光动力疗法作用于肝癌细胞的实验研究
|
摘要
目的:
全世界43.7%的肝癌患者在中国,严重威胁着我国人民的健康。原发性肝癌发病比较隐匿,临床诊疗中存在早期诊断难、复发转移率高、治疗缺乏针对性、治疗手段少等问题。因此对肝癌诊治新技术、新疗法的研究尤为重要和迫切。光动力疗法(Photodynamic therapy,PDT)作为一种新的肿瘤治疗方法已应用于临床,对体表和脑内肿瘤等取得了比较理想的治疗效果,但对肝脏肿瘤等腹腔内肿瘤的相关研究较少。本实验拟在初步探讨photosan—PDT在不同实验条件下对人HepG2肝癌细胞增殖的抑制作用并初步探讨其作用机制,以期为PDT成为肝癌治疗新方法提供进一步的理论依据。
内容:
将体外培养的人HepG2肝癌细胞常规传代后,在无光照情况下,比较不同浓度光敏剂—photosan对肝癌细胞增殖的影响,然后以photosan为光敏剂、半导体激光器(波长630nm)为光源进行激光照射,在不同浓度光敏剂经不同剂量的光照后,用MTT法测定PDT后HepG2肝癌细胞的残存率,并用流式细胞分析检测PDT对肝癌细胞周期的影响,以Annexin V-FITC/PI检测细胞凋亡。
方法:
HepG2肝癌细胞复苏,常规培养并传代。实验主要分四部分:(1)无光照情况下,不同浓度光敏剂对肝癌细胞增殖的影响。将细胞按实验设计转移至96孔板,常规培养,细胞贴壁后,加入不同浓度的光敏剂,使B2至G2为空白对照,既不加细胞也不加光敏剂,只加培养基;B3至G3为实验对照,只加细胞不加光敏剂;B4至G11的矩形区域为光敏剂实验组,按列分别加入2,4,6,8,10,12,14,16mg/L的光敏剂,孵育4h后,弃去光敏剂,继续孵育24h,进行MTT比色实验。(2)在激光照射下,比较不同浓度光敏剂及不同光剂量对肝癌细胞残存率的影响,并粗略比较在光剂量一定的情况下,激光强度对肝癌细胞残存率的影响。将细胞按实验设计转移至96孔板,常规培养,细胞贴壁后,加入不同浓度的光敏剂,为照光方便,每板只接受一种浓度光敏剂(6mg/L或10mg/L)。孵育4h后,弃去光敏剂,进行激光照射。为照光方便,每板只接受一种激光强度(30mW/cm~2或50mW/cm~2),调整照光时间,使D2、D4、D6、D8、D10、F2、F4、F6、F8、F10孔分别接受2,4,6,8,10,12,14,16,18,20J/cm~2的光剂量。B2至B4为空白对照,B5至B7为实验对照,B8至B10为光敏剂实验组。照光后继续孵育24h,进行MTT比色实验。相同的实验重复六次。(3)流式细胞分析检测细胞周期。肝癌细胞行PDT处理后,继续孵育24h,常规消化处理培养细胞,收集细胞,以标准程序进行流式细胞分析,结果用细胞周期拟合软件进行分析。(4)Annexin V-FITC/PI检测细胞凋亡。肝癌细胞行PDT处理后,继续孵育24h,常规消化处理培养细胞,依照Annexin V-FITC/PI双染试剂盒染色说明检测细胞凋亡。
结果:
(1)MTT结果显示,无光照情况下,不同浓度光敏剂对肝癌细胞的增殖不产生明显影响(P＞0.05)。(2)MTT结果显示PDT对HepG2肝癌细胞有很强的抑制作用,在一定范围条件下,随着光敏剂浓度及激光剂量的增加,细胞残存率明显下降(P＜0.05);在光剂量大于12J/cm~2后细胞残存率下降趋于缓和甚至不再产生明显下降(P＞0.05)。此外,在光敏剂浓度及激光剂量一定的情况下,30mW/cm~2的激光强度似乎比50mW/cm~2的效果更优。(3)流式细胞分析显示PDT后肝癌细胞周期被阻滞于G1、G2期,正常的细胞复制被阻断。(4)AnnexinV-FITC/PI检测发现PDT诱导了明显的细胞凋亡。
结论:
体外实验显示:PDT对HepG2肝癌细胞有很强的抑制作用,在一定的范围条件下,随着光敏剂浓度及激光剂量的增加,抑制作用明显增强。这种抑制作用可能由于PDT后正常的肝癌细胞复制周期被阻断导致细胞凋亡而引起的。
Objective:
Primary liver cancer severely threatens the health of people,because 43.7%liver cancer in the world is in our country.The progress of liver cancer is relatively hiding, which results in many problems in clinical diagnosis and treatment that early diagnosis is hard,the rate of relapse and diversion is high,treatments lack pertinence and treatment means is few.So,it's important and urgent to study new technology and therapeutics for liver cancer.As a new tumor treatment modality,photodynamic therapy(PDT)has been applied to clinic and gained relatively perfect treatment effect. But,PDT is mostly applied to superficial tumor and brain tumor.The correlative study on intraperitoneal tumor such as liver cancer is rare.This experiment is to observe the inhibitory effect of PDT for hepatoma cell under different experiment conditions and to primarily observe the mechanism of PDT.
