扶正解毒含药血清对镍致癌干预作用的细胞分子机制研究
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摘要
镍(Nickel)是人类在生产和生活中广泛接触的一种金属元素。常接触镍的职业包括：镍的冶炼、提纯、电镀、制造不锈钢以及生产镍铬电池等。人群流行病学调查和动物实验均已证实镍的职业性接触可以引起癌症,主要是肺癌,国际癌症研究中心(IARC)1990年将镍及其化合物列为第一类致癌物,其中尤以镍尘NiS和Ni3S2致癌性最强,其次为Ni2O3。目前有关镍致癌机理的研究主要集中在四个方面：一是镍诱导ras、P53等基因突变的形成,继而导致癌相关基因表达异常,诱发肿瘤的形成；二是镍诱导细胞内信号转导的改变,引起转录因子的异常活化并导致基因表达谱的改变,继而导致肿瘤的形成；三是镍诱导细胞产生的氧自由基可以导致细胞内遗传物质的损伤,影响癌相关基因的表达,最终导致肿瘤的发生；四是镍除了直接诱导基因组的表达之外,还可通过表观遗传学的改变,继而影响癌相关基因的表达,促进肿瘤的发生。目前有关镍诱导肿瘤发生以及肺损伤的研究很多,但是由于镍在人体中代谢的复杂性,镍致癌的分子机制到目前为止并未完全阐明,因此本文将镍化物诱导活性氧产生、激活信号转导通路、诱导细胞中核转录因子的活性以及中医药的干预实验对镍致癌的分子机制进行初步探讨,试图为镍致职业性肺癌的预防和中药治疗予以理论上的探索。
方法
1.体外培养支气管上皮细胞(16HBE),分别用无菌的MEM、不同浓度的NiS (1.0-8.0μmol/L)、NiS (2.0μmol/L)+扶正解毒(FJD)含药血清(低、中、高剂量组、空白血清组)处理细胞24h,用台盼蓝法检测支气管上皮细胞的存活率。取对数生长期的16HBE细胞接种于24孔培养板内,6×106个／孔,每个剂量设5个复孔,按实验分组分别加入受试物,继续培养24h,各组细胞弃去上清液,每孔加入500μl细胞裂解液,冰上放置30min,将裂解液作用后的各组细胞转移至Eppendorf管,离心20min,取上清液进行谷胱甘肽过氧化酶(GSH-Px)、活性氧(ROS)活性检测。将受试物处理后的各组细胞以PBS反复洗涤细胞,直至显微镜下大部分细胞已超声破碎,进行超氧化歧化酶(SOD)活性的检测。用考马斯亮兰蛋白测定法,用分光光度计测定波长595nm处吸光度OD值,计算蛋白含量,结合蛋白浓度计算16HBE细胞(细胞处理方法同前)内丙二醛(MDA)含量。2.将16HBE细胞接种于25ml培养瓶中,每孔接种的细胞数为6×106,培养24h后,用NiS (1-5.0μmol/L)染毒,并用无菌的MEM、NiS (2.0μmol/L)+不同剂量FJD含药血清、空白血清、以及三种抑制剂,磷酸化P38丝裂原活化蛋白激酶(P38MAPK)、磷酸化细胞外信号调节蛋白激酶(P-ERK)、磷酸化氨基未端蛋白激酶(P-JNK)处理细胞24h,无菌PBS洗涤细胞2次,胰酶消化后制成细胞悬液,用聚丙烯酰胺凝胶电泳和蛋白免疫印迹杂交(Western blot)法观察各组细胞中磷酸化ERK、P38和JNK的表达。3.将16HBE细胞用无菌的MEM、不同浓度的NiS (1.0-4.0μmol/L)、NiS (2.0μmol/L)+FJD含药血清(低、中、高剂量组)处理细胞24h后,用免疫组化法检测各组细胞中核转录因子(NF-κB)κB的活性,并进行半定量分析,用RT-PCR法测定支气管上皮细胞中NF-κB P65的表达。本次实验采用SPSS13.0统计软件进行相关分析,多组间比较采用方差分析,两组之间的比较采用T检验,P<0.05被认为有统计学意义。
结果
1.当NiS的浓度为1-8μmol/L时,细胞的存活率均低于阴性对照组(MEM培养液),且NiS的浓度越高,细胞存活率越低,分别为93.6%、92.8%、90.3%、70.3%；低、中、高剂量FJD含药血清和空白血清对16HBE细胞均无毒性,细胞存活率分别为99.1%、98.8%、99.55%、98.7%。2.NiS能明显激活细胞ROS、MDA的表达(P<0.05),NiS的浓度分别为1.0、2.0、4.0μmol/L时,16HBE细胞中ROS分别为31.68、33.21、35.26 U/ml,且存在剂量-效应关系(r=0.813)；16HBE细胞中MDA分别为0.9518、1.0503、1.1620 nmol/ml,且存在剂量-效应关系(r=0.7926)。低、中、高剂量FJD血清与NiS (2μmol/L)共同处理16HBE细胞,ROS、MDA活性均低于染镍组(NiS为2μmol/L),尤其中、高剂量血清组作用显著(P<0.05),空白血清对16HBE细胞中ROS, MDA没有影响。NiS能明显抑制SOD、GSH-Px的活性(P<0.05),NiS的浓度分别为1.0、2.0、4.0μmol/L时,16HBE细胞中SOD分别为11.99、7.89、5.24 NU/ml,且存在剂量-效应关系(r=0.875)；同时16HBE细胞中GSH-Px分别为22.65、15.75、10.38 U/ml,且存在剂量-效应关系(r=0.829)。低、中、高剂量FJD血清与NiS (2μmol/L)共同处理16HBE细胞中,SOD、GSH-Px活性均高于染镍组(NiS为2μmo1/L),尤其中、高剂量血清组作用显著(P<0.05),空白血清对16HBE细胞中SOD、GSH-Px活性未见影响。3.NiS能明显激活细胞磷酸P38MAPK的表达,NiS浓度越高,磷酸化P38MAPK的表达值越高,阴性对照组细胞中磷酸P38MAPK为0.2868,NiS的浓度分别为1.0、2.0、4.0、5.0μmol/L,染镍组细胞中磷酸P38MAPK分别为0.4112、0.5937、0.6968、0.8976,FJD含药血清干预组中磷酸P38MAPK分别为0.5868、0.4972、0.4869,我们可以看出FJD含药血清抑制了磷酸化P38MAPK的表达,尤以中、高剂量组为甚(P<0.01),空白血清组对染镍细胞中磷酸化P38MAPK的表达没有明显影响(P>0.05)。NiS能明显激活细胞P-JNK的表达(P<0.05),NiS浓度越高,P-JNK的表达值越高,阴性对照组细胞中P-JNK为0.041,NiS浓度分别为1、2、4.0μmo1/L,染毒组细胞中P-JNK分别为0.057、0.062、0.068,而且存在剂量-效应关系(r=0.7736),FJD高剂量含药血清组和SP600125(10μmol/L) (P-JNK抑制剂)明显干预P-JNK的表达(P<0.05),空白血清组对染镍细胞中P-JNK的表达未见影响。NiS不能明显激活P-ERK1的表达(P>0.05),阴性对照组细胞中P-ERK1表达值为0.210,NiS浓度分别为1、2、4.0μmol/L,染镍组细胞中P-ERK1表达值分别为0.216、0.219、0.226,FJD低、中、高剂量含药血清组和P-ERK1抑制剂对磷酸化P-ERK1的表达无明显影响(P>0.05)。4.NiS能明显激活NF-κB和NF-κB P65的表达(P<0.05),阴性对照组细胞中NF-κB强阳性例数为8,NiS浓度分别为1、2、4.0μmol/L,染镍组细胞中NF-κB强阳性例数分别为17、22、29,而中、高剂量的FJD含药血清能下调NF-κB在16HBE细胞中表达(P<0.05)。阴性对照组细胞中NF-κB p65光密度比值0.8079,NiS浓度分别为1、2、4.0μmol/L,染毒组细胞中NF-κB p65光密度比值分别为0.8558、0.8950、0.9937,FJD含药血清干预组中NF-κB p65光密度比值分别为0.8938、0.8337、0.8119,中、高剂量的FJD含药血清能下调细胞中NF-κB P65光密度比值(P<0.05)。
结论
1硫化镍能明显诱导支气管上皮细胞中活性氧浓度升高、细胞脂质过氧化作用增强及细胞存活率降低,一定浓度的FJD含药血清能抑制染镍细胞中脂质过氧化作用、降低活性氧浓度。
2硫化镍能明显激活细胞中磷酸化P38和JNK的表达,不能明显激活磷酸化ERK的表达,一定浓度的FJD含药血清对MAPK通路中细胞磷酸化P38和JNK有抑制作用。
3硫化镍能明显激活细胞中NF-κB和NF-κB P65的表达,一定浓度的FJD含药血清能下调细胞中NF-κB和NF-κB P65的表达。
4从细胞分子水平初步证明：扶正解毒汤对镍作业工人在防治癌症中可能有保护作用。
Nickel is a kind of metal which is widely contacted in production and life.Occupational exposed to human beings include:nickel of smelting metal purification、plating、making stainless steel and producing battery of nickel and chromium. Accumulated epidemiological evidence and animal experiments confirm that exposure to nickel compounds are associated with tumor, especially lung cancer,the International Agency for Research on Cancer (IARC)classified nickel compounds as group I carcinogen to human in 1990, the crystalline NiS and Ni3S2 are the highest carcinogenic activity among the nickel compounds and the Ni2O3 is second. The carcinogenic actions of nickel compounds are thought