幽门螺杆菌对胆管癌细胞增殖与凋亡及细胞核转录因子NF-κB表达的影响
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摘要
目的:探讨幽门螺杆菌(Hp)对人类胆管癌细胞增殖和凋亡的影响,以及该影响的细胞内信号转导途径。
方法:用CCK-8(Cell Counting Kit-8)法和流式细胞仪法检测Hp对胆管癌细胞增殖,凋亡及细胞周期分布情况的影响,利用免疫细胞化学和凝胶电泳迁移率变动实验(EMSA)分析细胞核转录因子NF-κB在Hp作用前后的表达活化情况。再使用特异性NF-κB阻断剂吡咯烷二硫氨基甲酸酯(PDTC),阻断NF-κB的活化,利用细胞免疫化学技术和RT-PCR半定量技术分析比较抗凋亡蛋白Bcl-2和Bcl-XL在转录因子NF-κB失活前后的表达情况。
结果:在MOI值(细菌与细胞浓度比)为0.5:1的状态下, Hp对QBC939细胞有促增殖作用,第1,3天的增殖率分别为15.24%±4.2%、34.18%±2.6%,t检验P<0.05,具有统计学意义。在该MOI值时无诱导细胞凋亡作用,但可推动细胞周期的进展;在此过程中伴有细胞NF-κB活化和抗凋亡蛋白Bcl-2,Bcl-XL表达上调;PDTC阻断实验证明抗凋亡蛋白Bcl-2,Bcl-XL的表达受到NF-κB活性的调控。
结论:幽门螺杆菌—核转录因子NF-κB—抗凋亡蛋白Bcl-2,Bcl-XL,这一传导通路的活化,在胆管癌细胞的增殖,凋亡及恶性转化中很可能具有重要作用。
Purpose: To discuss the influence on proliferation and apoptosis of human bile duct carcinoma cell by Helicobacter pylori (Hp); and signal transduction pathways in cells of this influence.
Methods: Apply the CCK-8 (Cell Counting Kit-8) method and flow cytometry to test the influence on proliferation and apoptosis of bile duct carcinoma cell by Helicobacter pylori and cell cycle distribution, utilize the immunocytochemistry and gel electrophoretic mobility shift assay ( EMSA)to analyze the expression and activation conditions of nuclear transcription factor NF-KB before and after Hp. And then use the specific NF-KB inhibitor pyrrolidine dithiocarbamate (PDTC) to inhibit the activation of NF-KB, and utilize the cellular immunity chemical technology and RT-PCR semi quantitative to analyze and compare the expression conditions of anti-apoptosis protein Bcl-2 and Bcl-XL before and after deactivation of NF-KB.
Result: At MOI (concentration ratio of bacteria to cell) of 0.5:1, Hp could facilitate the proliferation of QBC939 cells, the breeding ratios on the first and third day are 15.24%±4.2% and 34.18%±2.6% respectively, t test, P<0.05, having statistical significance. There is no induced Apoptosis at this MOI value, but could promote the development of cell cycle; the cell NF-KB is activated and the expression of anti-apoptosis protein Bcl-2 and Bcl-XL is up-regulated; the PDTC inhibition test proves that the expression of anti-apoptosis protein Bcl-2 and Bcl-XL is controlled by the activity of NF-KB.
Conclusion: Helicobacter pylori--nuclear factor NF-KB--anti-apoptosis protein Bcl-2, Bcl-XL, the activation of this pathway is likely to play a very important role in the proliferation, apoptosis and malignant transformation of bile duct carcinoma cell.
方法:用CCK-8(Cell Counting Kit-8)法和流式细胞仪法检测Hp对胆管癌细胞增殖,凋亡及细胞周期分布情况的影响,利用免疫细胞化学和凝胶电泳迁移率变动实验(EMSA)分析细胞核转录因子NF-κB在Hp作用前后的表达活化情况。再使用特异性NF-κB阻断剂吡咯烷二硫氨基甲酸酯(PDTC),阻断NF-κB的活化,利用细胞免疫化学技术和RT-PCR半定量技术分析比较抗凋亡蛋白Bcl-2和Bcl-XL在转录因子NF-κB失活前后的表达情况。
结果:在MOI值(细菌与细胞浓度比)为0.5:1的状态下, Hp对QBC939细胞有促增殖作用,第1,3天的增殖率分别为15.24%±4.2%、34.18%±2.6%,t检验P<0.05,具有统计学意义。在该MOI值时无诱导细胞凋亡作用,但可推动细胞周期的进展;在此过程中伴有细胞NF-κB活化和抗凋亡蛋白Bcl-2,Bcl-XL表达上调;PDTC阻断实验证明抗凋亡蛋白Bcl-2,Bcl-XL的表达受到NF-κB活性的调控。
结论:幽门螺杆菌—核转录因子NF-κB—抗凋亡蛋白Bcl-2,Bcl-XL,这一传导通路的活化,在胆管癌细胞的增殖,凋亡及恶性转化中很可能具有重要作用。
Purpose: To discuss the influence on proliferation and apoptosis of human bile duct carcinoma cell by Helicobacter pylori (Hp); and signal transduction pathways in cells of this influence.
