环孢霉素A诱导NIT-1胰岛β细胞基因的差异表达及有关分子机制
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摘要
环孢酶素A(cyclosporin A，CsA)是一种强效的免疫抑制剂，广泛地应用于器官、细胞移植后排斥反应的防治以及自身性免疫疾病的治疗。但是，不少临床观察显示CsA有影响胰岛β细胞功能、破坏糖耐量甚至导致糖尿病的副作用。研究分析CsA对胰岛功能的影响及其作用机制，对于指导临床合理用药及防治CsA的副作用具有较重要的现实意义。
目的：通过体外实验，观察CsA对NIT-1胰岛β细胞胰岛素释放的影响；同时，应用DNA芯片技术检测CsA作用于NIT-1细胞不同时间的基因差异表达，并结合生物信息学方法从基因水平上探讨CsA对胰岛素分泌影响的作用机制。
方法：以转基因NOD小鼠来源的NIT-1胰岛β细胞作为细胞模型，用不同浓度的CsA(0.05、0.1、0.5、1、5、10 Mm／L)分别处理24h、48h和72h后，分别检测胰岛素的累积释放；应用含有4096个小鼠基因的cDNA芯片分别检测以10μM／L的CsA作用于NIT-1细胞24h及48h后基因的差异表达，并应用RT-PCR方法对出现差异表达的6个基因进行了检测，验证了芯片检测结果；结合生物信息学方法对差异表达基因进行分析，筛选出20个与CsA对NIT-1细胞作用机制密切相关的基因，并对其有关分子机制进行了探讨。
结果：
1、CsA对胰岛素的累计释放具有抑制作用，且其抑制作用具有一定的剂量依赖性和时效性；
2、应用DNA芯片检测，在CsA作用于NIT-1细胞24h及48h后，分别
有84个基因(其中表达下调46个，表达上调38个)及710个基因(其中表
达下调366个，表达上调344个)出现差异表达;
3、结合生物信息学方法，从差异表达基因中筛选出20个与CsA对N工T一1
细胞作用机制密切相关的已知功能基因，它们都表达下调，其中包括氧化磷
酸化过程相关基因(共15个，eox7al、Cox7e、Coxsa;Atp6voe、Atp6vlgl;
Atplb3;AtP5k、Atpso;Mrps21、Mrpl23、MrP136、Mrp64、MrP13、MrPll3、
论pl一9)、SNAI花蛋白有关基因(共2个，Stxbpl、vamp4)、CaZ‘靶蛋白有关
基因(C aM kinasen一p亚单位类似蛋白)、G蛋白有关基因(Ar14因子)、
Pax6基因等。我们对其参与CSA抑制胰岛素分泌的有关分子机制进行了分
析、探讨。
结论:csA可抑制Nrr一1胰岛p细胞胰岛素的释放，其抑制作用
可能与抑制细胞氧化磷酸化、胰岛素胞吐分泌等代谢一分泌偶联过程中有关
功能性酶类或蛋白质mRNA的表达有关。
Cycloporin A (CsA) is widely used for therapeutic purposes for rejection after organ and cell transplantations and autoimmune diseases as a kind of potent immunosurpressant. But recently, quite a lot of clinical observations revealed that CsA has some side-effects on pancreatic ?cells such as impaired glucose tolerance and can even lead to diabetes mellitus. So, to study and analyze the effects and mechanism of the effect of CsA on pancreatic ? cells is of great importance for the rational drug usage and prevention of the side-effects of CsA.
Objective: To observe the impact of CsA on the insulin release of pancreatic ? cells. And to investigate the gene differential expression of NIT-1 cells treated by CsA at different time intervals. Then to explore the possible mechanism of the effects of CsA on insulin release combined with Bioinformatics.