to involve four aspects. The first is genomic mutation including ras and p53 induced by nickel compounds, which leads to change the tumor related gene expression pattern and result in carcinogenesis. The second is that nickel induces the signaling pathway in cell leading to transcription factor transactiration by which nickel regulate the expression of specific genes related to tumor development. The third is generation of reactive Oxygen Species (ROS) induced by nickel exposure which leads to DNA and chromatin damage, tumor related gene expression is changed to cause cancer. The fourth, besides nickel induce directly the expression of gene, nickel can change apparent genetics and effect on gene expression of cancer, and at least it will promote cancer development. Recently, numerous epidemiologic and experimental investigations about carcinogenesis and acute lung injury induced by nickel compounds have been reported and some hypotheses have been proposed. However, the exact mechanisms are not known due to its human body. In the present work, the molecular mechanism of lung cancer induced by nickel compounds has been studied on production of ROS, the signaling pathway in cell leading to transcription, inducing NF-κB in cell, intervention effects of Fuzheng Jiedu decoction on molecular mechanism of lung cancer induced by nickel, we try to obtain more understanding on prevention and treatment of occupational lung cancer.
1 Human bronchus epithelial(16HBE)cells in vitro were treated with EME、different concentrations of nickel purite(NiS)(1-8.0μmol/L)、NiS(2.0μmol/L)、Fuzheng Jiedu Decoction(FJD)(low middle high group)、blank serum in 24 hours. We detected survivor rate of 16HBE cells with Trypan—blue tinctured method. 16HBE cells were cultured with culture medium of 24 holes, every hole was 6×106 cells, every dose had five times,16HBE cells were added in all kinds of subjects by different experiment groups and went on culturing 24 hours, group cell was thrown away supernatant fluid, every hole was added in 500μl lytic factor of cells and was put up 30 minutes on the ice,each group cell which was effect on lytic factor was diverted to Eppendorf tube,it was centrifugated 20 minutes,supernatant fluid was detected the expression of glutathione peroxidase (GSH-Px)and reactive oxygen species(ROS). Each group cell which subjects were treated was washed with PBS again and again, until the most cells were broken under microscope, cells were detected activities of superoxide dismutase (SOD).16HBE cells were measured with coomassie brilliant blue,16HBE cells were measured OD of absorbance in 595nm air way with apeotrophotometer and calculated content of protein,16HBE cells were counted content of malondialdehyde (MDA) which was combined with chroma of protein.216HBE cells were inocukated into 25ml culture flask, every hole was inocukated 6×106 cells and was trained for 24 hours,16HBE cells were treated with NiS(1.0μmol/L、2.0μmol/L、4.0μmol/L、5.0μmol/L)、MEM of asepsis、NiS(2.0μmol/L)+different FJD serum containing Chinese medicine、blank serum and three kinds of depressant (p38MAPK P-ERK、P-JNK) in 24 hours,16HBE cells were washed two times with PBS and were made in cell suspension after cells were digested with enzyme,16HBE cells were detected the expression of p38MAPK P-ERK、P-JNK with western blot.316HBE cells were treated with different concentration NiS(1.0μmol/L、2.0μ.mol/L、4.0μmol/L)、NiS(2.0μmol/L)+different FJD serum containing Chinese medicine in 24 hours,16HBE cells were measured the expression of NF-κB with immunohistochemistry and were analyzed with semiquantitative, 16HBE cells were measured the expression of NF-κB P65 with RT-PCR. This test was analyzed with statistical software of SPSS 13.0,a lot of groups were compared with rariance analysis, two groups were compared with T examination, P<0.05 was regarded as meaning of statistics.
Results
1 When chroma of NiS was 1.0μmol/L、2.Oμmol/L、4.0μmol/L、.8.0μmol/L, persistence of 16HBE cells were lower than the group of EME, chroma of NiS was higher, persistence of cell was lower, it was 93.6%、92.8%、90.3%、70.3%; the different FJD serum containing Chinese medicine and blank serum were not poisonous to 16HBE cells, persistence of cell was 99.1%、98.8%、99.6%、98.7%.
