幽门螺杆菌促HepG2细胞增殖及其机制研究
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摘要
目的:探究幽门螺杆菌(Hp)是否具有促肝细胞增殖的作用,并揭示促增殖的可能机制,为进一步研究幽门螺杆菌致肝癌的机制奠定基础。
方法: 1.幽门螺杆菌对HepG2细胞增殖的影响:将不同量的幽门螺杆菌(国际标准株NCTC11637)作用于HepG2肝细胞,以同批次的同步化后未加细菌的HepG2细胞作为对照组,用CCK-8法,通过酶标仪读取各孔的OD值,检测细胞的增殖情况并确定最佳的幽门螺杆菌作用剂量。2.基因芯片检测:应用Affymetrix人类基因表达谱芯片,检测幽门螺杆菌促HepG2细胞增殖时细胞基因表达谱的变化。3.RT-PCR半定量技术验证部分基因芯片结果:将具有促进HepG2细胞增殖浓度的幽门螺杆菌与HepG2细胞共浴24小时,提取HepG2细胞的RNA,未经幽门螺杆菌处理的HepG2细胞作为对照组,用半定量RT-PCR的方法检测HepG2细胞部分基因表达情况(依据基因芯片结果挑选出五对基因),以验证基因芯片的结果。
结果:1、幽门螺杆菌在一定的MOI比值下(0.30:1～0.075:1), HepG2细胞增殖活性明显增强。2、基因芯片的结果显示,HepG2细胞经Hp作用后(MOI比值:0.15:1)时有19个表达上调基因,下调基因16个,其中与细胞骨架/基质有关的上调基因6个,包括intercellular adhesion molecule 1(ICAM-1)、transgelin、talin 1、tubulin、Integrin及keratin 23。DNA结合蛋白基因8个,上调4个为tubulin beta polypeptide paralog、Cell division cycle 2-like 5、myelin expression factor 2、V-erb-a erythroblastic leukemia viral oncogene homolog 4;下调基因有4个分别为zinc finger protein 513、nuclear receptor subfamily 1(group I, member 3)、Clock homolog、SRY (sex determining region Y)-box 5。细胞因子相关基因下调2个为interleukin 8及interleukin 1 family, member 9。3、半定量RT-PCR分析结果显示,以未加幽门螺杆菌处理的HepG2细胞作为对照组,加入幽门螺杆菌处理的HepG2细胞基因的Tubulin、ICAM、ARTS、Akeratin、ARHGDIA等转录水平显著增高,经两样本T检验具有显著性差别(P<0.05),该结果与之前的基因芯片的结果相符合。
结论: 1、幽门螺杆菌在适当的MOI比值下(0.30～0.075:1)下,对HepG2细胞具有明显的促增殖作用。2、基因芯片结果显示幽门螺杆菌在促进HepG2细胞增殖的过程中,HepG2细胞基因表达有明显改变,这些异常改变的基因可能参与了幽门螺杆菌致肝癌的过程。其中与细胞骨架/基质有关的上调基因有6个。DNA结合蛋白基因上调8个,其中上调基因4个、下调基因有4个。细胞因子相关基因下调2个这些异常改变的基因可能参与了幽门螺杆菌致肝癌的过程。3、半定量RT-PCR分析结果显示,幽门螺杆菌处理组比对照组的HepG2细胞某些基因转录水平显著增高。这些基因可能参与了幽门螺杆菌对细胞的促增殖过程甚至在肝癌的发生中发挥作用,通过这些基因的研究可能有助于进一步明确幽门螺杆菌与人类肝脏疾病的关系。
Objectives:To testify whether the Helicobacter pylori could promote the cell proliferation of HepG2 cells and then discover the mechanisms in order kown the progress of Helicobacter pylori participate the hepatoeellular carcinoma incur.
Method:1. HepG2 cells and H.pylori Was co-cultured in several concentration and compound the Cell Counting Kit-8 agent with it after 24 hours to Measure the absorbance at 450 nm using a microplate reader to inspect the change of HepG2 cells proliferation and discover the best dose。2. inspect the change of the gene that pick out from the expression in HepG2 cells by the Affymetrix GENE CHIPS.3.Add the H.pylori in the concentration that could promote the cells proliferation of HepG2 cells and co-cultured it and 24h.the total RNA of untreated and H.pylori treated HepG2 cells RNA were separated and RT-PCR was performed to detect that the change of genes that pick out from GENE CHIPS expression to confirm the result of it.