Methods: NIT-1 cells were exposed to CsA at various concentrations(0.05.. 0.1. 0.5. 1. 5.10 礛/L) at different time intervals(24h, 48h and 72h). And the insulin release was investigated. Differential gene expression was observed and analyzed using DNA microarrays after NIT-1 cells was treated with CsA (lOMm/L) for 24h and 48h respectively. The DNA microarray results were conformed by RT-PCR with 6 differentially expressed genes. We analyzed the differentially expressed genes by bioinformatic analysis and finally screened out 20 genes that have close relationships with CsA's effects on NIT-1 cells, and explored their related molecular mechanisms.
Results:
1. CsA can inhibit insulin release, and its inhibitory effects are concentration-dependent and time-dependent
2. By DNA microarray hibrization and analysis, 84 genes were differentially expressed at 24h. Among them, 46 genes were down-regulated and 38 genes were
up-regulated. 710 genes were differentially expressed at 48h. Among them, 366 genes were down-regulated and 344 genes were up-regulated.
3. We screened out 20 genes (functions already known) that exhibit close relationship to CsA's effects on NIT-1 cells. All of them were down-regulated. These genes include: genes related to oxidative phosphorylation (15 genes, Cox7al . Cox7c. Cox8a, Atp6vOe. Atp6vlgl. Atplb3, Atp5k. Atp5o, Mrps21 . Mrpl23, Mrpl36. Mrp64. Mrpl3. Mrpll3. Mrpll9), SNARE protein related genes (Stxbpl. Vamp4), Ca target protein (similar to CaM kinase II beta) gene, G protein related gene (Arl4), and Pax6. We explored their related molecular mechanisms with respect of CsA's inhibitory effects.
onclusion: CsA can inhibit insulin release of NIT-1 pancreatic P cells. Its inhibitory effects are probably related to its inhibition of genes expression of some functional enzymes and proteins in the process of metabolization-secretion couplet mechanism such as oxidative phosphorylation and insulin exocytosis.
目的：通过体外实验，观察CsA对NIT-1胰岛β细胞胰岛素释放的影响；同时，应用DNA芯片技术检测CsA作用于NIT-1细胞不同时间的基因差异表达，并结合生物信息学方法从基因水平上探讨CsA对胰岛素分泌影响的作用机制。
方法：以转基因NOD小鼠来源的NIT-1胰岛β细胞作为细胞模型，用不同浓度的CsA(0.05、0.1、0.5、1、5、10 Mm／L)分别处理24h、48h和72h后，分别检测胰岛素的累积释放；应用含有4096个小鼠基因的cDNA芯片分别检测以10μM／L的CsA作用于NIT-1细胞24h及48h后基因的差异表达，并应用RT-PCR方法对出现差异表达的6个基因进行了检测，验证了芯片检测结果；结合生物信息学方法对差异表达基因进行分析，筛选出20个与CsA对NIT-1细胞作用机制密切相关的基因，并对其有关分子机制进行了探讨。
结果：
1、CsA对胰岛素的累计释放具有抑制作用，且其抑制作用具有一定的剂量依赖性和时效性；
2、应用DNA芯片检测，在CsA作用于NIT-1细胞24h及48h后，分别
有84个基因(其中表达下调46个，表达上调38个)及710个基因(其中表
达下调366个，表达上调344个)出现差异表达;
3、结合生物信息学方法，从差异表达基因中筛选出20个与CsA对N工T一1
细胞作用机制密切相关的已知功能基因，它们都表达下调，其中包括氧化磷
酸化过程相关基因(共15个，eox7al、Cox7e、Coxsa;Atp6voe、Atp6vlgl;
Atplb3;AtP5k、Atpso;Mrps21、Mrpl23、MrP136、Mrp64、MrP13、MrPll3、
论pl一9)、SNAI花蛋白有关基因(共2个，Stxbpl、vamp4)、CaZ‘靶蛋白有关
基因(C aM kinasen一p亚单位类似蛋白)、G蛋白有关基因(Ar14因子)、
Pax6基因等。我们对其参与CSA抑制胰岛素分泌的有关分子机制进行了分
析、探讨。
结论:csA可抑制Nrr一1胰岛p细胞胰岛素的释放，其抑制作用
可能与抑制细胞氧化磷酸化、胰岛素胞吐分泌等代谢一分泌偶联过程中有关
功能性酶类或蛋白质mRNA的表达有关。
Cycloporin A (CsA) is widely used for therapeutic purposes for rejection after organ and cell transplantations and autoimmune diseases as a kind of potent immunosurpressant. But recently, quite a lot of clinical observations revealed that CsA has some side-effects on pancreatic ?cells such as impaired glucose tolerance and can even lead to diabetes mellitus. So, to study and analyze the effects and mechanism of the effect of CsA on pancreatic ? cells is of great importance for the rational drug usage and prevention of the side-effects of CsA.