2 NiS can increase obviously the expression of ROS and MDA in 16HBE cells(P< 0.05), chroma of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L, ROS was 31.68U/ml、33.21U/ml、35.76U/ml in 16HBE cells, it existed dose—effect relationship (r=0.8130); the expression of MDA was 0.9518nmol/ml、1.0503nmol/ml、1.1620nmol/ml, it existed dose—effect relationship (r=0.7926), the different FJD serum containing Chinese medicine and NiS (2.0μmol/L)were together treated with 16HBE cells, the expression of ROS and MDA were lower than dyeing nickel group (NiS 2.0μmol/L),middle and high dose FJD were higher than control group(P< 0.05),the blank serum group was not effect on the expression of ROS and MDA in 16HBE cells; NiS inhibited obviously the expression of GSH-Px and SOD (P<0.05), chroma of NiS was 1.0μmol/L、2.0μmol/L、.4.Oμmol/L, the expression of SOD was 11.99 NU/ml、7.89 NU/ml、5.24 NU/ml in 16HBE cells, it existed dose—effect relationship (r=0.8750); the expression of GSH-Px was 22.65 U/ml、15.75 U/ml、10.38 U/ml in 16HBE cells, it existed dose—effect relationship (r=0.8290), the different FJD serum containing Chinese medicine and NiS (2.Oμmol/L)were together treated with 16HBE cells, the expression of GSH-Px and SOD were higher than dyeing nickel group (NiS 2.0μmol/L),middle and high dose FJD were distinguished (P<0.05), the blank serum group was not effect on the expression of GSH-Px and SOD in 16HBE cells.3 NiS can increase obviously the expression of P38MAPK, dose of NiS was higher, the expression of P38MAPK was higher too, the expression of P38MAPK was 0.2868, chroma of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L、5.0μmol/L, the expression of P38MAPK was 0.4112、0.5937、0.6968、0.8976 in dyeing nickel groups.we showed:FJD serum containing Chinese medicine restrained the expression of P38MAPK; middle and high dose FJD were distinguished (P<0.01),but the blank serum group was not any effect on the expression of P38MAPK in 16HBE cells (P>0.05). NiS can increase obviously the expression of P-JNK(P<0.05),the dose of was higher, the expression of P-JNK was higher too,the P-JNK of control group was 0.0410, chroma of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L, the expression of P-JNK was 0.057、0.062、0.068 in dyeing nickel groups, it existed dose—effect relationship (r=0.7736), high dose FJD and SP600125(10μmol/L) restrained the expression of P-JNK(P<0.05), but the blank serum group was not any effect on the expression of P-JNK in 16HBE cells. NiS could not increase obviously the expression of P-ERK1(P>0.05), the expression of P-ERK1 was 0.210 in control group, chroma of NiS was 1.0μmol/L、2.0μmol/L、4.0μmol/L, the expression of P-ERK1 was 0.216、0.219、0.226 in dyeing nickel groups, the different FJD serum containing Chinese medicine and inhibitor of P-ERK1 were not effected on the expression of P-ERK1 (P>0.05).4 NiS could increase obviously the expression of NF-κB and NF-κB P65 (P<0.05),the strong masculine examples of NF-κB in control group were 8, the dose of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L, the strong masculine examples of NF-κB were 17、22、29 in dyeing nickel groups, the middle and high dose FJD serum containing Chinese medicine could descend the expression of NF-κB in 16HBE cells(P<0.05).The optical density of NF-κB P65 in control group was 0.8079, the dose of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L, The optical density of NF-κB P65 in dyeing nickel groups were 0.8558、0.8950、0.9937, the middle and high dose FJD serum containing Chinese medicine could fall the optical density of NF-κB P65(P<0.05).
1 NiS could induce the expression of ROS in 16HBE cells、increase lipid peroxidation and reduce the persistence of cells, the fitting FJD serum containing Chinese medicine could inhibit lipid peroxidation and cut down the expression of ROS in 16HBE cells.
2 NiS could induce the expression of P38MAPK and P-JNK in 16HBE cells, but it could not induce the expression of P-ERK1, the fitting FJD serum containing Chinese medicine could reduce the expression of P38MAPK and P-JNK in 16HBE cells.
3 NiS could induce the expression of NF-κB and NF-κB P65 in 16HBE cells, the fitting FJD serum containing Chinese medicine could reduce the expression of in 16HBE cells.
4 We could show from molecular mechanism of cells:the fitting FJD may be the prevention of the cancer in occupational workers of nickel.
方法
1.体外培养支气管上皮细胞(16HBE),分别用无菌的MEM、不同浓度的NiS (1.0-8.0μmol/L)、NiS (2.0μmol/L)+扶正解毒(FJD)含药血清(低、中、高剂量组、空白血清组)处理细胞24h,用台盼蓝法检测支气管上皮细胞的存活率。取对数生长期的16HBE细胞接种于24孔培养板内,6×106个／孔,每个剂量设5个复孔,按实验分组分别加入受试物,继续培养24h,各组细胞弃去上清液,每孔加入500μl细胞裂解液,冰上放置30min,将裂解液作用后的各组细胞转移至Eppendorf管,离心20min,取上清液进行谷胱甘肽过氧化酶(GSH-Px)、活性氧(ROS)活性检测。将受试物处理后的各组细胞以PBS反复洗涤细胞,直至显微镜下大部分细胞已超声破碎,进行超氧化歧化酶(SOD)活性的检测。用考马斯亮兰蛋白测定法,用分光光度计测定波长595nm处吸光度OD值,计算蛋白含量,结合蛋白浓度计算16HBE细胞(细胞处理方法同前)内丙二醛(MDA)含量。2.将16HBE细胞接种于25ml培养瓶中,每孔接种的细胞数为6×106,培养24h后,用NiS (1-5.0μmol/L)染毒,并用无菌的MEM、NiS (2.0μmol/L)+不同剂量FJD含药血清、空白血清、以及三种抑制剂,磷酸化P38丝裂原活化蛋白激酶(P38MAPK)、磷酸化细胞外信号调节蛋白激酶(P-ERK)、磷酸化氨基未端蛋白激酶(P-JNK)处理细胞24h,无菌PBS洗涤细胞2次,胰酶消化后制成细胞悬液,用聚丙烯酰胺凝胶电泳和蛋白免疫印迹杂交(Western blot)法观察各组细胞中磷酸化ERK、P38和JNK的表达。3.将16HBE细胞用无菌的MEM、不同浓度的NiS (1.0-4.0μmol/L)、NiS (2.0μmol/L)+FJD含药血清(低、中、高剂量组)处理细胞24h后,用免疫组化法检测各组细胞中核转录因子(NF-κB)κB的活性,并进行半定量分析,用RT-PCR法测定支气管上皮细胞中NF-κB P65的表达。本次实验采用SPSS13.0统计软件进行相关分析,多组间比较采用方差分析,两组之间的比较采用T检验,P<0.05被认为有统计学意义。
结果
