人JNK3真核表达载体的构建及稳定表达SHSY5Y细胞系的建立
|
摘要
凋亡是由基因调控的细胞自主性、程序性死亡。在机体的生命活动中,细胞凋亡和抗凋亡呈现出统一的动态平衡,其间各种信号转导途径和信号分子之间存在着复杂关系,调控着细胞的生存和死亡。研究表明,细胞的凋亡和抗凋亡失衡与肿瘤的发生发展有关。c-Jun氨基末端激酶(c-Jun N-terminal kinases,JNKs)为丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPKs)超家族成员之一。现已证明MAPK通路可转导并调控细胞凋亡信号,而JNK信号通路则在其中起着十分重要的作用。
在脊椎动物,JNK由jnk1、jnk2、jnk3三种基因编码,表达的JNK1、JNK2、JNK3蛋白主要位于细胞质,为丝氨酸/苏氨酸蛋白激酶。这3种基因编码的蛋白产物可通过选择性剪接产生了10种转录本。JNK1和JNK2在全身各组织广泛表达,JNK3仅在脑、心脏、睾丸等组织特异表达。和JNK1、JNK2不同,只有JNK3具有一个含38个氨基酸残基的延长的N末端特殊区域。现有研究发现,JNK3与神经退行性病变、神经细胞的增殖和凋亡有密切联系,可促进缺血、缺氧和有毒化学物质导致的神经细胞凋亡。但目前国内外对JNK3与肿瘤之间关系的研究尚不明确。因此,本课题应用DNA重组技术构建人JNK3真核表达载体,再稳定转染人神经母细胞瘤(SHSY5Y)细胞,分析其JNK3的表达,并探讨JNK3与肿瘤细胞凋亡的联系,为深入研究JNK3的功能奠定基础。研究内容主要包括以下两个部分:
一、JNK3真核表达载体的构建及鉴定。方法:以pDBLeu-JNK3为模板,PCR扩增获得人JNK3基因全长,目的片段定向插入至真核表达载体pEGFP-C3中,并转化大肠杆菌,经抗生素平板筛选获得阳性克隆,再分别进行PCR、酶切及测序鉴定。结果:获得了长为1269bp的人JNK3编码区全长DNA片段,并成功地将其定向插入到真核表达载体pEGFP-C3的CMV启动子下游,构建成为包含人JNK3基因的重组质粒pEGFP-C3-JNK3,PCR和酶切鉴定证实该重组质粒带有JNK3目的基因片段,DNA测序确认插入片段的序列准确无误。结论:真核表达载体pEGFP-C3-JNK3构建成功。
二、稳定表达人JNK3的SHSY5Y细胞系的建立及其在细胞凋亡方面的研究。方法:通过脂质体LipofectamineTM 2000将重组质粒转染SHSY5Y细胞,经G418筛选,建立稳定转染人JNK3的SHSY5Y细胞系。荧光显微镜下观察重组绿色荧光蛋白的表达,并采用RT-PCR、Western blot检测JNK3的表达。以过氧化氢(H_2O_2)为促凋亡因素,MMT和流式细胞仪检测各细胞系的细胞增殖及凋亡情况。结果:经脂质体转染和G418抗性筛选6~8周,建立了稳定转染pEGFP-C3-JNK3质粒的SHSY5Y细胞系。在该细胞系中JNK3基因能被有效地转录和翻译,且在H_2O_2诱导下,JNK3可明显抑制细胞增殖及促进细胞凋亡。
结论:稳定表达人JNK3的SHSY5Y细胞系建立成功。在H_2O_2诱导下,JNK3可明显抑制细胞增殖及促进细胞凋亡。该细胞系的建立为进一步研究JNK3的功能提供了重要的实验基础。
Apoptosis is the cellular autonomously and orderly death regulating by gene. In the activity of organism, apoptosis and anti-apoptosis present the unified dynamic equilibrium. Between signal transduction pathways and signal molecules, there may exist complicated relationship to regulate the living and death of cells. Many research indicated that disequilibrium between apoptosis and anti-apoptosis may relate to occurrence and development of tumor. The c-Jun N-terminal kinases (JNKs) are members of the mitogen activated protein kinases (MAPKs) superfamily. Studies have verificated that MAPK signaling pathway can transduce and regulate cellular apoptosis, and JNKs may play an important role in the process.