Content:
HepG2 human hepatoma cells were routinely cultured.The cell viability was determined by MTT assay after being treated with different concentration photosan without irradiation and being treated by PDT with different concentration photosan and different light doses and the cell cycle and cell apoptosis was detected by flow cytometer(FCM)after PDT.
Methods:
The HepG2 cells were recovered and cultured routinely.This experiment was divided four parts:(1)comparing the survival of hepatoma cell after being treated with different concentration photosan without irradiation.The cells were added into 96 wells culture plate according to experiment design making that the well from B2 to G2 was blank control added in only culture medium,the well from B3 to G3 was experiment control added in only cells and the rectangle area from B4 to G11 was photosensitizer experiment added in photosan by column with the different concentrations of 2,4,6,8,10,12,14,16mg/L,when the cells attached to the substratum.The experiment cells were incubated with photosan for 4 hours,then the photosan was abandoned and the cells continued to be incubated for 24 hours before the cell survival was determined by MTT assay.(2)Comparing the survival of hepatoma cell after PDT under different experiment conditions.The cells were added into 96 wells culture plate according to experiment design making that the well from B2 to B4 was blank control added in only culture medium,the well from B5 to B7 was experiment control added in only cells,the well from B8 to B10 was photosensitizer experiment added in photosan and cells without irradiation and D2、D4、D6、D8、D10、F2、F4、F6、F8、F10 wells was treated by PDT with different light doses of 2,4,6,8,10,12,14,16,18,20J/cm~2,respectively.The cells in one plate were treated with one concentration(6mg/L or 10mg/L)and one light intensity(30mW/cm~2 or 50mW/cm~2).The same experiment was repeated six times. The cell survival was determined by MTT assay,too.(3)Detecting the cell cycle after PDT by FCM.The cells were incubated for 24 hours after PDT,then collected and the cell cycle was detected by FCM.(4)Detecting the cell apoptosis after PDT by Annexin V-FITC/PI method.The cells were incubated for 24 hours after PDT,and the cell apoptosis was detected by Annexin V-FITC/PI method.
Results:
(1)MTT test showed that the cell viability had no significant difference(P＞0.05)after being treated with different concentration photosan without irradiation.(2) MTT test showed that the inhibitory effect of PDT for hepatoma cell was strong.The survival was descending obviously(P＜0.05)along with the increasing of the light dose and the photosan concentration in some condition.When the light dose was beyond 12J/cm~2,the descending of the survival was slow even no further change(P＞0.05).Besides,the light intensity of 30mW/cm~2 was superior to 50mW/cm~2,when the photosan concentration and the light dose were same.(3)FCM test showed that the cell cycle was blocked to G1、G2 phase after PDT.(4)Annexin V-FITC /PI test showed that the cell apoptosis was induced by PDT.
Conclusion:
The inhibitory effect of PDT for hepatoma cell is strong.The survival is descending obviously along with the increasing of the light dose and the photosan concentration in some condition.This inhibitory effect may be due to that the cell cycle is blocked after PDT,which induces the cell apoptosis.