1.当NiS的浓度为1-8μmol/L时,细胞的存活率均低于阴性对照组(MEM培养液),且NiS的浓度越高,细胞存活率越低,分别为93.6%、92.8%、90.3%、70.3%；低、中、高剂量FJD含药血清和空白血清对16HBE细胞均无毒性,细胞存活率分别为99.1%、98.8%、99.55%、98.7%。2.NiS能明显激活细胞ROS、MDA的表达(P<0.05),NiS的浓度分别为1.0、2.0、4.0μmol/L时,16HBE细胞中ROS分别为31.68、33.21、35.26 U/ml,且存在剂量-效应关系(r=0.813)；16HBE细胞中MDA分别为0.9518、1.0503、1.1620 nmol/ml,且存在剂量-效应关系(r=0.7926)。低、中、高剂量FJD血清与NiS (2μmol/L)共同处理16HBE细胞,ROS、MDA活性均低于染镍组(NiS为2μmol/L),尤其中、高剂量血清组作用显著(P<0.05),空白血清对16HBE细胞中ROS, MDA没有影响。NiS能明显抑制SOD、GSH-Px的活性(P<0.05),NiS的浓度分别为1.0、2.0、4.0μmol/L时,16HBE细胞中SOD分别为11.99、7.89、5.24 NU/ml,且存在剂量-效应关系(r=0.875)；同时16HBE细胞中GSH-Px分别为22.65、15.75、10.38 U/ml,且存在剂量-效应关系(r=0.829)。低、中、高剂量FJD血清与NiS (2μmol/L)共同处理16HBE细胞中,SOD、GSH-Px活性均高于染镍组(NiS为2μmo1/L),尤其中、高剂量血清组作用显著(P<0.05),空白血清对16HBE细胞中SOD、GSH-Px活性未见影响。3.NiS能明显激活细胞磷酸P38MAPK的表达,NiS浓度越高,磷酸化P38MAPK的表达值越高,阴性对照组细胞中磷酸P38MAPK为0.2868,NiS的浓度分别为1.0、2.0、4.0、5.0μmol/L,染镍组细胞中磷酸P38MAPK分别为0.4112、0.5937、0.6968、0.8976,FJD含药血清干预组中磷酸P38MAPK分别为0.5868、0.4972、0.4869,我们可以看出FJD含药血清抑制了磷酸化P38MAPK的表达,尤以中、高剂量组为甚(P<0.01),空白血清组对染镍细胞中磷酸化P38MAPK的表达没有明显影响(P>0.05)。NiS能明显激活细胞P-JNK的表达(P<0.05),NiS浓度越高,P-JNK的表达值越高,阴性对照组细胞中P-JNK为0.041,NiS浓度分别为1、2、4.0μmo1/L,染毒组细胞中P-JNK分别为0.057、0.062、0.068,而且存在剂量-效应关系(r=0.7736),FJD高剂量含药血清组和SP600125(10μmol/L) (P-JNK抑制剂)明显干预P-JNK的表达(P<0.05),空白血清组对染镍细胞中P-JNK的表达未见影响。NiS不能明显激活P-ERK1的表达(P>0.05),阴性对照组细胞中P-ERK1表达值为0.210,NiS浓度分别为1、2、4.0μmol/L,染镍组细胞中P-ERK1表达值分别为0.216、0.219、0.226,FJD低、中、高剂量含药血清组和P-ERK1抑制剂对磷酸化P-ERK1的表达无明显影响(P>0.05)。4.NiS能明显激活NF-κB和NF-κB P65的表达(P<0.05),阴性对照组细胞中NF-κB强阳性例数为8,NiS浓度分别为1、2、4.0μmol/L,染镍组细胞中NF-κB强阳性例数分别为17、22、29,而中、高剂量的FJD含药血清能下调NF-κB在16HBE细胞中表达(P<0.05)。阴性对照组细胞中NF-κB p65光密度比值0.8079,NiS浓度分别为1、2、4.0μmol/L,染毒组细胞中NF-κB p65光密度比值分别为0.8558、0.8950、0.9937,FJD含药血清干预组中NF-κB p65光密度比值分别为0.8938、0.8337、0.8119,中、高剂量的FJD含药血清能下调细胞中NF-κB P65光密度比值(P<0.05)。
结论
1硫化镍能明显诱导支气管上皮细胞中活性氧浓度升高、细胞脂质过氧化作用增强及细胞存活率降低,一定浓度的FJD含药血清能抑制染镍细胞中脂质过氧化作用、降低活性氧浓度。
2硫化镍能明显激活细胞中磷酸化P38和JNK的表达,不能明显激活磷酸化ERK的表达,一定浓度的FJD含药血清对MAPK通路中细胞磷酸化P38和JNK有抑制作用。
3硫化镍能明显激活细胞中NF-κB和NF-κB P65的表达,一定浓度的FJD含药血清能下调细胞中NF-κB和NF-κB P65的表达。
4从细胞分子水平初步证明：扶正解毒汤对镍作业工人在防治癌症中可能有保护作用。
Nickel is a kind of metal which is widely contacted in production and life.Occupational exposed to human beings include:nickel of smelting metal purification、plating、making stainless steel and producing battery of nickel and chromium. Accumulated epidemiological evidence and animal experiments confirm that exposure to nickel compounds are associated with tumor, especially lung cancer,the International Agency for Research on Cancer (IARC)classified nickel compounds as group I carcinogen to human in 1990, the crystalline NiS and Ni3S2 are the highest carcinogenic activity among the nickel compounds and the Ni2O3 is second. The carcinogenic actions of nickel compounds are thought to involve four aspects. The first is genomic mutation including ras and p53 induced by nickel compounds, which leads to change the tumor related gene expression pattern and result in carcinogenesis. The second is that nickel induces the signaling pathway in cell leading to transcription factor transactiration by which nickel regulate the expression of specific genes related to tumor development. The third is generation of reactive Oxygen Species (ROS) induced by nickel exposure which leads to DNA and chromatin damage, tumor related gene expression is changed to cause cancer. The fourth, besides nickel induce directly the expression of gene, nickel can change apparent genetics and effect on gene expression of cancer, and at least it will promote cancer development. Recently, numerous epidemiologic and experimental investigations about carcinogenesis and acute lung injury induced by nickel compounds have been reported and some hypotheses have been proposed. However, the exact mechanisms are not known due to its human body. In the present work, the molecular mechanism of lung cancer induced by nickel compounds has been studied on production of ROS, the signaling pathway in cell leading to transcription, inducing NF-κB in cell, intervention effects of Fuzheng Jiedu decoction on molecular mechanism of lung cancer induced by nickel, we try to obtain more understanding on prevention and treatment of occupational lung cancer.
1 Human bronchus epithelial(16HBE)cells in vitro were treated with EME、different concentrations of nickel purite(NiS)(1-8.0μmol/L)、NiS(2.0μmol/L)、Fuzheng Jiedu Decoction(FJD)(low middle high group)、blank serum in 24 hours. We detected survivor rate of 16HBE cells with Trypan—blue tinctured method. 16HBE cells were cultured with culture medium of 24 holes, every hole was 6×106 cells, every dose had five times,16HBE cells were added in all kinds of subjects by different experiment groups and went on culturing 24 hours, group cell was thrown away supernatant fluid, every hole was added in 500μl lytic factor of cells and was put up 30 minutes on the ice,each group cell which was effect on lytic factor was diverted to Eppendorf tube,it was centrifugated 20 minutes,supernatant fluid was detected the expression of glutathione peroxidase (GSH-Px)and reactive oxygen species(ROS). Each group cell which subjects were treated was washed with PBS again and again, until the most cells were broken under microscope, cells were detected activities of superoxide dismutase (SOD).16HBE cells were measured with coomassie brilliant blue,16HBE cells were measured OD of absorbance in 595nm air way with apeotrophotometer and calculated content of protein,16HBE cells were counted content of malondialdehyde (MDA) which was combined with chroma of protein.216HBE cells were inocukated into 25ml culture flask, every hole was inocukated 6×106 cells and was trained for 24 hours,16HBE cells were treated with NiS(1.0μmol/L、2.0μmol/L、4.0μmol/L、5.0μmol/L)、MEM of asepsis、NiS(2.0μmol/L)+different FJD serum containing Chinese medicine、blank serum and three kinds of depressant (p38MAPK P-ERK、P-JNK) in 24 hours,16HBE cells were washed two times with PBS and were made in cell suspension after cells were digested with enzyme,16HBE cells were detected the expression of p38MAPK P-ERK、P-JNK with western blot.316HBE cells were treated with different concentration NiS(1.0μmol/L、2.0μ.mol/L、4.0μmol/L)、NiS(2.0μmol/L)+different FJD serum containing Chinese medicine in 24 hours,16HBE cells were measured the expression of NF-κB with immunohistochemistry and were analyzed with semiquantitative, 16HBE cells were measured the expression of NF-κB P65 with RT-PCR. This test was analyzed with statistical software of SPSS 13.0,a lot of groups were compared with rariance analysis, two groups were compared with T examination, P<0.05 was regarded as meaning of statistics.