In vertebrate, JNKs are incoded by gene jnk1, jnk2 and jnk3. And the expressed protein including JNK1, JNK2 and JNK3 are Ser/Thr protein kinases, which are mostly found in cytoplasm. There are 10 JNK isoforms expressed in mammalian cells, which are derived from the alternative splicing of three genes. JNK1 and JNK2 isoforms are ubiquitous while JNK3 isoforms are expressed only in brain and testis and, to a lesser extent, heart. Unlike JNK1 and JNK2, JNK3 is unique in having an extended N-terminus of 38 amino acids. In recent years, studies have found that JNK3 is a mediator of nerve cell apoptosis and is consistently associated with nerve cell apoptosis induced by ischemic, hypoxic and poisonous chemical materials. However, researches on the relationships between JNK3 and tumors are not enough at present.
In this research, we used DNA recombination technique to construct a new eukaryotic expression vector of human JNK3. Then, we transfected SHSY5Y cell with the vector and established the stably-transfectnd SHSY5Y cell line. Finally we analysed the expression of JNK3, and investigated its relevance to cellular apoptosis to evaluate the physiological function of JNK3.
1. Construction and identification of eukaryotic expression vector of human JNK3. Methods: The full-length JNK3 cDNA fragment was amplified by PCR from eukaryotic expression vector pDBLeu-JNK3. The cDNA fragment was oriently inserted into another eukaryotic expression vector——pEGFP-C3. The positive clone was obtained after transformation and antibiotic screening,and it was identified by restriction endonuclease digestion, PCR, and DNA sequencing. Results: The JNK3 cDNA fragment was obtained and cloned into the downstream of CMV promoter of pEGFP-C3. The recombinant plasmid was identified by restriction PCR and endonuclease digestion. DNA sequencing confirmed that the sequence of the inserted element was correct. Conclusion: The eukaryotic expression vector pEGFP-C3-JNK3 was constructed successfully.
2. Establishment of stably-transfected SHSY5Y cell line and research on the relationship between JNK3 and apoptosis. Methods: the recombinant eukaryotic expression vector pEGFP-C3-JNK3 was transfected into SHSY5Y cells by lipofectamineTM 2000. After selected by G418, stably-transfected SHSY5Y cell line was establish. The expression of JNK3 was observed by fluorescence microscope and identified by RT-PCR and Western Blot. Then we discovered that high express JNK3 could restrain proliferation and promote apoptosis induced by hydrogen dioxide(H_2O_2). It was verificated by MTT and flow cytometer(FCM). Results: SHSY5Y cell line stably transfected with pEGFP-C3-JNK3 vector was established after liposome transfection and G418 selection. JNK3 gene was efficiently transcribed and translated. And high expression of JNK3 could significantly restrain proliferation and promote apoptosis induced by H_2O_2. Conclusion: Stably-transfected SHSY5Y cell line was established successfully. High expression of JNK3 could obviously restrain proliferation and promote apoptosis induced by H_2O_2. The stably-transfected cell line provides a solid experimental foundation for further studies on the function of JNK3.