Result: 1. When HepG2 cells and H.pylori Was co-cultured in the MOI of (0.3:1～0.075:1), the the HepG2 cells proliferation was great promoted by H.pylori.2.The GENE CHIPS shows that there are 19 genes up-regulation and 16 genes down-regulation after the H.pylori was compound in the HepG2 cells. They were contain 6 cytoskeleton up genes that intercellular adhesion molecule 1(ICAM-1)、transgelin、talin 1、tubulin、Integrin及keratin 23。Also they contain 8 DNA binding protein genes that tubulin beta polypeptide paralog、Cell division cycle 2-like 5、myelin expression factor 2、V-erb-a erythroblastic leukemia viral oncogene homolog 4 were up regulation,and 4 down regulation gene they are zinc finger protein 513、nuclear receptor subfamily 1(group I, member 3)、Clock homolog、SRY (sex determining region Y)-box 5。At last 2 cytokine up regulation genes were interleukin 8 and interleukin 1 family, member 9。3.The RT-PCR result reveal that there are many difference between the five differential GENE expression were successfully identified, These genes included Tubulin、ICAM、ARTS、Akeratin、ARHGDIA.
Conclusions:1.The HepG2 cells proliferation was great enhance after compound with the H.pylori in a suitable MOI。2.The GENE CHIPS shows that there are many genetic expression change after HepG2 cells was compound with the H.pylori in an suitable MOI. They may include 6 cytoskeleton up genes,8 DNA binding protein genes ,2 cytokine up regulation genes and they may inclosed the progress to the hepatoeellular carcinoma incur。3.There is a significant difference at the genes expression when the hepG2 cells and H.pylori Was co-cultured ,this genes may be include the progress of H.pylori promote the HepG2 cells proliferation and even enforce the progress of the carcinogenesis of hepatoeellular carcinoma.
方法: 1.幽门螺杆菌对HepG2细胞增殖的影响:将不同量的幽门螺杆菌(国际标准株NCTC11637)作用于HepG2肝细胞,以同批次的同步化后未加细菌的HepG2细胞作为对照组,用CCK-8法,通过酶标仪读取各孔的OD值,检测细胞的增殖情况并确定最佳的幽门螺杆菌作用剂量。2.基因芯片检测:应用Affymetrix人类基因表达谱芯片,检测幽门螺杆菌促HepG2细胞增殖时细胞基因表达谱的变化。3.RT-PCR半定量技术验证部分基因芯片结果:将具有促进HepG2细胞增殖浓度的幽门螺杆菌与HepG2细胞共浴24小时,提取HepG2细胞的RNA,未经幽门螺杆菌处理的HepG2细胞作为对照组,用半定量RT-PCR的方法检测HepG2细胞部分基因表达情况(依据基因芯片结果挑选出五对基因),以验证基因芯片的结果。
结果:1、幽门螺杆菌在一定的MOI比值下(0.30:1～0.075:1), HepG2细胞增殖活性明显增强。2、基因芯片的结果显示,HepG2细胞经Hp作用后(MOI比值:0.15:1)时有19个表达上调基因,下调基因16个,其中与细胞骨架/基质有关的上调基因6个,包括intercellular adhesion molecule 1(ICAM-1)、transgelin、talin 1、tubulin、Integrin及keratin 23。DNA结合蛋白基因8个,上调4个为tubulin beta polypeptide paralog、Cell division cycle 2-like 5、myelin expression factor 2、V-erb-a erythroblastic leukemia viral oncogene homolog 4;下调基因有4个分别为zinc finger protein 513、nuclear receptor subfamily 1(group I, member 3)、Clock homolog、SRY (sex determining region Y)-box 5。细胞因子相关基因下调2个为interleukin 8及interleukin 1 family, member 9。3、半定量RT-PCR分析结果显示,以未加幽门螺杆菌处理的HepG2细胞作为对照组,加入幽门螺杆菌处理的HepG2细胞基因的Tubulin、ICAM、ARTS、Akeratin、ARHGDIA等转录水平显著增高,经两样本T检验具有显著性差别(P<0.05),该结果与之前的基因芯片的结果相符合。