Objective: To observe the impact of CsA on the insulin release of pancreatic ? cells. And to investigate the gene differential expression of NIT-1 cells treated by CsA at different time intervals. Then to explore the possible mechanism of the effects of CsA on insulin release combined with Bioinformatics.
Methods: NIT-1 cells were exposed to CsA at various concentrations(0.05.. 0.1. 0.5. 1. 5.10 礛/L) at different time intervals(24h, 48h and 72h). And the insulin release was investigated. Differential gene expression was observed and analyzed using DNA microarrays after NIT-1 cells was treated with CsA (lOMm/L) for 24h and 48h respectively. The DNA microarray results were conformed by RT-PCR with 6 differentially expressed genes. We analyzed the differentially expressed genes by bioinformatic analysis and finally screened out 20 genes that have close relationships with CsA's effects on NIT-1 cells, and explored their related molecular mechanisms.
Results:
1. CsA can inhibit insulin release, and its inhibitory effects are concentration-dependent and time-dependent
2. By DNA microarray hibrization and analysis, 84 genes were differentially expressed at 24h. Among them, 46 genes were down-regulated and 38 genes were
up-regulated. 710 genes were differentially expressed at 48h. Among them, 366 genes were down-regulated and 344 genes were up-regulated.
3. We screened out 20 genes (functions already known) that exhibit close relationship to CsA's effects on NIT-1 cells. All of them were down-regulated. These genes include: genes related to oxidative phosphorylation (15 genes, Cox7al . Cox7c. Cox8a, Atp6vOe. Atp6vlgl. Atplb3, Atp5k. Atp5o, Mrps21 . Mrpl23, Mrpl36. Mrp64. Mrpl3. Mrpll3. Mrpll9), SNARE protein related genes (Stxbpl. Vamp4), Ca target protein (similar to CaM kinase II beta) gene, G protein related gene (Arl4), and Pax6. We explored their related molecular mechanisms with respect of CsA's inhibitory effects.
onclusion: CsA can inhibit insulin release of NIT-1 pancreatic P cells. Its inhibitory effects are probably related to its inhibition of genes expression of some functional enzymes and proteins in the process of metabolization-secretion couplet mechanism such as oxidative phosphorylation and insulin exocytosis.
引文
1. Borel JF, Feurer C, et al. Biological effects of cyclosporin A: a new antilymphocytic agents. Agents, 1976 Jul, 6(4):468-75
2. Calne RY, White DJ, et al. Cyclosporin A in patients receiving renal allografts from cadaver donors. Laancet, 1978 Dec, 2(8104-5):1323-7
3. AM, Awthomsen. Fk—506: an immunosuppressant for the 1990s? lancet 1991;337:25
4. Borel JF. The history of cyclosporin A and its significance. White DJG. ed,Amsterdam, 1983:5-18
5. Bullingham R, Monroe S, Nicholls A, et al. Pharmacokinetics and bioavailability of mycophenolate mofetil in healthy subjects after single-dose oral and intravenous administration. J Clin Pharmacol, 1996;36(4):315
6. Bullingham RE, Nicholls A, Hale M. Pharmacokinetics of mycophenolate mofetil (RS61443): a short revie. Transplant Proc, 1996; 28(2):925
7. Kohsaka M et al. In Okani Y ed. Biology of Actinomycetes'88 Tokyo: Janpan Scientific Societies Press; 1988:197
8. Lauerma AI, Surber C, Maibach H, et al. Absorption of topical tacrolimus(FK506) in vitro through human skin comparison with cyclosporin A. J Pharmacol, 1997;49:230～234
9. Hahn HJ, Laube F, et al. Toxic effects of cyclosporine on the endocrine pancreas of Wistar rats. Transplantation, 1986 Jan; 41(1):44-7
10. Yamanoto H, Akazawa S, et al. Effects of cyclosporin A and low dosages of steroid on posttransplantation diabetes in kidney transplant recipients. Diabetes Care, 1991,14(10):867-70
11. Robertson PR. Cyclosporin-induced inhibition of insulin secretion in isolated rat islets and HIT cells. Diabetes, 1986,35(9):1016-19
12. Tokumaru H, Umayahara K, Pellegrini L L, et al. SNARE complex oligomerization by synaphin/ complexin is essential for synaptic vesicle exocytosis.