在脊椎动物,JNK由jnk1、jnk2、jnk3三种基因编码,表达的JNK1、JNK2、JNK3蛋白主要位于细胞质,为丝氨酸/苏氨酸蛋白激酶。这3种基因编码的蛋白产物可通过选择性剪接产生了10种转录本。JNK1和JNK2在全身各组织广泛表达,JNK3仅在脑、心脏、睾丸等组织特异表达。和JNK1、JNK2不同,只有JNK3具有一个含38个氨基酸残基的延长的N末端特殊区域。现有研究发现,JNK3与神经退行性病变、神经细胞的增殖和凋亡有密切联系,可促进缺血、缺氧和有毒化学物质导致的神经细胞凋亡。但目前国内外对JNK3与肿瘤之间关系的研究尚不明确。因此,本课题应用DNA重组技术构建人JNK3真核表达载体,再稳定转染人神经母细胞瘤(SHSY5Y)细胞,分析其JNK3的表达,并探讨JNK3与肿瘤细胞凋亡的联系,为深入研究JNK3的功能奠定基础。研究内容主要包括以下两个部分:
一、JNK3真核表达载体的构建及鉴定。方法:以pDBLeu-JNK3为模板,PCR扩增获得人JNK3基因全长,目的片段定向插入至真核表达载体pEGFP-C3中,并转化大肠杆菌,经抗生素平板筛选获得阳性克隆,再分别进行PCR、酶切及测序鉴定。结果:获得了长为1269bp的人JNK3编码区全长DNA片段,并成功地将其定向插入到真核表达载体pEGFP-C3的CMV启动子下游,构建成为包含人JNK3基因的重组质粒pEGFP-C3-JNK3,PCR和酶切鉴定证实该重组质粒带有JNK3目的基因片段,DNA测序确认插入片段的序列准确无误。结论:真核表达载体pEGFP-C3-JNK3构建成功。
二、稳定表达人JNK3的SHSY5Y细胞系的建立及其在细胞凋亡方面的研究。方法:通过脂质体LipofectamineTM 2000将重组质粒转染SHSY5Y细胞,经G418筛选,建立稳定转染人JNK3的SHSY5Y细胞系。荧光显微镜下观察重组绿色荧光蛋白的表达,并采用RT-PCR、Western blot检测JNK3的表达。以过氧化氢(H_2O_2)为促凋亡因素,MMT和流式细胞仪检测各细胞系的细胞增殖及凋亡情况。结果:经脂质体转染和G418抗性筛选6~8周,建立了稳定转染pEGFP-C3-JNK3质粒的SHSY5Y细胞系。在该细胞系中JNK3基因能被有效地转录和翻译,且在H_2O_2诱导下,JNK3可明显抑制细胞增殖及促进细胞凋亡。
结论:稳定表达人JNK3的SHSY5Y细胞系建立成功。在H_2O_2诱导下,JNK3可明显抑制细胞增殖及促进细胞凋亡。该细胞系的建立为进一步研究JNK3的功能提供了重要的实验基础。
Apoptosis is the cellular autonomously and orderly death regulating by gene. In the activity of organism, apoptosis and anti-apoptosis present the unified dynamic equilibrium. Between signal transduction pathways and signal molecules, there may exist complicated relationship to regulate the living and death of cells. Many research indicated that disequilibrium between apoptosis and anti-apoptosis may relate to occurrence and development of tumor. The c-Jun N-terminal kinases (JNKs) are members of the mitogen activated protein kinases (MAPKs) superfamily. Studies have verificated that MAPK signaling pathway can transduce and regulate cellular apoptosis, and JNKs may play an important role in the process.
In vertebrate, JNKs are incoded by gene jnk1, jnk2 and jnk3. And the expressed protein including JNK1, JNK2 and JNK3 are Ser/Thr protein kinases, which are mostly found in cytoplasm. There are 10 JNK isoforms expressed in mammalian cells, which are derived from the alternative splicing of three genes. JNK1 and JNK2 isoforms are ubiquitous while JNK3 isoforms are expressed only in brain and testis and, to a lesser extent, heart. Unlike JNK1 and JNK2, JNK3 is unique in having an extended N-terminus of 38 amino acids. In recent years, studies have found that JNK3 is a mediator of nerve cell apoptosis and is consistently associated with nerve cell apoptosis induced by ischemic, hypoxic and poisonous chemical materials. However, researches on the relationships between JNK3 and tumors are not enough at present.
In this research, we used DNA recombination technique to construct a new eukaryotic expression vector of human JNK3. Then, we transfected SHSY5Y cell with the vector and established the stably-transfectnd SHSY5Y cell line. Finally we analysed the expression of JNK3, and investigated its relevance to cellular apoptosis to evaluate the physiological function of JNK3.