结论: 1、幽门螺杆菌在适当的MOI比值下(0.30～0.075:1)下,对HepG2细胞具有明显的促增殖作用。2、基因芯片结果显示幽门螺杆菌在促进HepG2细胞增殖的过程中,HepG2细胞基因表达有明显改变,这些异常改变的基因可能参与了幽门螺杆菌致肝癌的过程。其中与细胞骨架/基质有关的上调基因有6个。DNA结合蛋白基因上调8个,其中上调基因4个、下调基因有4个。细胞因子相关基因下调2个这些异常改变的基因可能参与了幽门螺杆菌致肝癌的过程。3、半定量RT-PCR分析结果显示,幽门螺杆菌处理组比对照组的HepG2细胞某些基因转录水平显著增高。这些基因可能参与了幽门螺杆菌对细胞的促增殖过程甚至在肝癌的发生中发挥作用,通过这些基因的研究可能有助于进一步明确幽门螺杆菌与人类肝脏疾病的关系。
Objectives:To testify whether the Helicobacter pylori could promote the cell proliferation of HepG2 cells and then discover the mechanisms in order kown the progress of Helicobacter pylori participate the hepatoeellular carcinoma incur.
Method:1. HepG2 cells and H.pylori Was co-cultured in several concentration and compound the Cell Counting Kit-8 agent with it after 24 hours to Measure the absorbance at 450 nm using a microplate reader to inspect the change of HepG2 cells proliferation and discover the best dose。2. inspect the change of the gene that pick out from the expression in HepG2 cells by the Affymetrix GENE CHIPS.3.Add the H.pylori in the concentration that could promote the cells proliferation of HepG2 cells and co-cultured it and 24h.the total RNA of untreated and H.pylori treated HepG2 cells RNA were separated and RT-PCR was performed to detect that the change of genes that pick out from GENE CHIPS expression to confirm the result of it.
Result: 1. When HepG2 cells and H.pylori Was co-cultured in the MOI of (0.3:1～0.075:1), the the HepG2 cells proliferation was great promoted by H.pylori.2.The GENE CHIPS shows that there are 19 genes up-regulation and 16 genes down-regulation after the H.pylori was compound in the HepG2 cells. They were contain 6 cytoskeleton up genes that intercellular adhesion molecule 1(ICAM-1)、transgelin、talin 1、tubulin、Integrin及keratin 23。Also they contain 8 DNA binding protein genes that tubulin beta polypeptide paralog、Cell division cycle 2-like 5、myelin expression factor 2、V-erb-a erythroblastic leukemia viral oncogene homolog 4 were up regulation,and 4 down regulation gene they are zinc finger protein 513、nuclear receptor subfamily 1(group I, member 3)、Clock homolog、SRY (sex determining region Y)-box 5。At last 2 cytokine up regulation genes were interleukin 8 and interleukin 1 family, member 9。3.The RT-PCR result reveal that there are many difference between the five differential GENE expression were successfully identified, These genes included Tubulin、ICAM、ARTS、Akeratin、ARHGDIA.
Conclusions:1.The HepG2 cells proliferation was great enhance after compound with the H.pylori in a suitable MOI。2.The GENE CHIPS shows that there are many genetic expression change after HepG2 cells was compound with the H.pylori in an suitable MOI. They may include 6 cytoskeleton up genes,8 DNA binding protein genes ,2 cytokine up regulation genes and they may inclosed the progress to the hepatoeellular carcinoma incur。3.There is a significant difference at the genes expression when the hepG2 cells and H.pylori Was co-cultured ,this genes may be include the progress of H.pylori promote the HepG2 cells proliferation and even enforce the progress of the carcinogenesis of hepatoeellular carcinoma.
