Notch1在胆管癌细胞光动力疗法中的作用研究
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摘要
目的:1、研究Photosan介导光动力疗法(Photodynamic Therapy,PDT)对胆管癌细胞的杀伤作用。2、探讨Notch1在胆管癌发生中的表达情况以及Notch1在PDT治疗胆管癌细胞中的作用。方法:1、体外培养人胆管癌QBC939细胞株,在细胞对数生长期用不同浓度Photosan处理,并用半导体激光治疗仪不同强度光照后,采用MTT法检测PDT对QBC939细胞的杀伤作用。观察不同的光敏剂孵育时间、光敏剂浓度及光照剂量对PDT效果的影响。2、采用免疫细胞化学方法和蛋白印迹法检测胆管癌细胞Notch1表达,经Photosan介导光动力作用胆管癌细胞后,检测胆管癌细胞内Notch1表达变化。结果:1、MTT结果显示Photosan介导的PDT对胆管癌细胞有杀伤作用(P<0.05),而且这种杀伤作用在一定范围内随着光敏剂浓度增加、光敏剂孵育时间、光照剂量呈正相关。2、免疫细胞学检查发现Notch1在胆管癌细胞中高表达,其表达主要位于细胞膜及胞浆,并且经PDT处理后Notch1表达量较前减少(P<0.05)。结论:Notch1与胆管癌细胞生长、增殖密切相关,且Notch1在PDT对胆管癌细胞产生的抑制、促凋亡和杀伤作用中有重要作用。
Objective: 1. Research the killing effect of Photosan mediated photodynamic therapy( PDT) for cholangiocarcinoma cells. 2. Investigate the expression of Notch1 protein in cholangiocarcinoma cells and the function of Notch1 when deal with PDT. Methods: 1. Cholangiocarcinoma cells line QBC939 was cultured in vitro,and incubated with different concentration of Photosan when QBC939 growth is in the logarithmic phase,then irradiated with semi-conducted laser device with different light doses. Detect the killing effects of PDT for cholangiocarcinoma with MTT assay when applying with different incubation time and changable concentrations of photosensitizer and variable doses of light. 2. Detect the expression of Notch1 protein in cholangiocarcinoma cells by using immunofluoresence method and Western-blot method,and detect the variation of Notch1 after PDT. Result: 1. MTT assay show that Photosan mediated PDT have killing effect to cholangiocarcinoma cells( P < 0. 05). Under certain circumstance,this result positively correlate to the longer incubation time,higher photosensitizer concentration and larger dose of light. 2. Notch1 was highly expressed in membrane and cytoplasm in cholangiocarcinoma which was detected by immunochemistry assay. The rate of Notch1 was decreassed when deal using different parameters for PDT. Conclusions: Notch1 protein was highly related to the development and proliferation of cholangiocarcinoma cells and plays important role in killing and hampering the growth of cholangiocarcinoma cells while treating with PDT.
Objective: 1. Research the killing effect of Photosan mediated photodynamic therapy( PDT) for cholangiocarcinoma cells. 2. Investigate the expression of Notch1 protein in cholangiocarcinoma cells and the function of Notch1 when deal with PDT. Methods: 1. Cholangiocarcinoma cells line QBC939 was cultured in vitro,and incubated with different concentration of Photosan when QBC939 growth is in the logarithmic phase,then irradiated with semi-conducted laser device with different light doses. Detect the killing effects of PDT for cholangiocarcinoma with MTT assay when applying with different incubation time and changable concentrations of photosensitizer and variable doses of light. 2. Detect the expression of Notch1 protein in cholangiocarcinoma cells by using immunofluoresence method and Western-blot method,and detect the variation of Notch1 after PDT. Result: 1. MTT assay show that Photosan mediated PDT have killing effect to cholangiocarcinoma cells( P < 0. 05). Under certain circumstance,this result positively correlate to the longer incubation time,higher photosensitizer concentration and larger dose of light. 2. Notch1 was highly expressed in membrane and cytoplasm in cholangiocarcinoma which was detected by immunochemistry assay. The rate of Notch1 was decreassed when deal using different parameters for PDT. Conclusions: Notch1 protein was highly related to the development and proliferation of cholangiocarcinoma cells and plays important role in killing and hampering the growth of cholangiocarcinoma cells while treating with PDT.
引文
[1]GATTO M,ALVARO D.New insights on cholangiocarcinoma[J].World J Gastrointest Oncol,2010,2(3):136-145.
[2]黄志强.肝胆管外科的发展方向[J].外科理论与实践,2011,16(4):329-331.HUANG Zhiqiang.Developing direction of hepatobiliary surgery[J].Journal of Surgery Concepts&Practice,2011,16(4):329-331.
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[4]MFOUO-TYNGA I,ABRAHAMSE H.Cell death pathways and phthalocyanine as an efficient agent for photodynamic cancer therapy[J].Int J Mol Sci,2015,16(5):10228-10241.