1. Construction and identification of eukaryotic expression vector of human JNK3. Methods: The full-length JNK3 cDNA fragment was amplified by PCR from eukaryotic expression vector pDBLeu-JNK3. The cDNA fragment was oriently inserted into another eukaryotic expression vector——pEGFP-C3. The positive clone was obtained after transformation and antibiotic screening,and it was identified by restriction endonuclease digestion, PCR, and DNA sequencing. Results: The JNK3 cDNA fragment was obtained and cloned into the downstream of CMV promoter of pEGFP-C3. The recombinant plasmid was identified by restriction PCR and endonuclease digestion. DNA sequencing confirmed that the sequence of the inserted element was correct. Conclusion: The eukaryotic expression vector pEGFP-C3-JNK3 was constructed successfully.
2. Establishment of stably-transfected SHSY5Y cell line and research on the relationship between JNK3 and apoptosis. Methods: the recombinant eukaryotic expression vector pEGFP-C3-JNK3 was transfected into SHSY5Y cells by lipofectamineTM 2000. After selected by G418, stably-transfected SHSY5Y cell line was establish. The expression of JNK3 was observed by fluorescence microscope and identified by RT-PCR and Western Blot. Then we discovered that high express JNK3 could restrain proliferation and promote apoptosis induced by hydrogen dioxide(H_2O_2). It was verificated by MTT and flow cytometer(FCM). Results: SHSY5Y cell line stably transfected with pEGFP-C3-JNK3 vector was established after liposome transfection and G418 selection. JNK3 gene was efficiently transcribed and translated. And high expression of JNK3 could significantly restrain proliferation and promote apoptosis induced by H_2O_2. Conclusion: Stably-transfected SHSY5Y cell line was established successfully. High expression of JNK3 could obviously restrain proliferation and promote apoptosis induced by H_2O_2. The stably-transfected cell line provides a solid experimental foundation for further studies on the function of JNK3.
引文
[1] Chang L, Karin M. Mammalian MAP kinase signaling cascades[J]. Nature.2001, 410: 37-40.
[2] Prasad KN, Kumar B, Yan XD, et al. alpha-Tocopheryl succinate, the most effectiveform of vitamin E for adjuvant cancer treatment: a review[J]. J Am Coll Nutr. 2003, 22(2): 108-117.
[3] Verheij M, Bose R, Lin XH, et al. Requirement for ceramide-initiatied SAPK/JNK signaling in stress-induced apoptosis[J]. Nature.1996, 380(6569): 75-79.
[4] Parola M, Robino G, Marra F, et al. HNE interacts directly with JNK isoforms in human hepatic stellate cells[J]. J Clin Invest. l998, 102: 1942-1950.
[5] Constant SL, Dong C, Yang DD, et al. JNK1 is required for T cell-mediated immunity against Leishmania major infection[J]. J Immunol. 2000, 165(5): 2671-2676.
[6] Ip YT, Davis R J. Signal transduction by the c-Jun N-terminal kinase (JNK) from inflammation to development[J]. Curr Opin Cell Biol. 1998, 10 (2): 205-219.
[7] Davis RJ. Signal transduction by the JNK group of MAPK kinases[J]. Cell. 2000, 103: 239-252.
[8] Gupta S, Barrett T, Whitmarsh AJ, et al. Selective interaction of JNK protein kinase isoforms with transcription factors[J]. EMBO J. 1996, 15(11): 2760-2770.
[9] Yang DD, Kuan CY, Whitmarsh AJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene[J]. Nature. 1997, 389(6653): 865-870.
[10] Morishima Y, Gotoh Y, Zieg J, et al. Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of F as ligand[J]. Neurosci. 2001, 21: 7551-7560.
[11] Waetzig V, Herdegen T. A single c-jun N-terminal kinase isoform(JNK3-p54) is an effector in both neuronal differentiation and cell death[J]. J Biol Chem. 2003, 278(1): 576-572.
[12] Namgung U, Xia Z. Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase[J]. J Neurosci. 2000, 20(17): 6442-6451.
[13] Waido GS, Standish BM, Berendzen J, et al. Rapid protein-folding assay using green fluorescent protein[J]. Nat Bioteehnol. 1999, 17(7): 691-695.
[1] Miloso M, Scuteri A, Foudah D, et al. MAPKs as mediators of cell fate determination:an approach to neurodegenerative diseases[J]. Curr Med Chem. 2008, 15(6): 538-48.