Results
1 When chroma of NiS was 1.0μmol/L、2.Oμmol/L、4.0μmol/L、.8.0μmol/L, persistence of 16HBE cells were lower than the group of EME, chroma of NiS was higher, persistence of cell was lower, it was 93.6%、92.8%、90.3%、70.3%; the different FJD serum containing Chinese medicine and blank serum were not poisonous to 16HBE cells, persistence of cell was 99.1%、98.8%、99.6%、98.7%.
2 NiS can increase obviously the expression of ROS and MDA in 16HBE cells(P< 0.05), chroma of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L, ROS was 31.68U/ml、33.21U/ml、35.76U/ml in 16HBE cells, it existed dose—effect relationship (r=0.8130); the expression of MDA was 0.9518nmol/ml、1.0503nmol/ml、1.1620nmol/ml, it existed dose—effect relationship (r=0.7926), the different FJD serum containing Chinese medicine and NiS (2.0μmol/L)were together treated with 16HBE cells, the expression of ROS and MDA were lower than dyeing nickel group (NiS 2.0μmol/L),middle and high dose FJD were higher than control group(P< 0.05),the blank serum group was not effect on the expression of ROS and MDA in 16HBE cells; NiS inhibited obviously the expression of GSH-Px and SOD (P<0.05), chroma of NiS was 1.0μmol/L、2.0μmol/L、.4.Oμmol/L, the expression of SOD was 11.99 NU/ml、7.89 NU/ml、5.24 NU/ml in 16HBE cells, it existed dose—effect relationship (r=0.8750); the expression of GSH-Px was 22.65 U/ml、15.75 U/ml、10.38 U/ml in 16HBE cells, it existed dose—effect relationship (r=0.8290), the different FJD serum containing Chinese medicine and NiS (2.Oμmol/L)were together treated with 16HBE cells, the expression of GSH-Px and SOD were higher than dyeing nickel group (NiS 2.0μmol/L),middle and high dose FJD were distinguished (P<0.05), the blank serum group was not effect on the expression of GSH-Px and SOD in 16HBE cells.3 NiS can increase obviously the expression of P38MAPK, dose of NiS was higher, the expression of P38MAPK was higher too, the expression of P38MAPK was 0.2868, chroma of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L、5.0μmol/L, the expression of P38MAPK was 0.4112、0.5937、0.6968、0.8976 in dyeing nickel groups.we showed:FJD serum containing Chinese medicine restrained the expression of P38MAPK; middle and high dose FJD were distinguished (P<0.01),but the blank serum group was not any effect on the expression of P38MAPK in 16HBE cells (P>0.05). NiS can increase obviously the expression of P-JNK(P<0.05),the dose of was higher, the expression of P-JNK was higher too,the P-JNK of control group was 0.0410, chroma of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L, the expression of P-JNK was 0.057、0.062、0.068 in dyeing nickel groups, it existed dose—effect relationship (r=0.7736), high dose FJD and SP600125(10μmol/L) restrained the expression of P-JNK(P<0.05), but the blank serum group was not any effect on the expression of P-JNK in 16HBE cells. NiS could not increase obviously the expression of P-ERK1(P>0.05), the expression of P-ERK1 was 0.210 in control group, chroma of NiS was 1.0μmol/L、2.0μmol/L、4.0μmol/L, the expression of P-ERK1 was 0.216、0.219、0.226 in dyeing nickel groups, the different FJD serum containing Chinese medicine and inhibitor of P-ERK1 were not effected on the expression of P-ERK1 (P>0.05).4 NiS could increase obviously the expression of NF-κB and NF-κB P65 (P<0.05),the strong masculine examples of NF-κB in control group were 8, the dose of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L, the strong masculine examples of NF-κB were 17、22、29 in dyeing nickel groups, the middle and high dose FJD serum containing Chinese medicine could descend the expression of NF-κB in 16HBE cells(P<0.05).The optical density of NF-κB P65 in control group was 0.8079, the dose of NiS was 1.Oμmol/L、2.0μmol/L、4.0μmol/L, The optical density of NF-κB P65 in dyeing nickel groups were 0.8558、0.8950、0.9937, the middle and high dose FJD serum containing Chinese medicine could fall the optical density of NF-κB P65(P<0.05).
1 NiS could induce the expression of ROS in 16HBE cells、increase lipid peroxidation and reduce the persistence of cells, the fitting FJD serum containing Chinese medicine could inhibit lipid peroxidation and cut down the expression of ROS in 16HBE cells.
2 NiS could induce the expression of P38MAPK and P-JNK in 16HBE cells, but it could not induce the expression of P-ERK1, the fitting FJD serum containing Chinese medicine could reduce the expression of P38MAPK and P-JNK in 16HBE cells.
3 NiS could induce the expression of NF-κB and NF-κB P65 in 16HBE cells, the fitting FJD serum containing Chinese medicine could reduce the expression of in 16HBE cells.
4 We could show from molecular mechanism of cells:the fitting FJD may be the prevention of the cancer in occupational workers of nickel.
引文
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40 赵万红,曹永寿,袁泽飞。中药血清药理学的方法探讨[J]。中药新药与临床药理,2002,13(2)：122-124
1 Bennett BG. Environmental nickel pathways to man. Nickel in the human environment. IARC Scientific Publications,1984,53:487-495.
2 Costa M.Molecular targets of nickel carcinogenesis, epigenetic mechanisms. Environ Sic,1996,4:163-175.
3 刘艺敏,张敬,徐曼妮等。三氧化二镍诱导人肺成纤维细胞恶性转化的实验研究。中国公共卫生,2000,16(8)：694-696
4 Costa M, Salnikow K, Sutherland J, et al. The role of oxidative stress in nickel and chromate genotoxicity.Mol Cell Biol,2002,234:265-275
5 Denissenko MF, Salnikow K, et al. Nickel essentiality, toxicity and carcinoma gentility. Toxically Apple Pharmacology,2004,194:60-68
6 Costa M, et al.Molecular targets of nickel carcinogenesis.Mol Cell Biochem, 2001,222:205-211
7 Kasprzak KS, Sunderman FW, Salnikow K. Nickel carcinogenesis. Mutat Res,2003,533:67-97
8 Bal W, Kozlowski H, Kasprzale KS.Molecular models in nickel carcinogenesis. Inorg Biochem,2000,79:213-218
9 Sheth SG, Coudon FU, Chopua S. Nonalcoholic steatohepatitis. Ann Intern Med, 1997,126:137-138.
10 郑仲承。研究蛋白组学的新工具[J]。生命的化学,2001,21(4)：314-315。
11 To disco A, Takeuchi Y, Sadoshima JI, et al. Molecular mechanisms for somatost at inhibition of c-fos gene expression[J].Am I Physiol,1997,272:721-726.