引文
1.Warren JR. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet,1983;1:1273
2. Uhl M, Helma C, Knasmuller S. Evaluation of the single cell gel electrophoresis assay with human hepatoma (HepG2) cells. Mutat Res 2000;468:213-225
3. Xu MH, Qu Q, Zhang GY,et al,H.pylori in patients with cirrhosis and liver cirrhosis with hepatocellular. carcinoma epartment of Gastroenterology 2007 Oct;32(5):917-20
4. Xuan SY, Xin YN, Chen AJ ,et al ,Association between the presence of H pylori in the liver and hepatocellular carcinoma: A meta analysis,World J Gastroenterol. 2008 Jan 14;14(2):307-12
5. 杨艺 邓长生 彭俊忠 幽门螺杆菌对体外培养的胃上皮细胞增殖与凋亡的影响 中华微生物和免疫杂志 2002 年 1 月第 22 卷第一期
6. Avenaud P, marais,Monteirol,et al,etdction of Helicobacter species in the liver of patients with and without primary liver carcinoma .Cancer ,2000,89:1431-1439
7. Leelawat K, Suksumek N, Leelawat S,et al, Detection of VacA gene specific for Helicobactor pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients, Southeast Asian J Trop Med Public Health. 2007 Sep;38(5):881-5
8. 汪泳. 影响胃癌预后的基因的研究进展.国外医学分子生物学分册,2002, 24: 245-247
9. Torimura T, Takati U. Integrin α6β1 plays a significant role in the attachment of hepatoma cells to laminin. J Hepatol, 1999, 31:734-740.
10 孙婧璟,周信达,周铬,等. 血清中细胞间粘附分子 1 与肝癌侵袭转移关系的初步观察. 中华医学杂志,1998,5:383.
11.孙婧璟,周信达,刘银坤,等. 肝癌组织中细胞间粘附分子 1 与肝癌侵袭转移关系的研究. 中国癌症杂志,1997,7:161-163
12. Johnson JP, Stade BG ,Holzmann B, et al. De novo expression of intercellular adhesion molecule 1 in melanoma correlate with increased risk of metastasis[J]. Proc Natl Acad Sci USA, 1989,86(2):641
13. 王慧 , 牛昀 中心体微管蛋白异常与肿瘤研究进展 诊断学理论与实践 2006 年 04 期 263
14. 聂轶飞,王凯娟,代丽萍,徐学琴,张建营肝癌相关抗原血清学自身抗体反应分析 中国公共卫生 2007 年 7 月 22 卷 7 期 132
15. Burridge K, Wennerberg K, Rho and rac take center stage[J].Cell, 2004, 116(2):167-197.
16 Wang W,Yang LY,Huang GW,et al.Genomic analysis reveals RhoC as a potenitail marker in hepatocelular carcinoma with poor prognosis[J].Br J Cancer,2004,90(12):2 3492 355.
17. Wang W,Yang LY,Yang ZL,et al.Expression and signifificance of RhoC gene in hepatocellular carcinoma[J].World J Gasstorenterol,2003,9(9):1 9501 953.
18. Yoshioka K,Matsumura F,Akedo H,et al.Small GTP-binding protein Rho stim ulates the actomyosin system,leading to invasion of tumor cells.J Biol Chem,1998 ;273(9):5146
19. Takamura M,Sakamoto M,Genda T,et al.Inhibition of intrahepatic metastasis of human heatocellular carcinom by Rhoassociated protein kinase inhibitor Y27632[J].Hepatology,2001,33(3):577581.