[2] Yang DD, Kuan CY, Whitmarsh AJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene[J]. Nature. 1997, 389 (6653): 865-70.
[3] Kuan CY, Whitmarsh AJ, Yang DD, et al. A critical role of neural-specific JNK3 for ischemic apoptosis[J]. Proc Natl Acad Sci U S A. 2003, 100(25): 15184-9
[4] Pirianov G, Brywe KG, Mallard C, et al. Deletion of the c-Jun N-terminal kinase 3 gene protects neonatal mice against cerebral hypoxic-ischaemic injury[J]. J Cereb Blood Flow Metab. 2007, 27(5): 1022-32. .
[1] Li BS, Wang J, Zeng YJ, et al. Exterimental study on serum fibrosis markers and liver tissue pathology and hepatic fibrosis in inmunodamaged rats [J]. Word J Gastroenterol. 1999, 7(12):1031-1034.
[2] Chang L, Karin M. Mammalian MAP kinase signaling cascades[J]. Nature. 2001, 410: 37-40.
[3] Prasad KN, Kumar B, Yan XD, et al. alpha-Tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment: a review[J]. J Am Coll Nutr. 2003, 22(2): 108-117.
[4] Verheij M, Bose R, Lin XH, et al. Requirement for ceramide-initiatied SAPK/JNK signaling in stress-induced apoptosis[J]. Nature. 1996, 380(6569): 75-79.
[5] Parola M, Robino G, Marra F, et al. HNE interacts directly with JNK isoforms in human hepatic stellate cells[J]. J Clin Invest. l998, 102: 1942-1950.
[6] Lin H, Lu M, Zhang YX, et al. Induce of a rat model of acetaldehyde liver diseases[J]. Word J Gastroenterol. 2001, 9(1): 24-28.
[7] Lawler S, Fleming Y, Goedert M, et al. Synergistic activation of SAPK1-JNK1 by two MAP kinase kinases in vitro [J]. Curr Biol. 1998, 8: 1387-90.
[8] Constant SL, Dong C, Yang DD, et al. JNK1 is required for T cell-mediatedimmunity against Leishmania major infection[J]. J Immunol, 2000, 165(5): 2671-2676.
[9] Davis RJ. Signal transduction by the JNK group of MAPK kinases[J]. Cell. 2000, 103: 239-252.
[10] Gupta S, Barrett T, Whitmarsh AJ, et al. Selective interaction of JNK protein kinase isoforms with transcription factors[J]. EMBO J. 1996, 15(11): 2760-2770.
[11] Tsuruta F, Sunayama J, Mori Y, et al. JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins[J]. EMBO J. 2004, 23(8): 1889-99.
[12] Kim BJ, Ryu SW, Song BJ. JNK-and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells[J]. J Biol Chem. 2006, 281(30): 21256-65.
[13] Lei K, Davis RJ. JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis[J]. Proc Natl Acad Sci USA. 2003, 100(5): 2432-7.
[14] Putcha GV, Le S, Frank S, et.al. JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis[J]. Neuron. 2003, 38(6): 899-914.
[15] Yang DD, Kuan CY, Whitmarsh AJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene[J]. Nature. 1997, 389(6653): 865-870.
[16] Morishima Y, Gotoh Y, Zieg J, et al. Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of F as ligand[J]. Neurosci. 2001, 21: 7551-7560.
[17] Waetzig V, Herdegen T. A single c-jun N-terminal kinase isoform(JNK3-p54) is an effector in both neuronal differentiation and cell death[J]. J Biol Chem. 2003, 278(1): 576-572.
[18] Namgung U, Xia Z. Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase[J]. J Neurosci, 2000, 20(17): 6442-6451.
[1] Li BS, Wang J, Zeng YJ, et al. Exterimental study on serum fibrosis markers and liver tissue pathology and hepatic fibrosis in inmunodamaged rats[J]. Word J Gastroenterol. 1999, 7(12): 1031-1034.
[2] Chang L, Karin M. Mammalian MAP kinase signaling cascades[J]. Nature. 2001, 410: 37-40.
[3] Prasad KN, Kumar B, Yan XD, et al. alpha-Tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment: a review[J]. J Am Coll Nutr. 2003, 22(2): 108-117.