12 Fan HY, Tong CH, Mitogen-activated protein kinase (MAPK) signaling pathways. State of the art [J], Chin J Zoology,2002,37:98-102.
13 Salnikow K, Davidson T, Zhang QW, et al. The involvement of hypoxia inducible transcription factor-1 dependent pathway in nickel carcinogenesis [J]. Cancer Res,2003,63:3524-3532.
14 Brewster JL, Devaloir T, Dwyer N C, et al.An osmosensing signal transduction pathway in yeast [J]. Science 1993,259:1760-1766.
15 Lee JC, Laydon JT, Medonnell PC, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis [J]. Nature 1994, 372:739-746.
16 Hebelhah H,Shah K, Huang L, et al. Identification of new JNK substrate using ATP pocket mutant JNK and a corresponding ATP analogue [J]. J Boil Chem., 2001,276:18090-18099.
17 张璐,姜勇,张琳等。丝裂原活化蛋白激酶研究进展[J]。国外医学生理、病理科学与临床分册,1999,19(2)；84-87。
18 朱敏,孙振学等。丝裂原活化蛋白激酶信号转导通路。武警医学院学报,2006,15(5)：500-503。
19 Jlindk T, Catling AD, Reuter CW, et al. Ras and Raf-1 form a signaling complex with MEKl.but not MEK7 [J]. Mol Cell Boil,2001,14:8211-8213.
20 Sutherland CL, Heath AW, Pelech SL, et al.Differential activation of the ERK, JNK and P38 mitogen activated protein kinase by CD40 and the B cell antigen receptor [J]. J Immune,1996,157:3381-3383.
21 Dziareke R, Jin YP, Gupta D. Differential activation of extra cellular signal regulated kinase ERK2 P38 and C-Jun N-terminal kinase mitogen activated protein kinases by bacterial peptidoglycan [J]. J Infect Dis,1996,174:777-785.
22 Waddick KG, Uckum FM. Innovative treatment programs against cancer nuclear factor kappa B as a molecular target.Biochem Pharm.1999,57:9-17.
23 Sen. R, Baltimore D, Multiple nuclear factors interact with the immunoglubin enhancer sequences. Cell,1986,46:205-209.
24 Blackwell TS, Chrisman JW. The role of nuclear factor kappa B in cytokine gene regulation.Cell Mol Boil,1997,17:3-9.
25 Beg AA, Sha WC, Bronson RT, Ghoshs SH, et al. Embryonic lethality and liver degeneration in mice lacking the role a component of Nuclear factor kappa B. Nature,1995,376:167-170.
26 Royds JA, Dower SK,Qwarnstrom EE,et al. Response of tumor cells to hypoxia role of P53 and Nuclear factor kappa B. Mol Pathol,1998,51:55-61.
27 Wu H, Lozano G, Nuclear factor kappa B activation of P53, A potential mechanism for suppressing cell growth in response to stress. Biol Chem.,1994, 269:67-74.
28 Reuther JY, Reuther GW, Cortez D, et al. A requirement for nuclear factor kappa B activation in mediated transformation. Genes Dev.,1998,12:968-981.
29 Mukhopadhyay T, Roth JA, Maxwell SA. Altered expression of the P50 subunit of the nuclear factor kappa B, transcription factor complex in non-small cell lung carcinoma. Oncogene,1995,11:999-1003.
30 Bargou RC, Wagener C, Bommer K, et al. Over expression of the death-promoting gene box-alpha which is down regulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.Clin Invest,1996,97:2651-2659.
31 Gilmore TD, Koedood M, Piffat KA, et al. Rel nuclear factor kappa B proteins and cancer. Oncogene,1996,13:1367-1378.
32 Nast G, Muller-Rober B. Molecular characterization of potato fumarate hydrates and functional expression in Escherichia coli.Plant Physiol,1996, 112:1219-1227.
33 Nakshatri H, Bhat-Nakshatri P, Martin DA, et al. Constitutive activation of nuclear factor kappa B during progression of breast cancer to hormone-independent growth. Mol Cell Biol,1997,17:3629-3639.
34 涂刚,姚楱祥,董蒲江。核因子KB在人乳腺癌组织中的表达及意义。中国普通外科杂志,2003,12(5)：348-350。
35 Cogswell PC, Gultridge DC, Funkhouser WK, et al. Selective activation of Nuclear factor kappa B subunits in human breast cancer potential roles for NF-κB/P52 and for Bcl-3. Oncogene,2000,19:1123-1131.
36 Shah N, Thomas TJ, Lewis JS, et al.Regulation of estrogenic and nuclear factor kappa B functions by polyamines and their role in polyamine analogy-induced apoptosis of breast cancer cells. Oncogene,2001,20:1715-1729.
37 Chen F, Cast ramova V, Shi X, et al. New insights into the role of nuclear factor kappa B, a ubiquitous transcription factor in the initiation of diseases.Clin Chem.1999,45:7-17.
38 胡海燕,邹典武,孙惠等。NF-κB与肿瘤细胞增殖及转移关系的研究进展。生命科学,2003,15(50：293-298。
39 Klein CB,et al.[J]. Environ Health Perspect,1999,107:438-439.
40 Salnikow K,et al. [J]. Mol Cell Biochem,2002,234:265-275.
41 sunderman FW. Metal and lipid peroxidation. Act Pharmacol Toxicol. 1986,59(17):248-255.
42 Aleksandra WK, Wojciech B. Damage of zinc fingers in DNA repair proteins a novel molecular mechanism in carcinogenesis. Toxicol Let.2006,162:29-42.
43 顾望松。镍的致癌作用。实用癌症杂志,1995,10(1)：71-72。
44 Ciccare Lli, KE Welter Hahn. Nickel-bound chromatin, nucleic acids and nuclear proteins from kidney and liver of rats treated with nickel carbonate in viro.Cancer Res.1984,44:3893-3897.
45 Farrah Clemens and Joseph R. Genotoxicity of samples of nickel refinery dust. Landolph Toxicol. Sci.2003,73:114-123.
46 Patierno SR, Costa M. Effects of nickel on nuclear protein hinding to DNA in intact mammalian cells.Cancer Biochem Biophys.l987,97:113-126.
2 Cruz MT, Concalo M, Fifueirexo A, et al.Contact sensitizer nickel sulfate activates the transcription factors nuclear factor kappa B and AP-1 and increases the expression of nitric oxide syntheses in a skin dendritic cell line.Exp Dematol, 2004,13:18-26.
3 Ernst MK, Dunn LL, Rice NR, et al. The pest like sequence of IKB is responsible for inhibition of DNA binding but not for cytoplasmic retention of C-REL or RelA homodimers.Mol Cell Biol,1995,15:872-882.