20: Cui X, Rouhani FN, Hawari F, Levine SJ Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding J Immunol. 2003 Dec 15;171(12):6814-9
1. Avenaud P, marais,Monteirol,et al,etdction of Helicobacter species in the liver of patients with and without primary liver carcinoma .Cancer ,2000,89:1431-1439
2. Xuan SY, Li N, Qiang X,et al ,Helicobacter infection in hepatocellular carcinoma tissue,World J Gastroenterol. 2006 Apr 21;12(15):2335-40
3. Leelawat K, Suksumek N, Leelawat S,et al, Detection of VacA gene specific for Helicobactor pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients, Southeast Asian J Trop Med Public Health. 2007 Sep;38(5):881-5
4.Xu MH, Qu Q, Zhang GY,et al,H.pylori in patients with cirrhosis and liver cirrhosis with hepatocellular. carcinoma epartment of Gastroenterology 2007 Oct;32(5):917-20
5. Xuan SY, Xin YN, Chen AJ ,et al ,Association between the presence of H pylori in the liver and hepatocellular carcinoma: A meta analysis,World J Gastroenterol. 2008 Jan 14;14(2):307-12
6. Ito K, Yamaoka Y, Ota H,et al,Adherence, Internalization, and Persistence of Helicobacter pylori in Hepatocytes. Dig Dis Sci. 2008 Mar 5[Epub ahead of print]
7. Chmiela M, Paziak-Domanska B, Rudnicka W,et al ,The role of heparan sulphate-binding activity of Helicobacter pylori bacteria in their adhesion to murine macrophages. APMIS. 1995 Jun;103(6):469-74.
8. Huang B, Zhao J, Shen S, et al,Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor 2 signaling. Cancer Res. 2007 May 1;67(9):4346-52
9.张艳,范学工,田雪飞 等 ,幽门螺杆菌对肝细胞系HepG2 cyclin D1,PCNA mRNA表达的影响,世界华人消化杂志2004 January 12 93—96
10. 张艳,范学工,陈仁等 幽门螺杆菌对人肝细胞系HepG2原癌基因C-fos表达的影响 中国人兽共患病杂志 2004 20(8)690-693
11. Zhang Y, Fan XG, Chen R ,et al ,Comparative proteome analysis of untreated and Helicobacter pylori-treated HepG2. World J Gastroenterol. 2005 Jun 14;11(22):3485-9
12. Fox JG, Li X, Cahill RJ,et al,Hypertrophic gastropathy in Helicobacter felis-infected wild-type C57BL/6 mice and p53 hemizygous transgenic mice.,Gastroenterology. 1996 Jan;110(1):155-66
13. Nyska A, Maronpot RR, Eldridge SR, et al,Alteration in cell kinetics in control B6C3F1 mice infected with Helicobacter hepaticus, Toxicol Pathol. 1997 Nov-Dec;25(6):591-6
14. Racha M Avenaud P Menard A, et al ,Association of Helicobacter species with hepatitis C cirrhosis with or without hepatocellular carcinoma. Gut. 2005 Mar;54(3):396-401
15. 张世强 鲍勇 祖茂衡等 幽门螺杆菌与原发性肝癌关系的相关性研究 中国肿瘤杂志 2004. 31. 761-763
16..Fagoonee S, Pellicano R, Rizzetto M,et al,The journey from hepatitis to hepatocellular carcinoma. Bridging role of Helicobacter species,Panminerva Med. 2001 Dec;43(4):279-82
2. Uhl M, Helma C, Knasmuller S. Evaluation of the single cell gel electrophoresis assay with human hepatoma (HepG2) cells. Mutat Res 2000;468:213-225
3. Xu MH, Qu Q, Zhang GY,et al,H.pylori in patients with cirrhosis and liver cirrhosis with hepatocellular. carcinoma epartment of Gastroenterology 2007 Oct;32(5):917-20
4. Xuan SY, Xin YN, Chen AJ ,et al ,Association between the presence of H pylori in the liver and hepatocellular carcinoma: A meta analysis,World J Gastroenterol. 2008 Jan 14;14(2):307-12
5. 杨艺 邓长生 彭俊忠 幽门螺杆菌对体外培养的胃上皮细胞增殖与凋亡的影响 中华微生物和免疫杂志 2002 年 1 月第 22 卷第一期
6. Avenaud P, marais,Monteirol,et al,etdction of Helicobacter species in the liver of patients with and without primary liver carcinoma .Cancer ,2000,89:1431-1439
7. Leelawat K, Suksumek N, Leelawat S,et al, Detection of VacA gene specific for Helicobactor pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients, Southeast Asian J Trop Med Public Health. 2007 Sep;38(5):881-5
8. 汪泳. 影响胃癌预后的基因的研究进展.国外医学分子生物学分册,2002, 24: 245-247
9. Torimura T, Takati U. Integrin α6β1 plays a significant role in the attachment of hepatoma cells to laminin. J Hepatol, 1999, 31:734-740.