[4] Verheij M, Bose R, Lin XH, et al. Requirement for ceramide-initiatied SAPK/JNK signaling in stress-induced apoptosis[J]. Nature. 1996, 380(6569): 75-79.
[5] Parola M, Robino G, Marra F, et al. HNE interacts directly with JNK isoforms in human hepatic stellate cells[J]. J Clin Invest. l998, 102: 1942-1950.
[6] Davis RJ. Signal transduction by the JNK group of MAPK kinases[J]. Cell. 2000, 103: 239-252.
[7] Gupta S, Barrett T, Whitmarsh AJ, et al. Selective interaction of JNK protein kinase isoforms with transcription factors[J]. EMBO J. 1996, 15(11): 2760-2770.
[8] Lin H, Lu M, Zhang YX, et al. Induce of a rat model of acetaldehyde liver diseases[J]. Word J Gastroenterol. 2001, 9(1): 24-28.
[9] Lawler S, Fleming Y, Goedert M, et al. Synergistic activation of SAPK1-JNK1 by two MAP kinase kinases in vitro [J]. Curr Biol. 1998, 8: 1387-90.
[10] Davis RJ. Signal transduction by the JNK group of MAPK kinases[J]. Cell. 2000, 103: 239-252.
[11] Constant SL, Dong C, Yang DD, et al. JNK1 is required for T cell-mediated immunity against Leishmania major infection[J]. J Immunol, 2000, 165(5): 2671-2676.
[12] Serge YF et al. [J]. Biol Chem. 1997, 272(51): 32163.
[13] Chen YR, Tan TH. The c-Jun N-teruunal kinase pathway and apoptotic signaling (review)[J]. 1nt J Oncol. 2000, 16(4): 651-662.
[14] Xia Z et al. [J].Science. 1995, 270: 326-1331.
[15] Yang DD, Kuan CY, Whitmarsh AJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene[J]. Nature. 1997, 389(6653): 865-870.
[16] Morishima Y, Gotoh Y, Zieg J, et al. Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of F as ligand[J]. Neurosci. 2001, 21: 7551-7560.
[17] Waetzig V, Herdegen T. A single c-jun N-terminal kinase isoform(JNK3-p54) is an effector in both neuronal differentiation and cell death[J]. J Biol Chem. 2003, 278(1): 576-572.
[18] Namgung U, Xia Z. Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase[J]. J Neurosci. 2000, 20(17): 6442-6451.
[19] Constant SL, Dong C, Yang DD, et al. JNK1 is required for T cell-mediated immunity against Leishmania major infection[J]. J Immunol. 2000, 165: 2671-2676.
[20] Yoshida S, K Fukino, H Harada, et al. The c-Jun N-terminal kinase3 ( JNK3 ) gene: genomic structure, chromosomal assignment, and loss of expression in brain tumors [J]. J Hum Genet. 2001, 46(4): 182.
[2] Prasad KN, Kumar B, Yan XD, et al. alpha-Tocopheryl succinate, the most effectiveform of vitamin E for adjuvant cancer treatment: a review[J]. J Am Coll Nutr. 2003, 22(2): 108-117.
[3] Verheij M, Bose R, Lin XH, et al. Requirement for ceramide-initiatied SAPK/JNK signaling in stress-induced apoptosis[J]. Nature.1996, 380(6569): 75-79.
[4] Parola M, Robino G, Marra F, et al. HNE interacts directly with JNK isoforms in human hepatic stellate cells[J]. J Clin Invest. l998, 102: 1942-1950.
[5] Constant SL, Dong C, Yang DD, et al. JNK1 is required for T cell-mediated immunity against Leishmania major infection[J]. J Immunol. 2000, 165(5): 2671-2676.
[6] Ip YT, Davis R J. Signal transduction by the c-Jun N-terminal kinase (JNK) from inflammation to development[J]. Curr Opin Cell Biol. 1998, 10 (2): 205-219.
[7] Davis RJ. Signal transduction by the JNK group of MAPK kinases[J]. Cell. 2000, 103: 239-252.