4 Waddick KG, Uckum FM. Innovative treatment programs against cancer nuclear factor kappa B as a molecular target.Biochem Pharm.1999,1,57:9-17.
5 Sen R, Baltimore D, Multiple nuclear factors interact with the immunoglubin enhancer sequences. Cell,1986,46:205-209.
6 Epinat JC, Gilmocr TD. Diverse agents act at multiple levels to inhibit the nuclear factor kappa B signal transduction pathway.Oncogene,1999,18:6896-6909.
7 Batkelt M, Gilmore TD. Control of apoptosis by nuclear factor kappa B transcription factors..Oncogene,1999,18:6910-6924.
8 Stancoraski I, Baltimore D. Nuclear factor kappa B activation:The 1KB kinase revealed.Cell,1997,91:299-302.
9 Blackwell TS, Christman JW. The role of nuclear factor kappa B in cytokine gene regulation.Cell Mol Biol,1997,17:3-9.
10 Beg AA, Sha WC, Bronson RT, Ghoshs SH, et al. Embryonic lethality and liver degeneration in mice lacking the role a component of Nuclear factor kappa B. Nature,1995,376:167-170.
11 Royds JA, Dower SK,Qwarnstrom EE,et al. Response of tumor cells to hypoxia role of P53 and Nuclear factor kappa B. Mol Pathol,1998,51:55-61.
12 Wu H, Lozano G, Nuclear factor kappa B activation of P53, A potential mechanism for suppressing cell growth in response to stress. Biol Chem.,1994, 269:67-74.
13 Reuther JY, Reuther GW, Cortez D, et al. A requirement for nuclear factor kappa B activation in mediated transformation. Genes Dev.,1998,12:968-981.
14 Mukhopadhyay T, Roth JA, Maxwell SA. Altered expression of the P50 subunit of the nuclear factor kappa B, transcription factor complex in non-small cell lung carcinoma. Oncogene,1995,11:999-1003.
15 Bargou RC, Wagener C, Bommer K, et al. Over expression of the death-promoting gene box-alpha which is down regulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.Clin Invest,1996,97:2651-2659.
16 Gilmore TD, Koedood M, Piffat KA, et al. Rel nuclear factor kappa B proteins and cancer. Oncogene,1996,13:1367-1378.
17 Nast G, Muller-Rober B. Molecular characterization of potato fumarate hydrates and functional expression in Escherichia coli.Plant Physiol,1996,112:1219-1227.
18 Nakshatri H, Bhat-Nakshatri P, Martin DA, et al. Constitutive activation of nuclear factor kappa B during progression of breast cancer to hormone-independent growth. Mol Cell Biol,1997,17:3629-3639.
19 涂刚,姚楱祥,董蒲江。核因子KB在人乳腺癌组织中的表达及意义。中国普通外科杂志,2003,12(5)：348-350。
20 Cogswell PC, Gultridge DC, Funkhouser WK, et al. Selective activation of Nuclear factor kappa B subunits in human breast cancer potential roles for NF-κB/P52 and for Bcl-3. Oncogene,2000,19:1123-1131.
21 Shah N, Thomas TJ, Lewis JS, et al.Regulation of estrogenic and nuclear factor kappa B functions by polyamines and their role in polyamine analogy-induced apoptosis of breast cancer cells. Oncogene,2001,20:1715-1729.
22 Chen F, Cast ramova V, Shi X, et al. New insights into the role of nuclear factor kappa B, a ubiquitous transcription factor in the initiation of diseases.Clin Chem.1999,45:7-17.
23 胡海燕,邹典武,孙惠等。NF-κB与肿瘤细胞增殖及转移关系的研究进展。生命科学,2003,15(5)：293-298。
24 朱茂祥,龚治芬,葛桂秀等。镍对作业工人脂质过氧化的损伤及硒的保护效果观察。职业医学,1995,22(3)：10-12。
25 Tkeshelashivli LK, Reid TM, Mcbride TJ, Loeb LA:Nickel induces a signature mutation for oxygen free radical damage. Cancer Res,1993,53:4172-4174.
26 Yin Z, Iranov VN, Habelhah H, Tewk, Ronaiz:Glutathione transfers pelicits protection against H2O 2-induced cell death via coordinated regulation of stress kinases. Cancer Res,2000,60:4053-4057.
27 Chen CY, Wang YF, Lin YH. Nickel-induced oxidative stress and effect of antioxidants in human lymphocytes.Arch Toxicol,2003,77:123-130.
28 Sunderman FW, Hopfer SM, Lin SM, Ploeman WC. Toxicity to alveolar macrophages in rats following paremteral injection of nickel chloride. Toxicol Appl Pharmacol,1989,100:107-118.
29 Bal W, Kozlow ski H, Kasprzak KS:Molecular models in nickel carcinogenesis.Inorg Biochem,2000,79:213-218.
30 Lin X, Costa M. Transform motion of human osteoblasts to anchorage independent growth by insoluble nickel parties.Environ health prospect,1994, 102(supple3):289-292.
31 申晓东,苏忆兰,蒋至诚等。氯化镍对大鼠脂质过氧化和抗氧化酶活性的影响[J]。卫生毒理学杂志,1998,12(1)：42-43.
32 周晔,李一俊,江雷军等。示波极谱法分析16种中药对活性氧自由基的清除能力[J]。天津医科大学学报,2003,9(2)：177-179.
33 谢大泽,湛学军,章涛等。枸杞等5种多糖清除氧自由基的实验研究[J]。江西医学检验,2002,20(4)：195-196.
34 袁新初,张端莲。当归注射液对更年期大鼠超氧化物歧化酶和脂质过氧化物的影响[J]。中草药,2001,32(9)：822-823.
35 李国莉,杨建军,任彬彬等。宁夏枸杞对密闭缺氧小鼠自由基防御体系的影 响[J]。卫生研究,2002,31(1)：30-31.
36 杨建雄,朱淑云,李发荣.连翘叶的体外抗氧化活性研究[J]。食品科学,2002,23(12)：120-123.
37 李凯霞,秦世武,付纯芳。茯苓合剂对冻融蟾蜍肝脏组织的生物化学研究[J]。黑龙江医药科学,2002,25(3)：11-13
38 宋剑涛,宋红梅。活血化瘀中药抗衰老作用机理研究进展[J]。山西中医,2002,18(6)：53-55
39 汪德清,田亚平,宋淑珍等。黄芪总黄酮抗突变作用实验研究[J]。中国中药杂志,2003,(28)：1164-1167
40 赵万红,曹永寿,袁泽飞。中药血清药理学的方法探讨[J]。中药新药与临床药理,2002,13(2)：122-124
1 Bennett BG. Environmental nickel pathways to man. Nickel in the human environment. IARC Scientific Publications,1984,53:487-495.