Cell, 2001,104(3):421～432
13. Xu T, Ashery U, Burgoyne R D, et al. Early requirement for α—SNAP and NSF in the secretory cascade in chromaffin cells. EMBO J, 1999, 18(12):3293～3304
14. Kowluru A, Li G, Rabaglia M E, et al. Evidence for differential roles of the Rho subfamily of GTP2binding proteins in glucose- and calcium- induced insulin secretion from pancreatic beta cells. Biochem Pharmacol,1997,54(10):1097～1108
15. Iezzi M, Escher G, Meda P, et al. Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin2secreting cells. Mol Endocrinol, 1999, 13(2):202～212
16. Makino S, Kunimoto K, et al. Breeding of a non-obese, diabetic strain of mice. Jikken Dobutsu. 1980;29(1):1-13
17. Hamaguchi K, Gaskins HR, et al. NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse. Diabetes 1991 Jul;40(7):842-9
18. Martin F, Bedoya FJ. Mechanism of action of cyclosporin A on islet alfa- and beta-cells. Effects on camp-and calcium-dependent pathways. Life Sci. 1991;49(25):1915-21
19. Nielsen JH, Mandrup-Poulsen T, et al. Direct effects of cyclosporin A on human pancreatic beta-cells. Diabetes. 1986 Sep; 35(9):1049-52
20. Schwaninger M, Blume R, et al. Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK506 after membrane depolarization. J Biol Chem, 1993 Nov 5; 268(31):23111-5
21. Schwaninger M, Blume R, et al. The immunosuppressive drugs cyclosporin A and FK506 inhibit calcineurin phosphatase activity and gene transcription mediated through the cAMP-responsive element in a nonimmune cell line. Naunyn Schiedebergs Arch Pharmacl. 1993 Nov; 348(5):541-5
22. Oetjen E, Baun D, et al. Inhibition of human insulin gene transcription by immunosuppressive drugs cyclosporin A and tacrolimus in primary, mature islets
of transgenic mice. Mol Pharmacol.