[8] Gupta S, Barrett T, Whitmarsh AJ, et al. Selective interaction of JNK protein kinase isoforms with transcription factors[J]. EMBO J. 1996, 15(11): 2760-2770.
[9] Yang DD, Kuan CY, Whitmarsh AJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene[J]. Nature. 1997, 389(6653): 865-870.
[10] Morishima Y, Gotoh Y, Zieg J, et al. Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of F as ligand[J]. Neurosci. 2001, 21: 7551-7560.
[11] Waetzig V, Herdegen T. A single c-jun N-terminal kinase isoform(JNK3-p54) is an effector in both neuronal differentiation and cell death[J]. J Biol Chem. 2003, 278(1): 576-572.
[12] Namgung U, Xia Z. Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase[J]. J Neurosci. 2000, 20(17): 6442-6451.
[13] Waido GS, Standish BM, Berendzen J, et al. Rapid protein-folding assay using green fluorescent protein[J]. Nat Bioteehnol. 1999, 17(7): 691-695.
[1] Miloso M, Scuteri A, Foudah D, et al. MAPKs as mediators of cell fate determination:an approach to neurodegenerative diseases[J]. Curr Med Chem. 2008, 15(6): 538-48.
[2] Yang DD, Kuan CY, Whitmarsh AJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene[J]. Nature. 1997, 389 (6653): 865-70.
[3] Kuan CY, Whitmarsh AJ, Yang DD, et al. A critical role of neural-specific JNK3 for ischemic apoptosis[J]. Proc Natl Acad Sci U S A. 2003, 100(25): 15184-9
[4] Pirianov G, Brywe KG, Mallard C, et al. Deletion of the c-Jun N-terminal kinase 3 gene protects neonatal mice against cerebral hypoxic-ischaemic injury[J]. J Cereb Blood Flow Metab. 2007, 27(5): 1022-32. .
[1] Li BS, Wang J, Zeng YJ, et al. Exterimental study on serum fibrosis markers and liver tissue pathology and hepatic fibrosis in inmunodamaged rats [J]. Word J Gastroenterol. 1999, 7(12):1031-1034.
[2] Chang L, Karin M. Mammalian MAP kinase signaling cascades[J]. Nature. 2001, 410: 37-40.
[3] Prasad KN, Kumar B, Yan XD, et al. alpha-Tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment: a review[J]. J Am Coll Nutr. 2003, 22(2): 108-117.
[4] Verheij M, Bose R, Lin XH, et al. Requirement for ceramide-initiatied SAPK/JNK signaling in stress-induced apoptosis[J]. Nature. 1996, 380(6569): 75-79.
[5] Parola M, Robino G, Marra F, et al. HNE interacts directly with JNK isoforms in human hepatic stellate cells[J]. J Clin Invest. l998, 102: 1942-1950.
[6] Lin H, Lu M, Zhang YX, et al. Induce of a rat model of acetaldehyde liver diseases[J]. Word J Gastroenterol. 2001, 9(1): 24-28.
[7] Lawler S, Fleming Y, Goedert M, et al. Synergistic activation of SAPK1-JNK1 by two MAP kinase kinases in vitro [J]. Curr Biol. 1998, 8: 1387-90.
[8] Constant SL, Dong C, Yang DD, et al. JNK1 is required for T cell-mediatedimmunity against Leishmania major infection[J]. J Immunol, 2000, 165(5): 2671-2676.
[9] Davis RJ. Signal transduction by the JNK group of MAPK kinases[J]. Cell. 2000, 103: 239-252.
[10] Gupta S, Barrett T, Whitmarsh AJ, et al. Selective interaction of JNK protein kinase isoforms with transcription factors[J]. EMBO J. 1996, 15(11): 2760-2770.
[11] Tsuruta F, Sunayama J, Mori Y, et al. JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins[J]. EMBO J. 2004, 23(8): 1889-99.
[12] Kim BJ, Ryu SW, Song BJ. JNK-and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells[J]. J Biol Chem. 2006, 281(30): 21256-65.
[13] Lei K, Davis RJ. JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis[J]. Proc Natl Acad Sci USA. 2003, 100(5): 2432-7.