2 Costa M.Molecular targets of nickel carcinogenesis, epigenetic mechanisms. Environ Sic,1996,4:163-175.
3 刘艺敏,张敬,徐曼妮等。三氧化二镍诱导人肺成纤维细胞恶性转化的实验研究。中国公共卫生,2000,16(8)：694-696
4 Costa M, Salnikow K, Sutherland J, et al. The role of oxidative stress in nickel and chromate genotoxicity.Mol Cell Biol,2002,234:265-275
5 Denissenko MF, Salnikow K, et al. Nickel essentiality, toxicity and carcinoma gentility. Toxically Apple Pharmacology,2004,194:60-68
6 Costa M, et al.Molecular targets of nickel carcinogenesis.Mol Cell Biochem, 2001,222:205-211
7 Kasprzak KS, Sunderman FW, Salnikow K. Nickel carcinogenesis. Mutat Res,2003,533:67-97
8 Bal W, Kozlowski H, Kasprzale KS.Molecular models in nickel carcinogenesis. Inorg Biochem,2000,79:213-218
9 Sheth SG, Coudon FU, Chopua S. Nonalcoholic steatohepatitis. Ann Intern Med, 1997,126:137-138.
10 郑仲承。研究蛋白组学的新工具[J]。生命的化学,2001,21(4)：314-315。
11 To disco A, Takeuchi Y, Sadoshima JI, et al. Molecular mechanisms for somatost at inhibition of c-fos gene expression[J].Am I Physiol,1997,272:721-726.
12 Fan HY, Tong CH, Mitogen-activated protein kinase (MAPK) signaling pathways. State of the art [J], Chin J Zoology,2002,37:98-102.
13 Salnikow K, Davidson T, Zhang QW, et al. The involvement of hypoxia inducible transcription factor-1 dependent pathway in nickel carcinogenesis [J]. Cancer Res,2003,63:3524-3532.
14 Brewster JL, Devaloir T, Dwyer N C, et al.An osmosensing signal transduction pathway in yeast [J]. Science 1993,259:1760-1766.
15 Lee JC, Laydon JT, Medonnell PC, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis [J]. Nature 1994, 372:739-746.
16 Hebelhah H,Shah K, Huang L, et al. Identification of new JNK substrate using ATP pocket mutant JNK and a corresponding ATP analogue [J]. J Boil Chem., 2001,276:18090-18099.
17 张璐,姜勇,张琳等。丝裂原活化蛋白激酶研究进展[J]。国外医学生理、病理科学与临床分册,1999,19(2)；84-87。
18 朱敏,孙振学等。丝裂原活化蛋白激酶信号转导通路。武警医学院学报,2006,15(5)：500-503。
19 Jlindk T, Catling AD, Reuter CW, et al. Ras and Raf-1 form a signaling complex with MEKl.but not MEK7 [J]. Mol Cell Boil,2001,14:8211-8213.
20 Sutherland CL, Heath AW, Pelech SL, et al.Differential activation of the ERK, JNK and P38 mitogen activated protein kinase by CD40 and the B cell antigen receptor [J]. J Immune,1996,157:3381-3383.
21 Dziareke R, Jin YP, Gupta D. Differential activation of extra cellular signal regulated kinase ERK2 P38 and C-Jun N-terminal kinase mitogen activated protein kinases by bacterial peptidoglycan [J]. J Infect Dis,1996,174:777-785.
22 Waddick KG, Uckum FM. Innovative treatment programs against cancer nuclear factor kappa B as a molecular target.Biochem Pharm.1999,57:9-17.
23 Sen. R, Baltimore D, Multiple nuclear factors interact with the immunoglubin enhancer sequences. Cell,1986,46:205-209.
24 Blackwell TS, Chrisman JW. The role of nuclear factor kappa B in cytokine gene regulation.Cell Mol Boil,1997,17:3-9.
25 Beg AA, Sha WC, Bronson RT, Ghoshs SH, et al. Embryonic lethality and liver degeneration in mice lacking the role a component of Nuclear factor kappa B. Nature,1995,376:167-170.
26 Royds JA, Dower SK,Qwarnstrom EE,et al. Response of tumor cells to hypoxia role of P53 and Nuclear factor kappa B. Mol Pathol,1998,51:55-61.
27 Wu H, Lozano G, Nuclear factor kappa B activation of P53, A potential mechanism for suppressing cell growth in response to stress. Biol Chem.,1994, 269:67-74.
28 Reuther JY, Reuther GW, Cortez D, et al. A requirement for nuclear factor kappa B activation in mediated transformation. Genes Dev.,1998,12:968-981.
29 Mukhopadhyay T, Roth JA, Maxwell SA. Altered expression of the P50 subunit of the nuclear factor kappa B, transcription factor complex in non-small cell lung carcinoma. Oncogene,1995,11:999-1003.
30 Bargou RC, Wagener C, Bommer K, et al. Over expression of the death-promoting gene box-alpha which is down regulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice.Clin Invest,1996,97:2651-2659.
31 Gilmore TD, Koedood M, Piffat KA, et al. Rel nuclear factor kappa B proteins and cancer. Oncogene,1996,13:1367-1378.
32 Nast G, Muller-Rober B. Molecular characterization of potato fumarate hydrates and functional expression in Escherichia coli.Plant Physiol,1996, 112:1219-1227.
33 Nakshatri H, Bhat-Nakshatri P, Martin DA, et al. Constitutive activation of nuclear factor kappa B during progression of breast cancer to hormone-independent growth. Mol Cell Biol,1997,17:3629-3639.
34 涂刚,姚楱祥,董蒲江。核因子KB在人乳腺癌组织中的表达及意义。中国普通外科杂志,2003,12(5)：348-350。
35 Cogswell PC, Gultridge DC, Funkhouser WK, et al. Selective activation of Nuclear factor kappa B subunits in human breast cancer potential roles for NF-κB/P52 and for Bcl-3. Oncogene,2000,19:1123-1131.
36 Shah N, Thomas TJ, Lewis JS, et al.Regulation of estrogenic and nuclear factor kappa B functions by polyamines and their role in polyamine analogy-induced apoptosis of breast cancer cells. Oncogene,2001,20:1715-1729.
37 Chen F, Cast ramova V, Shi X, et al. New insights into the role of nuclear factor kappa B, a ubiquitous transcription factor in the initiation of diseases.Clin Chem.1999,45:7-17.
38 胡海燕,邹典武,孙惠等。NF-κB与肿瘤细胞增殖及转移关系的研究进展。生命科学,2003,15(50：293-298。
39 Klein CB,et al.[J]. Environ Health Perspect,1999,107:438-439.
40 Salnikow K,et al. [J]. Mol Cell Biochem,2002,234:265-275.
41 sunderman FW. Metal and lipid peroxidation. Act Pharmacol Toxicol. 1986,59(17):248-255.
42 Aleksandra WK, Wojciech B. Damage of zinc fingers in DNA repair proteins a novel molecular mechanism in carcinogenesis. Toxicol Let.2006,162:29-42.
43 顾望松。镍的致癌作用。实用癌症杂志,1995,10(1)：71-72。
44 Ciccare Lli, KE Welter Hahn. Nickel-bound chromatin, nucleic acids and nuclear proteins from kidney and liver of rats treated with nickel carbonate in viro.Cancer Res.1984,44:3893-3897.
45 Farrah Clemens and Joseph R. Genotoxicity of samples of nickel refinery dust. Landolph Toxicol. Sci.2003,73:114-123.
46 Patierno SR, Costa M. Effects of nickel on nuclear protein hinding to DNA in intact mammalian cells.Cancer Biochem Biophys.l987,97:113-126.