10 孙婧璟,周信达,周铬,等. 血清中细胞间粘附分子 1 与肝癌侵袭转移关系的初步观察. 中华医学杂志,1998,5:383.
11.孙婧璟,周信达,刘银坤,等. 肝癌组织中细胞间粘附分子 1 与肝癌侵袭转移关系的研究. 中国癌症杂志,1997,7:161-163
12. Johnson JP, Stade BG ,Holzmann B, et al. De novo expression of intercellular adhesion molecule 1 in melanoma correlate with increased risk of metastasis[J]. Proc Natl Acad Sci USA, 1989,86(2):641
13. 王慧 , 牛昀 中心体微管蛋白异常与肿瘤研究进展 诊断学理论与实践 2006 年 04 期 263
14. 聂轶飞,王凯娟,代丽萍,徐学琴,张建营肝癌相关抗原血清学自身抗体反应分析 中国公共卫生 2007 年 7 月 22 卷 7 期 132
15. Burridge K, Wennerberg K, Rho and rac take center stage[J].Cell, 2004, 116(2):167-197.
16 Wang W,Yang LY,Huang GW,et al.Genomic analysis reveals RhoC as a potenitail marker in hepatocelular carcinoma with poor prognosis[J].Br J Cancer,2004,90(12):2 3492 355.
17. Wang W,Yang LY,Yang ZL,et al.Expression and signifificance of RhoC gene in hepatocellular carcinoma[J].World J Gasstorenterol,2003,9(9):1 9501 953.
18. Yoshioka K,Matsumura F,Akedo H,et al.Small GTP-binding protein Rho stim ulates the actomyosin system,leading to invasion of tumor cells.J Biol Chem,1998 ;273(9):5146
19. Takamura M,Sakamoto M,Genda T,et al.Inhibition of intrahepatic metastasis of human heatocellular carcinom by Rhoassociated protein kinase inhibitor Y27632[J].Hepatology,2001,33(3):577581.
20: Cui X, Rouhani FN, Hawari F, Levine SJ Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding J Immunol. 2003 Dec 15;171(12):6814-9
1. Avenaud P, marais,Monteirol,et al,etdction of Helicobacter species in the liver of patients with and without primary liver carcinoma .Cancer ,2000,89:1431-1439
2. Xuan SY, Li N, Qiang X,et al ,Helicobacter infection in hepatocellular carcinoma tissue,World J Gastroenterol. 2006 Apr 21;12(15):2335-40
3. Leelawat K, Suksumek N, Leelawat S,et al, Detection of VacA gene specific for Helicobactor pylori in hepatocellular carcinoma and cholangiocarcinoma specimens of Thai patients, Southeast Asian J Trop Med Public Health. 2007 Sep;38(5):881-5
4.Xu MH, Qu Q, Zhang GY,et al,H.pylori in patients with cirrhosis and liver cirrhosis with hepatocellular. carcinoma epartment of Gastroenterology 2007 Oct;32(5):917-20
5. Xuan SY, Xin YN, Chen AJ ,et al ,Association between the presence of H pylori in the liver and hepatocellular carcinoma: A meta analysis,World J Gastroenterol. 2008 Jan 14;14(2):307-12
6. Ito K, Yamaoka Y, Ota H,et al,Adherence, Internalization, and Persistence of Helicobacter pylori in Hepatocytes. Dig Dis Sci. 2008 Mar 5[Epub ahead of print]
7. Chmiela M, Paziak-Domanska B, Rudnicka W,et al ,The role of heparan sulphate-binding activity of Helicobacter pylori bacteria in their adhesion to murine macrophages. APMIS. 1995 Jun;103(6):469-74.
8. Huang B, Zhao J, Shen S, et al,Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor 2 signaling. Cancer Res. 2007 May 1;67(9):4346-52
9.张艳,范学工,田雪飞 等 ,幽门螺杆菌对肝细胞系HepG2 cyclin D1,PCNA mRNA表达的影响,世界华人消化杂志2004 January 12 93—96
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