Methods: Apply the CCK-8 (Cell Counting Kit-8) method and flow cytometry to test the influence on proliferation and apoptosis of bile duct carcinoma cell by Helicobacter pylori and cell cycle distribution, utilize the immunocytochemistry and gel electrophoretic mobility shift assay ( EMSA)to analyze the expression and activation conditions of nuclear transcription factor NF-KB before and after Hp. And then use the specific NF-KB inhibitor pyrrolidine dithiocarbamate (PDTC) to inhibit the activation of NF-KB, and utilize the cellular immunity chemical technology and RT-PCR semi quantitative to analyze and compare the expression conditions of anti-apoptosis protein Bcl-2 and Bcl-XL before and after deactivation of NF-KB.
Result: At MOI (concentration ratio of bacteria to cell) of 0.5:1, Hp could facilitate the proliferation of QBC939 cells, the breeding ratios on the first and third day are 15.24%±4.2% and 34.18%±2.6% respectively, t test, P<0.05, having statistical significance. There is no induced Apoptosis at this MOI value, but could promote the development of cell cycle; the cell NF-KB is activated and the expression of anti-apoptosis protein Bcl-2 and Bcl-XL is up-regulated; the PDTC inhibition test proves that the expression of anti-apoptosis protein Bcl-2 and Bcl-XL is controlled by the activity of NF-KB.
Conclusion: Helicobacter pylori--nuclear factor NF-KB--anti-apoptosis protein Bcl-2, Bcl-XL, the activation of this pathway is likely to play a very important role in the proliferation, apoptosis and malignant transformation of bile duct carcinoma cell.
引文
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[3] Jerrold M. Ward ,Miriam R. Anver ,Diana C. Haines ,et al. Chronic active hepatitis in mice caused by Helicobacter hepaticus. Am J Pathol ,1994 ,145 :959-967.
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[31]陈辉星,陈燕凌.胆管癌组织螺杆菌16S2rRNA基因的检测及意义[J].中华普通外科杂志,2003,18(12):733-735.
[32] Leelawat K,Suksumek N,Leelawat S,etal.Detection of Vac gene specific for Helicobacter pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients, Southeast[J].Asian J Trop Med Public Health,2007,38:881-885.
[33] Kuroki T,Fukuda K,Yamanouchi K,eta1.Helicohacter pylori accelerates the biliary epitheLal cell proliferation activity in hepatolithiasis[J].Hepatogastroenterol,2002,49(45):648-651.
[2] Jerrold M. Ward ,James G. Fox ,Miriam R. Anver ,et al. Chronic active hepatitis and associated liver tumors in mice caused by a persis tent bacterial infection with a novel Helicobacter species. J N C I , 1994 ,86 :122221227.
[3] Jerrold M. Ward ,Miriam R. Anver ,Diana C. Haines ,et al. Chronic active hepatitis in mice caused by Helicobacter hepaticus. Am J Pathol ,1994 ,145 :959-967.
[4] JG,Li X,Yan L,et al:Chronic proliferative hepatitis in A/JCr mice associated with persistent helicobacter hepaticus inferction,Infect Immu,1996,61:1548-58.
[5]Ponzetto A,Pellicano R,Leone N,et al:Helicobacter infertion and cirrhosis in hepatitis C virus carriage:is it an innocent bystander or a troublemaker ? Med Hypotheses,2000,54:257-9.
[6]陈辉星陈燕凌,胆管癌组织螺杆菌16S2rRNA基因的检测及意义,中国普通外科学杂志, 2003 ,18(12):733-735.
[7] Chen Ren,Fan Xue-Gong,Huang Yan,Li Ning,et al,In vitro cytotoxicity of helicobacter pylori on hepatocarcinoma HepG2 cells ,Chinese Journal Of Cancer,2004,23(1):44-49.
[8] Leelawat K,Suksumek N,Leelawat S,et al,Detection of Vac gene specific for helicobacter pyloric in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients,Southeast Asian J TropMed Public Health,2007,38:881-885.