全世界43.7%的肝癌患者在中国,严重威胁着我国人民的健康。原发性肝癌发病比较隐匿,临床诊疗中存在早期诊断难、复发转移率高、治疗缺乏针对性、治疗手段少等问题。因此对肝癌诊治新技术、新疗法的研究尤为重要和迫切。光动力疗法(Photodynamic therapy,PDT)作为一种新的肿瘤治疗方法已应用于临床,对体表和脑内肿瘤等取得了比较理想的治疗效果,但对肝脏肿瘤等腹腔内肿瘤的相关研究较少。本实验拟在初步探讨photosan—PDT在不同实验条件下对人HepG2肝癌细胞增殖的抑制作用并初步探讨其作用机制,以期为PDT成为肝癌治疗新方法提供进一步的理论依据。
内容:
将体外培养的人HepG2肝癌细胞常规传代后,在无光照情况下,比较不同浓度光敏剂—photosan对肝癌细胞增殖的影响,然后以photosan为光敏剂、半导体激光器(波长630nm)为光源进行激光照射,在不同浓度光敏剂经不同剂量的光照后,用MTT法测定PDT后HepG2肝癌细胞的残存率,并用流式细胞分析检测PDT对肝癌细胞周期的影响,以Annexin V-FITC/PI检测细胞凋亡。
方法:
HepG2肝癌细胞复苏,常规培养并传代。实验主要分四部分:(1)无光照情况下,不同浓度光敏剂对肝癌细胞增殖的影响。将细胞按实验设计转移至96孔板,常规培养,细胞贴壁后,加入不同浓度的光敏剂,使B2至G2为空白对照,既不加细胞也不加光敏剂,只加培养基;B3至G3为实验对照,只加细胞不加光敏剂;B4至G11的矩形区域为光敏剂实验组,按列分别加入2,4,6,8,10,12,14,16mg/L的光敏剂,孵育4h后,弃去光敏剂,继续孵育24h,进行MTT比色实验。(2)在激光照射下,比较不同浓度光敏剂及不同光剂量对肝癌细胞残存率的影响,并粗略比较在光剂量一定的情况下,激光强度对肝癌细胞残存率的影响。将细胞按实验设计转移至96孔板,常规培养,细胞贴壁后,加入不同浓度的光敏剂,为照光方便,每板只接受一种浓度光敏剂(6mg/L或10mg/L)。孵育4h后,弃去光敏剂,进行激光照射。为照光方便,每板只接受一种激光强度(30mW/cm~2或50mW/cm~2),调整照光时间,使D2、D4、D6、D8、D10、F2、F4、F6、F8、F10孔分别接受2,4,6,8,10,12,14,16,18,20J/cm~2的光剂量。B2至B4为空白对照,B5至B7为实验对照,B8至B10为光敏剂实验组。照光后继续孵育24h,进行MTT比色实验。相同的实验重复六次。(3)流式细胞分析检测细胞周期。肝癌细胞行PDT处理后,继续孵育24h,常规消化处理培养细胞,收集细胞,以标准程序进行流式细胞分析,结果用细胞周期拟合软件进行分析。(4)Annexin V-FITC/PI检测细胞凋亡。肝癌细胞行PDT处理后,继续孵育24h,常规消化处理培养细胞,依照Annexin V-FITC/PI双染试剂盒染色说明检测细胞凋亡。
结果:
(1)MTT结果显示,无光照情况下,不同浓度光敏剂对肝癌细胞的增殖不产生明显影响(P＞0.05)。(2)MTT结果显示PDT对HepG2肝癌细胞有很强的抑制作用,在一定范围条件下,随着光敏剂浓度及激光剂量的增加,细胞残存率明显下降(P＜0.05);在光剂量大于12J/cm~2后细胞残存率下降趋于缓和甚至不再产生明显下降(P＞0.05)。此外,在光敏剂浓度及激光剂量一定的情况下,30mW/cm~2的激光强度似乎比50mW/cm~2的效果更优。(3)流式细胞分析显示PDT后肝癌细胞周期被阻滞于G1、G2期,正常的细胞复制被阻断。(4)AnnexinV-FITC/PI检测发现PDT诱导了明显的细胞凋亡。
结论:
体外实验显示:PDT对HepG2肝癌细胞有很强的抑制作用,在一定的范围条件下,随着光敏剂浓度及激光剂量的增加,抑制作用明显增强。这种抑制作用可能由于PDT后正常的肝癌细胞复制周期被阻断导致细胞凋亡而引起的。
Objective:
Primary liver cancer severely threatens the health of people,because 43.7%liver cancer in the world is in our country.The progress of liver cancer is relatively hiding, which results in many problems in clinical diagnosis and treatment that early diagnosis is hard,the rate of relapse and diversion is high,treatments lack pertinence and treatment means is few.So,it's important and urgent to study new technology and therapeutics for liver cancer.As a new tumor treatment modality,photodynamic therapy(PDT)has been applied to clinic and gained relatively perfect treatment effect. But,PDT is mostly applied to superficial tumor and brain tumor.The correlative study on intraperitoneal tumor such as liver cancer is rare.This experiment is to observe the inhibitory effect of PDT for hepatoma cell under different experiment conditions and to primarily observe the mechanism of PDT.