23. Schena M, Shalon D, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995, 270(20):467-470
24. Shalev A, Pise-Masison CA, et al. Oligonucleotide microarray analysis of intact human pancreatic islets: identification of glucose-responsive genes and a highly regulated TGFbeta signaling pathway. Endocrinology, 2002, Sep, 143(9):3695-8
25. Jenssen TK, Laegreid A, et al. A literature network of human genes for high-throughput analysis of gene expression. Nature Genet 2001, 28:21-28
26. Damien C, Alan S. Mining microarray expression data by literature profiling. Genome Biol, 2002;3(10):research0055.1-research 0055.16
27．黄仲曦等，用文献轮廓挖掘大肠癌芯片表达谱。第一军医大学学报，2003；23(11)，1195-1197
28. Duchen MR, Smith PA, et al. Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells. Biochem J, 1993, 294(Pt1):35-42
29. Dufer M, Krippeit-Drews P, et al. Diabetogenic effect of cyclosporin A is mediated by interference with mitochondrial function of pancreatic B-cells. Mol Pharmacol, 2001, 60(4):873-9
30. Xu T, Binz T, Niemann H, et al. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity [see comments]. Nat Neurosci, 1998, 1(3):192～200
31. Xu T, Rammner B, Margittai M, et al. Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis. Cell, 1999,99(7):713～722
32. Wheeler M B, Sheu L, Ghai M, et al. Characterization of SNARE protein expression in beta cell lines and pancreatic islets. Endocrinology, 1996,137(4):1340～1348
33. Zeng X H, Qu A L, Lou X L, et al. Ca~(2+) signals induced from calcium stores in
pancreatic islet βcells. Chin Sci Bull, 2000,45(1)51～56
34. Clapham DE, Neer EJ. G protein β γ subunits. Annu Rev Pharmacol Toxicol, 1997,37:167-203
35. De Vries L, Zheng B, et al. The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol,1997, 37:167-203
36. Iezzi M, Escher G, Meda P, et al. Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin2secreting cells. Mol Endocrinol, 1999,13(2):202～212
37. Yasuda T, Kajimoto Y, et al. PAX6 mutation as a genetic factor common to aniridia and glucose intolerance. Diabetes. 2002 Jan;51(1):224-30.
38. Hill ME, Asa SL, et al. Essential requirement for Pax6 in control of enteroendocrine proglucagon gene transcription. Mol Endocrinol. 1999 Sep;13(9):1474-86
2. Calne RY, White DJ, et al. Cyclosporin A in patients receiving renal allografts from cadaver donors. Laancet, 1978 Dec, 2(8104-5):1323-7
3. AM, Awthomsen. Fk—506: an immunosuppressant for the 1990s? lancet 1991;337:25
4. Borel JF. The history of cyclosporin A and its significance. White DJG. ed,Amsterdam, 1983:5-18
5. Bullingham R, Monroe S, Nicholls A, et al. Pharmacokinetics and bioavailability of mycophenolate mofetil in healthy subjects after single-dose oral and intravenous administration. J Clin Pharmacol, 1996;36(4):315
6. Bullingham RE, Nicholls A, Hale M. Pharmacokinetics of mycophenolate mofetil (RS61443): a short revie. Transplant Proc, 1996; 28(2):925
7. Kohsaka M et al. In Okani Y ed. Biology of Actinomycetes'88 Tokyo: Janpan Scientific Societies Press; 1988:197
8. Lauerma AI, Surber C, Maibach H, et al. Absorption of topical tacrolimus(FK506) in vitro through human skin comparison with cyclosporin A. J Pharmacol, 1997;49:230～234
9. Hahn HJ, Laube F, et al. Toxic effects of cyclosporine on the endocrine pancreas of Wistar rats. Transplantation, 1986 Jan; 41(1):44-7
10. Yamanoto H, Akazawa S, et al. Effects of cyclosporin A and low dosages of steroid on posttransplantation diabetes in kidney transplant recipients. Diabetes Care, 1991,14(10):867-70
11. Robertson PR. Cyclosporin-induced inhibition of insulin secretion in isolated rat islets and HIT cells. Diabetes, 1986,35(9):1016-19
12. Tokumaru H, Umayahara K, Pellegrini L L, et al. SNARE complex oligomerization by synaphin/ complexin is essential for synaptic vesicle exocytosis.
