoba,Satoaki MD,PhD;Kobara,Miyuki MD,PhD;Tanaka,Hideo MD,PhD;Shirayama,Takeshi MD,PhD;Takamatsu,Tetsuo MD,PhD;Nozawa,Yoshihisa PhD;Matsubara,Hiroaki MD,PhD Aldosterone Directly Induces Myocyte Apoptosis Through Calcineurin-Dependent Pathways.Circulation.110(3):317-323,July 20,2004。
19.Cooke,David Tom;Hoyt,E Grantl;Robbins,Robert C.Overexpression of Human Bcl-2 in Syngeneic Rat Donor Lungs Preserves Posttransplant Function and Reduces Intragraft Caspase Activity and Interleukin-1[beta]Production(?)2005 Lippincott Williams & Wilkins,Inc.Volume 79(7),15 April 2005,pp 762-767。
20.Chinnaiyan,Arul M.;O'Rourke,Karen;Lane,Brian R.;Dixit,Vishva M.Interaction of CED-4 with CED-3 and CED-9:A Molecular Framework for Cell Death.[Report]Science.275(5303):1122-1126,February 21,1997。
21.Chanan-Khan,Asher Bcl-2 antisense therapy in hematologic malignancies(?)2004 Lippincott Williams & Wilkins,Inc.Volume 16(6),November 2004,pp 581-585。
22.RICCIO,VERONICA DEL;VAN TUYL,MINKE;POST,MARTIN Apoptosis in Lung Development and Neonatal Lung Injury.Pediatric Research.55(2):183-189,February 20040
23.RICCIO,VERONICA DEL;VAN TUYL,MINKE;POST,MARTIN Apoptosis in Lung Development and Neonatal Lung Injury.[Review]Pediatric Research.55(2):183-189,February 2004。
24.Bank,Micha I MD ~*;Gudbrand,Charlotte MD ~*;Rengtved,Pia ~*;Carstensen,Henrik MD +;Fadeel,Bengt MD,PhD ++[S];Henter,Jan-Inge MD,PhD[S];Petersen,Bodil Laub MD,
PhD * Immunohistochemical Detection of the Apoptosis-Related Proteins FADD, FLICE, and FLIP in Langerhans Cell Histiocytosis. [Article] Journal of Pediatric Hematology/Oncology.27(6):301-306,June 2005。