Content:
HepG2 human hepatoma cells were routinely cultured.The cell viability was determined by MTT assay after being treated with different concentration photosan without irradiation and being treated by PDT with different concentration photosan and different light doses and the cell cycle and cell apoptosis was detected by flow cytometer(FCM)after PDT.
Methods:
The HepG2 cells were recovered and cultured routinely.This experiment was divided four parts:(1)comparing the survival of hepatoma cell after being treated with different concentration photosan without irradiation.The cells were added into 96 wells culture plate according to experiment design making that the well from B2 to G2 was blank control added in only culture medium,the well from B3 to G3 was experiment control added in only cells and the rectangle area from B4 to G11 was photosensitizer experiment added in photosan by column with the different concentrations of 2,4,6,8,10,12,14,16mg/L,when the cells attached to the substratum.The experiment cells were incubated with photosan for 4 hours,then the photosan was abandoned and the cells continued to be incubated for 24 hours before the cell survival was determined by MTT assay.(2)Comparing the survival of hepatoma cell after PDT under different experiment conditions.The cells were added into 96 wells culture plate according to experiment design making that the well from B2 to B4 was blank control added in only culture medium,the well from B5 to B7 was experiment control added in only cells,the well from B8 to B10 was photosensitizer experiment added in photosan and cells without irradiation and D2、D4、D6、D8、D10、F2、F4、F6、F8、F10 wells was treated by PDT with different light doses of 2,4,6,8,10,12,14,16,18,20J/cm~2,respectively.The cells in one plate were treated with one concentration(6mg/L or 10mg/L)and one light intensity(30mW/cm~2 or 50mW/cm~2).The same experiment was repeated six times. The cell survival was determined by MTT assay,too.(3)Detecting the cell cycle after PDT by FCM.The cells were incubated for 24 hours after PDT,then collected and the cell cycle was detected by FCM.(4)Detecting the cell apoptosis after PDT by Annexin V-FITC/PI method.The cells were incubated for 24 hours after PDT,and the cell apoptosis was detected by Annexin V-FITC/PI method.
Results:
(1)MTT test showed that the cell viability had no significant difference(P＞0.05)after being treated with different concentration photosan without irradiation.(2) MTT test showed that the inhibitory effect of PDT for hepatoma cell was strong.The survival was descending obviously(P＜0.05)along with the increasing of the light dose and the photosan concentration in some condition.When the light dose was beyond 12J/cm~2,the descending of the survival was slow even no further change(P＞0.05).Besides,the light intensity of 30mW/cm~2 was superior to 50mW/cm~2,when the photosan concentration and the light dose were same.(3)FCM test showed that the cell cycle was blocked to G1、G2 phase after PDT.(4)Annexin V-FITC /PI test showed that the cell apoptosis was induced by PDT.
Conclusion:
The inhibitory effect of PDT for hepatoma cell is strong.The survival is descending obviously along with the increasing of the light dose and the photosan concentration in some condition.This inhibitory effect may be due to that the cell cycle is blocked after PDT,which induces the cell apoptosis.
引文
[1]王宝恩,张定凤.现代肝脏病学[M].第一版,北京:科学出版社,2003:852-857.
[2]Parkin DM,Bray F,Ferlay J,et al.Global cancer statistics,2002[J].CA Cancer J Clin,2005,55:74-108.
[3]Sherman M.Hepatocellular carcinoma:epidemiology,risk factors,and screening[J].SeminLiver Dis,2005,25:143-154.
[4]王红阳,付静.肝癌研究现状与进展[J].中华肝脏病杂志,2007,15(8):561-562.
[5]李瑜元.光动力疗法诊治消化系疾病[J].现代消化及介入诊疗,2004,9(3):180-183.
[6]丁新民,徐勤枝,顾瑛,等.光动力学治疗肿瘤的简史和现状[J].中国肿瘤,2003,12(3):151-155.