Cell, 2001,104(3):421～432
13. Xu T, Ashery U, Burgoyne R D, et al. Early requirement for α—SNAP and NSF in the secretory cascade in chromaffin cells. EMBO J, 1999, 18(12):3293～3304
14. Kowluru A, Li G, Rabaglia M E, et al. Evidence for differential roles of the Rho subfamily of GTP2binding proteins in glucose- and calcium- induced insulin secretion from pancreatic beta cells. Biochem Pharmacol,1997,54(10):1097～1108
15. Iezzi M, Escher G, Meda P, et al. Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin2secreting cells. Mol Endocrinol, 1999, 13(2):202～212
16. Makino S, Kunimoto K, et al. Breeding of a non-obese, diabetic strain of mice. Jikken Dobutsu. 1980;29(1):1-13
17. Hamaguchi K, Gaskins HR, et al. NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse. Diabetes 1991 Jul;40(7):842-9
18. Martin F, Bedoya FJ. Mechanism of action of cyclosporin A on islet alfa- and beta-cells. Effects on camp-and calcium-dependent pathways. Life Sci. 1991;49(25):1915-21
19. Nielsen JH, Mandrup-Poulsen T, et al. Direct effects of cyclosporin A on human pancreatic beta-cells. Diabetes. 1986 Sep; 35(9):1049-52
20. Schwaninger M, Blume R, et al. Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK506 after membrane depolarization. J Biol Chem, 1993 Nov 5; 268(31):23111-5
21. Schwaninger M, Blume R, et al. The immunosuppressive drugs cyclosporin A and FK506 inhibit calcineurin phosphatase activity and gene transcription mediated through the cAMP-responsive element in a nonimmune cell line. Naunyn Schiedebergs Arch Pharmacl. 1993 Nov; 348(5):541-5
22. Oetjen E, Baun D, et al. Inhibition of human insulin gene transcription by immunosuppressive drugs cyclosporin A and tacrolimus in primary, mature islets
of transgenic mice. Mol Pharmacol.
23. Schena M, Shalon D, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995, 270(20):467-470
24. Shalev A, Pise-Masison CA, et al. Oligonucleotide microarray analysis of intact human pancreatic islets: identification of glucose-responsive genes and a highly regulated TGFbeta signaling pathway. Endocrinology, 2002, Sep, 143(9):3695-8
25. Jenssen TK, Laegreid A, et al. A literature network of human genes for high-throughput analysis of gene expression. Nature Genet 2001, 28:21-28
26. Damien C, Alan S. Mining microarray expression data by literature profiling. Genome Biol, 2002;3(10):research0055.1-research 0055.16
27．黄仲曦等，用文献轮廓挖掘大肠癌芯片表达谱。第一军医大学学报，2003；23(11)，1195-1197
28. Duchen MR, Smith PA, et al. Substrate-dependent changes in mitochondrial function, intracellular free calcium concentration and membrane channels in pancreatic beta-cells. Biochem J, 1993, 294(Pt1):35-42
29. Dufer M, Krippeit-Drews P, et al. Diabetogenic effect of cyclosporin A is mediated by interference with mitochondrial function of pancreatic B-cells. Mol Pharmacol, 2001, 60(4):873-9
30. Xu T, Binz T, Niemann H, et al. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity [see comments]. Nat Neurosci, 1998, 1(3):192～200
31. Xu T, Rammner B, Margittai M, et al. Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis. Cell, 1999,99(7):713～722
32. Wheeler M B, Sheu L, Ghai M, et al. Characterization of SNARE protein expression in beta cell lines and pancreatic islets. Endocrinology, 1996,137(4):1340～1348
33. Zeng X H, Qu A L, Lou X L, et al. Ca~(2+) signals induced from calcium stores in
pancreatic islet βcells. Chin Sci Bull, 2000,45(1)51～56
34. Clapham DE, Neer EJ. G protein β γ subunits. Annu Rev Pharmacol Toxicol, 1997,37:167-203
35. De Vries L, Zheng B, et al. The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol,1997, 37:167-203
36. Iezzi M, Escher G, Meda P, et al. Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin2secreting cells. Mol Endocrinol, 1999,13(2):202～212
37. Yasuda T, Kajimoto Y, et al. PAX6 mutation as a genetic factor common to aniridia and glucose intolerance. Diabetes. 2002 Jan;51(1):224-30.
38. Hill ME, Asa SL, et al. Essential requirement for Pax6 in control of enteroendocrine proglucagon gene transcription. Mol Endocrinol. 1999 Sep;13(9):1474-86