[5]WANG X,HU J,WANG P,et al.Analysis of the in vivo and in vitro effects of photodynamic therapy on breast cancer by using a sensitizer,sinoporphyrin sodium[J].Theranostics,2015.5(7):772-786.
[6]WU W R,ZHANG R,SHI X D,et al.Notch1 is overexpressed in human intrahepatic cholangiocarcinoma and is associated with its proliferation,invasiveness and sensitivity to 5-fluorouracil in vitro[J].Oncol Rep,2014,31(6):2515-2524.
[7]GRENDAR J,GRENDAROVA P,SINHA R,et al.Neoadjuvant therapy for downstaging of locally advanced hilar cholangiocarcinoma:a systematic review[J].HPB(Oxford),2014,16(4):297-303.
[8]RICHTER J A,KAHALEH M.Photodynamic therapy:Palliation and endoscopic technique in cholangiocarcinoma[J].World J Gastrointest Endosc,2010,2(11):357-361.
[9]BECHMANN L P,PHILIP HILGARD,ANDREA FRILLING,et al.Successful photodynamic therapy for biliary papillomatosis:a case report[J].World J Gastroenterol,2008,14(26):4234-4237.
[10]ZHOU J J,XIONG L,LI Q L,et al.Photodynamic therapy for high-grade dysplasia of bile duct via a choledochoscope[J].World J Gastroenterol,2013,19(33):5590-5592.
[11]姜海涛,曹景玉,韩瑞,等.光动力疗法促进胆管癌细胞QBC939的凋亡[J].世界华人消化杂志,2013,21(39):1853-1858.JIANG Haitao,CAO Jingyu,HAN Rui,et al.Photodynamic therapy promotes apoptosis of cholangiocarcinoma QBC939 cells[J].World Chinese Journal of Digestology,2013,21(39):1853-1858.
[12]KIM C H,CHUNG C W,CHOI K H,et al.Effect of 5-aminolevulinic acid-based photodynamic therapy via reactive oxygen species in human cholangiocarcinoma cells[J].Int J Nanomedicine,2011,6:1357-1363.
[13]WAGNER A,MAYR C.MicroRNAs associated with the efficacy of photodynamic therapy in biliary tract cancer cell lines[J].Int J Mol Sci,2014,15(11):20134-20157.
[14]刘园园,王逸飞,张春敏,等.ALA-PDT对皮肤鳞状细胞癌A431细胞Caspase-3及Caspase-7表达的影响[J].山东大学学报(医学版),2014,52(12):50-53,59.LIU Yuanyuan,WANG Yifei,ZHANG Chunmin,et al.Effects of ALA-PDT on Caspase-3 and Caspase-7 expressions incutaneous squamous cell carcinoma A431 cells[J].Journal of Shandong University(Health Sciences),2014,52(12):50-53,59.
[15]刘忠涛,刘志鹏,熊力,等.纳米氧化硅空心球负载光敏剂对肝癌体内外的杀伤作用研究[J].激光生物学报,2014,23(1):17-23.LIU Zhongtao,LIU Zhipeng,XIONG Li,et al.The killing effects of photosensitizer-loaded hollow silica nanoparticles on hepatoma cell in vitro and in vivo[J].Acta Laser Biology Sinica,2014,23(1):17-23.
[16]LI D,LI L,LI P,et al.Apoptosis of He La cells induced by a new targeting photosensitizer-based PDT via a mitochondrial pathway and ER stress[J].Onco Targets Ther,2015,8:703-711.
[17]FAN B,MALATO Y,CALVISI D F,et al.Cholangiocarcinomas can originate from hepatocytes in mice[J].J Clin Invest,2012,122(8):2911-2915.
[18]YOON H A,NOH M H,KIM B G,et al.Clinicopathological significance of altered Notch signaling in extrahepatic cholangiocarcinoma and gallbladder carcinoma[J].World J Gastroenterol,2011,17(35):4023-4030.
[19]LICCIULLI S,AVILA J L,HANLON L,et al.Notch1 is required for Kras-induced lung adenocarcinoma and controls tumor cell survival via p53[J].Cancer Res,2013,73(19):5974-5984.
[20]EL KHATIB M,BOZKO P,PALAGANI V,et al.Activation of Notch signaling is required for cholangiocarcinoma progression and is enhanced by inactivation of p53 in vivo[J].PLo S One,2013,8(10):e77433.
[21]KOFLER N M,SHAWBER C J,KANGSAMAKSIN T,et al.Notch signaling in developmental and tumor angiogenesis[J].Genes Cancer,2011,2(12):1106-1116.
[2]黄志强.肝胆管外科的发展方向[J].外科理论与实践,2011,16(4):329-331.HUANG Zhiqiang.Developing direction of hepatobiliary surgery[J].Journal of Surgery Concepts&Practice,2011,16(4):329-331.