[7]Dougherty T,Gomer C,Henderson B,et al.Photodynamic therapy[J].J Natl Cancer Inst,1998,90:889-905.
[8]Triesscheijn M,Baas P,Schellens JanH.M.,et al.Photodynamic therapy in oncology[J].The Oncologist,2006,11:1034-1044.
[9]肖卫东,葛海燕,陈祖林.光动力疗法治疗结肠癌肝转移的研究进展[J].中国激光医学杂志,2003,12(2):119-121.
[10]Van den Bergh H.On the evolution of some endoscopic light delivery systems for photodynamictherapy[J].Endoscopy,1998,30:392-407.
[11]Date M,Fukuchi K,Namiki Y,et al.Therapeutic effect of photodynamic therapy using PAD-S31 and diode laser on human liver cancer cells[J].Liver Int,2004,24(2):142-148.
[12]Yow CM,Wong CK,Huang Z,et al.Study of the efficacy and mechanism of ALA-mediated photodynamic therapy on human hepatocellular carcinoma cell[J].Liver Int,2007,27(2):201-208.
[13]Kessel D,Caruso JA.,Reiners JJ.,et al.Potentiation of photodynamic therapy by Ursodeoxycholic Acid[J].CANCER RESEARCH,2000,60:6985-6988.
[14]Kirveliene V,Grazeliene G,Dabkeviciene D,et al.Schedule-dependent interaction between Doxorubicin and mTHPC-mediated photodynamic therapy in murine hepatoma in vitro and in vivo[J].Cancer Chemother Pharrnacol,2006,57(1):65-72.
[15]李翔,易继林,万亚峰,等.光动力作用对人肝癌细胞HepG2的影响及其机制探讨[J].华中医学杂志,2007,31(3):198-200.
[16]韩锐.抗癌药物研究与实验技术[M].第1版,北京医科大学中国协和医科大学联合出版社,1997:285-286.
[17]崔巍.流式细胞术PI染色进行细胞周期分析的原理是什么?[J].中华检验医学杂志,2004,27(3):151-151.
[18]Baserga R.The Relationship of the Cell Cycle to Tumor Growth and Control of Cell Division:A Review[J].CANCER RESEARCH,1965,25(5):581-595.
[19]王敬春,苗术,黄海涛.流式细胞仪在细胞凋亡检测中的应用[J].齐齐哈尔医学院学报,2007,28(8):970-971.
[20]马鑫,孟志强.原发性肝癌的非手术治疗进展[J].世界肿瘤杂志,2007,6(2):133-136.
[21]Harrod-Kim P.Tumor ablation with photodynamic therapy:introduction to mechanism and clinical applications[J].JVIR,2006,17(9):1441-1448.
[22]Foote CS.Definition of type Ⅰ and type Ⅱ photosensitized oxidation[J].PhotochemPhotobiol,1991,54:659-659.
[23]Athar M,Elmets CA,Bickers DR,et al.A novel mechanism for the generation of superoxide anions in hematoporphyrin derivative-mediated cutaneous photosensitization.Activation of the xanthine oxidase pathway[J].J Clin Invest,1989,83:1137-1143.
[24]李立,富永明,董齐.光动力疗法治疗现状[J].中国误诊学杂志,2005,5(3):439-440.
[25]Johansen LS.Photodynamic therapy:A new method for the treatment of cancer[J].Ugeskr-Laeger,1999,161:3992-3995.
[26]Rhodes LE,de Rie M,Enstrom Y,et al.Photodynamic therapy using topical methyl aminolevulinate vs surgery for nodular basal cell carcinoma:results of a multicenter randomized prospective trial[J].Arch Dermatol,2004,140:17-23.
[27]Fien SM,Oseroff AR.Photodynamic therapy for non-melanoma skin cancer[J].J Natl Compr Canc Netw,2007,5(5):531-540.
[28]Manyak MJ,Ogan K.Photodynamic therapy for refractory superficial bladder cancer:long-term clinical outcomes of single treatment using intravesical diffusionmedium[J].J Endourol,2003,17(8):633-639.
[29]Moghissi K,Dixon K,Thorpe JAC,et al.Photodynamic therapy(PDT)in early central lung cancer:a treatment option for patients ineligible for surgical resection[J].Thorax,2007,62:391-395.
[30]Jang TW,Kim HK,Oak CH,et al.Photodynamic therapy in early lung cancer:a report of two cases[J].Korean J Intern Med,2006,21(3):178-182.