[9] Apostolov E,Al-Soud WA,Nilsson I,et al.Helicobacter pylori and other Helicohac.ter species in gallbladder and liver of patients with chronic cholecystitis detected by immunological and molecular methods[J]. Scand J Gastroenterol, 2005, 40(1): 96-102.
[10] Kuroki T,Fukuda K,Yamanouchi K,et a1.Helicoha cter pylori accelerates the biliary epitheLal cell proliferation activity in hepatolithiasis [J]. Hepatogastroenterology,2002,49(45):648.651.
[11] Lin 1vr,Yeh CT,Wn CS,et a1.Detection and partial sequence an alysis of helicohacter pylori DNA in the bile smples[J]. Dig DisSci,1995, 40(10): 2214-2219.
[12] Matsukura N,Yokomuro S,Yamads S,eta1.Association between Hdicobacter bilis in Kh and biliary tract malignancies: H. bilis in biles from Japanese and Thai patients with benign and malignant diseases in the biliary tract[J]. Jpn J Cancer Res, 2002, 93(7): 842—847.
[13] Fukuda K, Kuroki T, Tajima Y, et a1. Comparative analysis of Helicohacter DNAs and biliary pathology in patients with and without hepatobiliary cancer[J].Carcinogenesis,2002,23(11):1927—1931.
[14]郭成,车向明. NF—KB与胃肠道肿瘤的关系.中华腹部疾病杂志,2003,3(4),0302—04.
[15] Rayet B,Celinas C,Aberrant .Bcl/ NF—KappaB genes and activity in human cancer [J],Oncogene ,1999,18:6938-6947.
[16]张建淮,赵翰林,孙玉洁,韩晓.核因子-KB及Bcl-2基因在肝外胆管癌组织中表达及意义.现代肿瘤医学,2006,14(3):32-34.
[17] Chiao PJ, Na R, Niu J , et a1. Role of Rel/ NF—kappa B transcription factors in apoptosis of human hepatocellular carcinoma cells[J]. Cancer, 2002, 95(8): 1696—1705.
[18] Sasaki N, Morisaki T, Hashizume K, Yao T, Tsuneyoshi M ,Noshiro H,Nakamura K,Yanlanaka T,Uchiyama A,TanakaM,Katano M . Nuclear factor-kappaB p65(RelA)transcription factor is constitutivelv activated in human gastric carcinoma tissue . Clin CancerRes 2001,7: 4136-4142.
[19]Lawniczak M,Starzynska T.Helicobacter pylori CagA(+) infectionin gastric cancer patients .Pol Merkuriusz Lek, 2002,13: 216-220.
[20]EL—OmarEM, Oienk, MurrayLs et a1. Increased prevalence of precancerous change in Relatives of Gastric Cancer Patients: Critical Role of H. pylori. Gastroenterology, 2000,118: 306-308.
[21]Van Den Brink GR,Ten Kate FJ,Ponsioen CY et a1.Expression and activation of NF—KappaB in the antrum of the human stomach[J]. J Immunol, 2000,164(6): 3353～3359.
[22]李国平,吴灵飞,蒲泽锦,冯家琳,郑宗茂,王炳.CagA+幽门螺杆菌胃癌前及癌变中N F—KB的表达增强.世界华人消化杂志,2005 ,13(17):2064-2068.
[23]Ueda M,Kokura S,Imamoto E.Blocking of NF—kappa B activation enhances the tumor necrosis factor alpha—apoptosis of a human gastric cancer cell line [J]. Cancer Lett, 2003, 193(2): 177—182.
[24] Maeda S, Akanuma M , Mistsunoy et a1. Distinct mechanism of Helicobacter Pylori mediated NF—KappaB activation between gastric cancer cells and monolytic cells[J]. J Biol chem, 2001,276(48):44856-44864.
[25] Huan B,Zhao J,Shen S,et al,Listeria monocytogenes promotes tumor growth via tumor cell toll kike receptor 2 signaling.Cancer Res,2007, 5:45-48.
[26]金琳芳,陈同钰.Bcl-2基因蛋白家族与细胞凋亡.医学综述,2001,11(5):56-58.
[27] Tamatani M,Chen YH ,Matsuzaki H ,et al. Tumor necrosis factor induces Bcl-2 and Bcl-xl expression through NF kappa B activation in primary hippocampal neurons.J Biol Chem,1999,274:8531-85381.
[28] Cheng Q,Lee HH,Li Y,et a1.Upregulation of Bcl—xl and Bf1.1 as a potential mechanism of chemoresistance,which can be overcome by NF—kappaB inhibition [J]. Oncegene, 2000, 19(42): 4936-4940.
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