[3]HEZEL A F,ZHU A X.Systemic therapy for biliary tract cancers[J].Oncologist,2008,13(4):415-423.
[4]MFOUO-TYNGA I,ABRAHAMSE H.Cell death pathways and phthalocyanine as an efficient agent for photodynamic cancer therapy[J].Int J Mol Sci,2015,16(5):10228-10241.
[5]WANG X,HU J,WANG P,et al.Analysis of the in vivo and in vitro effects of photodynamic therapy on breast cancer by using a sensitizer,sinoporphyrin sodium[J].Theranostics,2015.5(7):772-786.
[6]WU W R,ZHANG R,SHI X D,et al.Notch1 is overexpressed in human intrahepatic cholangiocarcinoma and is associated with its proliferation,invasiveness and sensitivity to 5-fluorouracil in vitro[J].Oncol Rep,2014,31(6):2515-2524.
[7]GRENDAR J,GRENDAROVA P,SINHA R,et al.Neoadjuvant therapy for downstaging of locally advanced hilar cholangiocarcinoma:a systematic review[J].HPB(Oxford),2014,16(4):297-303.
[8]RICHTER J A,KAHALEH M.Photodynamic therapy:Palliation and endoscopic technique in cholangiocarcinoma[J].World J Gastrointest Endosc,2010,2(11):357-361.
[9]BECHMANN L P,PHILIP HILGARD,ANDREA FRILLING,et al.Successful photodynamic therapy for biliary papillomatosis:a case report[J].World J Gastroenterol,2008,14(26):4234-4237.
[10]ZHOU J J,XIONG L,LI Q L,et al.Photodynamic therapy for high-grade dysplasia of bile duct via a choledochoscope[J].World J Gastroenterol,2013,19(33):5590-5592.
[11]姜海涛,曹景玉,韩瑞,等.光动力疗法促进胆管癌细胞QBC939的凋亡[J].世界华人消化杂志,2013,21(39):1853-1858.JIANG Haitao,CAO Jingyu,HAN Rui,et al.Photodynamic therapy promotes apoptosis of cholangiocarcinoma QBC939 cells[J].World Chinese Journal of Digestology,2013,21(39):1853-1858.
[12]KIM C H,CHUNG C W,CHOI K H,et al.Effect of 5-aminolevulinic acid-based photodynamic therapy via reactive oxygen species in human cholangiocarcinoma cells[J].Int J Nanomedicine,2011,6:1357-1363.
[13]WAGNER A,MAYR C.MicroRNAs associated with the efficacy of photodynamic therapy in biliary tract cancer cell lines[J].Int J Mol Sci,2014,15(11):20134-20157.
[14]刘园园,王逸飞,张春敏,等.ALA-PDT对皮肤鳞状细胞癌A431细胞Caspase-3及Caspase-7表达的影响[J].山东大学学报(医学版),2014,52(12):50-53,59.LIU Yuanyuan,WANG Yifei,ZHANG Chunmin,et al.Effects of ALA-PDT on Caspase-3 and Caspase-7 expressions incutaneous squamous cell carcinoma A431 cells[J].Journal of Shandong University(Health Sciences),2014,52(12):50-53,59.
[15]刘忠涛,刘志鹏,熊力,等.纳米氧化硅空心球负载光敏剂对肝癌体内外的杀伤作用研究[J].激光生物学报,2014,23(1):17-23.LIU Zhongtao,LIU Zhipeng,XIONG Li,et al.The killing effects of photosensitizer-loaded hollow silica nanoparticles on hepatoma cell in vitro and in vivo[J].Acta Laser Biology Sinica,2014,23(1):17-23.
[16]LI D,LI L,LI P,et al.Apoptosis of He La cells induced by a new targeting photosensitizer-based PDT via a mitochondrial pathway and ER stress[J].Onco Targets Ther,2015,8:703-711.
[17]FAN B,MALATO Y,CALVISI D F,et al.Cholangiocarcinomas can originate from hepatocytes in mice[J].J Clin Invest,2012,122(8):2911-2915.
[18]YOON H A,NOH M H,KIM B G,et al.Clinicopathological significance of altered Notch signaling in extrahepatic cholangiocarcinoma and gallbladder carcinoma[J].World J Gastroenterol,2011,17(35):4023-4030.
[19]LICCIULLI S,AVILA J L,HANLON L,et al.Notch1 is required for Kras-induced lung adenocarcinoma and controls tumor cell survival via p53[J].Cancer Res,2013,73(19):5974-5984.
[20]EL KHATIB M,BOZKO P,PALAGANI V,et al.Activation of Notch signaling is required for cholangiocarcinoma progression and is enhanced by inactivation of p53 in vivo[J].PLo S One,2013,8(10):e77433.
[21]KOFLER N M,SHAWBER C J,KANGSAMAKSIN T,et al.Notch signaling in developmental and tumor angiogenesis[J].Genes Cancer,2011,2(12):1106-1116.