[31]Ha BW,Kim JI,Hwang EM,et al.A case of photodynamic therapy for early esophageal cancer recurred after esophagectomy[J].Korean J Gastroenterol,2007,49(5):331-335.
[32]Litle VR.,Luketich JD.,Christie NA.,et al.Photodynamic therapy as palliation for esophageal cancer:experience in 215 patients[J].Ann.Thorac.Surg.,2003,76:1687-1693.
[33]Lou PJ,Jager HR,Jones L,et al.Interstitial photodynamic therapy as salvage treatment for recurrent head and neck cancer[J].Br J Cancer,2004,91(3):441-446.
[34]Biel M.Advances in photodynamic therapy for the treatment of head and neck cancers[J].Lasers SurgMed,2006,38(5):349-355.
[35]Schmidt MH,Meyer GA,Reichert KW,et al.Evaluation of photodynamic therapy near functional brain tissue in patients with recurrent brain tumors[J].J Neurooncol,2004,67(1-2):201-207.
[36]Zoepf T,Jakobs R,Arnold JC,et al.Palliation of nonresectable bile duct cancer:improved survival after photodynamic therapy[J].Am J Gastroenterol,2005,100:2426-2430.
[37]陈杰波,欧敏锐,李忠琴,等.光动力治疗药物——光敏剂的研究进展[J].海峡药学,2003,15(6):8-12.
[38]杨小民,罗荣城,李黎波,等.血卟啉衍生物光动力效应对体外培养人鼻咽癌细胞株的杀伤作用[J].中国组织工程研究与临床康复,2007,11(9):1707-1709.
[39]龙朝钦,邓军,钟白玉,等.5—氨基酮戊酸光动力效应对体外培养人恶性黑素瘤细胞的物理学干预特征[J].中国临床康复,2005,9(10):133-135.
[40]刘慧龙,刘凡光,顾瑛.影响光动力疗法的几个主要因素[J].中国激光医学杂志,2002,11(2):121-124.
[41]Glenn F,Rex M.UVA radiation induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen[J].Free Radic Biol Med,1995,18:721-730.
[42]Foster TH,Murant RS,Bryant RG,et al.Oxygen consumption and diffusion effects in photodynamic therapy[J].Radiation Research,1991,126:296-303.
[43]Suzuki S,Nakamura S,Sakaguchi S,et al.Experimental study of intra abdominal photodynamic therapy[J].Lasers Medical Science,1987,2(2):195-203.
[44]Sun Cun-ying,Zhao Dao-mu,Zhou Lin-yun,et al.Quantum analysis of heat effect on laser-tissue interaction[J].Acta Photonica Sinica,2002,31(1):15-17.
[45]Star WM.Light dosimetry in vivo[J].Phys Med Biol,1997,42:763-787.
[46]Wyld L,Smith O,Lawry J,et al.Cell cycle phase influences tumour cell sensitivity to aminolaevulinic acid-induced photodynamic therapy in vitro[J].Br J Cancer,1998,78(1):50-55.
[47]Rebeiz N,Arkins S,Kelley KW,et al.Modulator of heme biosynthesis induces apoptosis in leukemia cell[J].J Clin Laser Med Surg,2001,19(2):59-67.
[48]Sano M,Furuta T,Takahira K,et al.Cell-cycle-dependent efficacy of photodynamic therapy with ATX-S10(Na)[J].Lasers Med Sci,2005,20(1):1-5.
[49]Cohen JJ.Apoptosis[J].Immunol Today,1993,14(3):126-130.
[50]Plaetzer K,Kiesslich T,Krammer B,et al.Characterization of the cell death modes and the associated changes in cellular energy supply in response to AIPcS4-PDT[J].Photochem Photobiol Sci,2002,1(3):172-177.
[51]Conlon KA,Zharkov DO,Berrios M.Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase(mOGG1):mOGG1 associates with microtubules during interphase and mitosis[J].DNA Repair,2004,12(3):1601-1615.
[2]Parkin DM,Bray F,Ferlay J,et al.Global cancer statistics,2002[J].CA Cancer J Clin,2005,55:74-108.
[3]Sherman M.Hepatocellular carcinoma:epidemiology,risk factors,and screening[J].SeminLiver Dis,2005,25:143-154.