[14] Putcha GV, Le S, Frank S, et.al. JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis[J]. Neuron. 2003, 38(6): 899-914.
[15] Yang DD, Kuan CY, Whitmarsh AJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene[J]. Nature. 1997, 389(6653): 865-870.
[16] Morishima Y, Gotoh Y, Zieg J, et al. Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of F as ligand[J]. Neurosci. 2001, 21: 7551-7560.
[17] Waetzig V, Herdegen T. A single c-jun N-terminal kinase isoform(JNK3-p54) is an effector in both neuronal differentiation and cell death[J]. J Biol Chem. 2003, 278(1): 576-572.
[18] Namgung U, Xia Z. Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase[J]. J Neurosci, 2000, 20(17): 6442-6451.
[1] Li BS, Wang J, Zeng YJ, et al. Exterimental study on serum fibrosis markers and liver tissue pathology and hepatic fibrosis in inmunodamaged rats[J]. Word J Gastroenterol. 1999, 7(12): 1031-1034.
[2] Chang L, Karin M. Mammalian MAP kinase signaling cascades[J]. Nature. 2001, 410: 37-40.
[3] Prasad KN, Kumar B, Yan XD, et al. alpha-Tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment: a review[J]. J Am Coll Nutr. 2003, 22(2): 108-117.
[4] Verheij M, Bose R, Lin XH, et al. Requirement for ceramide-initiatied SAPK/JNK signaling in stress-induced apoptosis[J]. Nature. 1996, 380(6569): 75-79.
[5] Parola M, Robino G, Marra F, et al. HNE interacts directly with JNK isoforms in human hepatic stellate cells[J]. J Clin Invest. l998, 102: 1942-1950.
[6] Davis RJ. Signal transduction by the JNK group of MAPK kinases[J]. Cell. 2000, 103: 239-252.
[7] Gupta S, Barrett T, Whitmarsh AJ, et al. Selective interaction of JNK protein kinase isoforms with transcription factors[J]. EMBO J. 1996, 15(11): 2760-2770.
[8] Lin H, Lu M, Zhang YX, et al. Induce of a rat model of acetaldehyde liver diseases[J]. Word J Gastroenterol. 2001, 9(1): 24-28.
[9] Lawler S, Fleming Y, Goedert M, et al. Synergistic activation of SAPK1-JNK1 by two MAP kinase kinases in vitro [J]. Curr Biol. 1998, 8: 1387-90.
[10] Davis RJ. Signal transduction by the JNK group of MAPK kinases[J]. Cell. 2000, 103: 239-252.
[11] Constant SL, Dong C, Yang DD, et al. JNK1 is required for T cell-mediated immunity against Leishmania major infection[J]. J Immunol, 2000, 165(5): 2671-2676.
[12] Serge YF et al. [J]. Biol Chem. 1997, 272(51): 32163.
[13] Chen YR, Tan TH. The c-Jun N-teruunal kinase pathway and apoptotic signaling (review)[J]. 1nt J Oncol. 2000, 16(4): 651-662.
[14] Xia Z et al. [J].Science. 1995, 270: 326-1331.
[15] Yang DD, Kuan CY, Whitmarsh AJ, et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene[J]. Nature. 1997, 389(6653): 865-870.
[16] Morishima Y, Gotoh Y, Zieg J, et al. Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of F as ligand[J]. Neurosci. 2001, 21: 7551-7560.
[17] Waetzig V, Herdegen T. A single c-jun N-terminal kinase isoform(JNK3-p54) is an effector in both neuronal differentiation and cell death[J]. J Biol Chem. 2003, 278(1): 576-572.
[18] Namgung U, Xia Z. Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase[J]. J Neurosci. 2000, 20(17): 6442-6451.
[19] Constant SL, Dong C, Yang DD, et al. JNK1 is required for T cell-mediated immunity against Leishmania major infection[J]. J Immunol. 2000, 165: 2671-2676.
[20] Yoshida S, K Fukino, H Harada, et al. The c-Jun N-terminal kinase3 ( JNK3 ) gene: genomic structure, chromosomal assignment, and loss of expression in brain tumors [J]. J Hum Genet. 2001, 46(4): 182.