[4]王红阳,付静.肝癌研究现状与进展[J].中华肝脏病杂志,2007,15(8):561-562.
[5]李瑜元.光动力疗法诊治消化系疾病[J].现代消化及介入诊疗,2004,9(3):180-183.
[6]丁新民,徐勤枝,顾瑛,等.光动力学治疗肿瘤的简史和现状[J].中国肿瘤,2003,12(3):151-155.
[7]Dougherty T,Gomer C,Henderson B,et al.Photodynamic therapy[J].J Natl Cancer Inst,1998,90:889-905.
[8]Triesscheijn M,Baas P,Schellens JanH.M.,et al.Photodynamic therapy in oncology[J].The Oncologist,2006,11:1034-1044.
[9]肖卫东,葛海燕,陈祖林.光动力疗法治疗结肠癌肝转移的研究进展[J].中国激光医学杂志,2003,12(2):119-121.
[10]Van den Bergh H.On the evolution of some endoscopic light delivery systems for photodynamictherapy[J].Endoscopy,1998,30:392-407.
[11]Date M,Fukuchi K,Namiki Y,et al.Therapeutic effect of photodynamic therapy using PAD-S31 and diode laser on human liver cancer cells[J].Liver Int,2004,24(2):142-148.
[12]Yow CM,Wong CK,Huang Z,et al.Study of the efficacy and mechanism of ALA-mediated photodynamic therapy on human hepatocellular carcinoma cell[J].Liver Int,2007,27(2):201-208.
[13]Kessel D,Caruso JA.,Reiners JJ.,et al.Potentiation of photodynamic therapy by Ursodeoxycholic Acid[J].CANCER RESEARCH,2000,60:6985-6988.
[14]Kirveliene V,Grazeliene G,Dabkeviciene D,et al.Schedule-dependent interaction between Doxorubicin and mTHPC-mediated photodynamic therapy in murine hepatoma in vitro and in vivo[J].Cancer Chemother Pharrnacol,2006,57(1):65-72.
[15]李翔,易继林,万亚峰,等.光动力作用对人肝癌细胞HepG2的影响及其机制探讨[J].华中医学杂志,2007,31(3):198-200.
[16]韩锐.抗癌药物研究与实验技术[M].第1版,北京医科大学中国协和医科大学联合出版社,1997:285-286.
[17]崔巍.流式细胞术PI染色进行细胞周期分析的原理是什么?[J].中华检验医学杂志,2004,27(3):151-151.
[18]Baserga R.The Relationship of the Cell Cycle to Tumor Growth and Control of Cell Division:A Review[J].CANCER RESEARCH,1965,25(5):581-595.
[19]王敬春,苗术,黄海涛.流式细胞仪在细胞凋亡检测中的应用[J].齐齐哈尔医学院学报,2007,28(8):970-971.
[20]马鑫,孟志强.原发性肝癌的非手术治疗进展[J].世界肿瘤杂志,2007,6(2):133-136.
[21]Harrod-Kim P.Tumor ablation with photodynamic therapy:introduction to mechanism and clinical applications[J].JVIR,2006,17(9):1441-1448.
[22]Foote CS.Definition of type Ⅰ and type Ⅱ photosensitized oxidation[J].PhotochemPhotobiol,1991,54:659-659.
[23]Athar M,Elmets CA,Bickers DR,et al.A novel mechanism for the generation of superoxide anions in hematoporphyrin derivative-mediated cutaneous photosensitization.Activation of the xanthine oxidase pathway[J].J Clin Invest,1989,83:1137-1143.
[24]李立,富永明,董齐.光动力疗法治疗现状[J].中国误诊学杂志,2005,5(3):439-440.
[25]Johansen LS.Photodynamic therapy:A new method for the treatment of cancer[J].Ugeskr-Laeger,1999,161:3992-3995.
[26]Rhodes LE,de Rie M,Enstrom Y,et al.Photodynamic therapy using topical methyl aminolevulinate vs surgery for nodular basal cell carcinoma:results of a multicenter randomized prospective trial[J].Arch Dermatol,2004,140:17-23.
[27]Fien SM,Oseroff AR.Photodynamic therapy for non-melanoma skin cancer[J].J Natl Compr Canc Netw,2007,5(5):531-540.
[28]Manyak MJ,Ogan K.Photodynamic therapy for refractory superficial bladder cancer:long-term clinical outcomes of single treatment using intravesical diffusionmedium[J].J Endourol,2003,17(8):633-639.
[29]Moghissi K,Dixon K,Thorpe JAC,et al.Photodynamic therapy(PDT)in early central lung cancer:a treatment option for patients ineligible for surgical resection[J].Thorax,2007,62:391-395.
[30]Jang TW,Kim HK,Oak CH,et al.Photodynamic therapy in early lung cancer:a report of two cases[J].Korean J Intern Med,2006,21(3):178-182.
[31]Ha BW,Kim JI,Hwang EM,et al.A case of photodynamic therapy for early esophageal cancer recurred after esophagectomy[J].Korean J Gastroenterol,2007,49(5):331-335.
[32]Litle VR.,Luketich JD.,Christie NA.,et al.Photodynamic therapy as palliation for esophageal cancer:experience in 215 patients[J].Ann.Thorac.Surg.,2003,76:1687-1693.
[33]Lou PJ,Jager HR,Jones L,et al.Interstitial photodynamic therapy as salvage treatment for recurrent head and neck cancer[J].Br J Cancer,2004,91(3):441-446.
[34]Biel M.Advances in photodynamic therapy for the treatment of head and neck cancers[J].Lasers SurgMed,2006,38(5):349-355.
[35]Schmidt MH,Meyer GA,Reichert KW,et al.Evaluation of photodynamic therapy near functional brain tissue in patients with recurrent brain tumors[J].J Neurooncol,2004,67(1-2):201-207.
[36]Zoepf T,Jakobs R,Arnold JC,et al.Palliation of nonresectable bile duct cancer:improved survival after photodynamic therapy[J].Am J Gastroenterol,2005,100:2426-2430.
[37]陈杰波,欧敏锐,李忠琴,等.光动力治疗药物——光敏剂的研究进展[J].海峡药学,2003,15(6):8-12.
[38]杨小民,罗荣城,李黎波,等.血卟啉衍生物光动力效应对体外培养人鼻咽癌细胞株的杀伤作用[J].中国组织工程研究与临床康复,2007,11(9):1707-1709.
[39]龙朝钦,邓军,钟白玉,等.5—氨基酮戊酸光动力效应对体外培养人恶性黑素瘤细胞的物理学干预特征[J].中国临床康复,2005,9(10):133-135.
[40]刘慧龙,刘凡光,顾瑛.影响光动力疗法的几个主要因素[J].中国激光医学杂志,2002,11(2):121-124.
[41]Glenn F,Rex M.UVA radiation induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen[J].Free Radic Biol Med,1995,18:721-730.
[42]Foster TH,Murant RS,Bryant RG,et al.Oxygen consumption and diffusion effects in photodynamic therapy[J].Radiation Research,1991,126:296-303.
[43]Suzuki S,Nakamura S,Sakaguchi S,et al.Experimental study of intra abdominal photodynamic therapy[J].Lasers Medical Science,1987,2(2):195-203.
[44]Sun Cun-ying,Zhao Dao-mu,Zhou Lin-yun,et al.Quantum analysis of heat effect on laser-tissue interaction[J].Acta Photonica Sinica,2002,31(1):15-17.
[45]Star WM.Light dosimetry in vivo[J].Phys Med Biol,1997,42:763-787.
[46]Wyld L,Smith O,Lawry J,et al.Cell cycle phase influences tumour cell sensitivity to aminolaevulinic acid-induced photodynamic therapy in vitro[J].Br J Cancer,1998,78(1):50-55.
[47]Rebeiz N,Arkins S,Kelley KW,et al.Modulator of heme biosynthesis induces apoptosis in leukemia cell[J].J Clin Laser Med Surg,2001,19(2):59-67.
[48]Sano M,Furuta T,Takahira K,et al.Cell-cycle-dependent efficacy of photodynamic therapy with ATX-S10(Na)[J].Lasers Med Sci,2005,20(1):1-5.
[49]Cohen JJ.Apoptosis[J].Immunol Today,1993,14(3):126-130.
[50]Plaetzer K,Kiesslich T,Krammer B,et al.Characterization of the cell death modes and the associated changes in cellular energy supply in response to AIPcS4-PDT[J].Photochem Photobiol Sci,2002,1(3):172-177.
[51]Conlon KA,Zharkov DO,Berrios M.Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase(mOGG1):mOGG1 associates with microtubules during interphase and mitosis[J].DNA Repair,2004,12(3):1601-1615.