摘要
藏族是对高原低氧环境适应最好的民族,研究藏族人组织细胞的基因表达特性对于揭示低氧适应机制具有重要理论意义,对于寻找加速高原低氧习服措施也具有指导意义。
本研究以藏汉族骨髓组织为材料,采用抑制削减杂交技术和基因芯片技术对藏汉族骨髓组织差异表达基因进行筛选,利用定量RT-PCR方法对部分基因进行验证,应用生物信息学方法对差异表达基因进行分析。
对部分藏汉族骨髓组织差异表达基因进行验证发现,藏族人血红蛋白(Hb)基因表达与平原汉族人接近,而胚胎和胎儿期Hb基因(HbE、Hbζ和Hbγ)表达上调,促红细胞生成素受体(EPOR)的表达下调;促凋亡基因(HIFl-β、TP53、BAX)表达下调、抑制凋亡基因Bcl-2表达上调,凋亡蛋白基因Caspase2和Caspase8表达下调;细胞因子基因CCL5、IL28RA、IGFBP1与TGFBR3表达上调,参与酸碱平衡的碳酸酐酶Ⅱ(CA2)和水通道蛋白1 (AQP1)基因表达下调。
对藏汉族骨髓组织差异表达基因谱综合分析发现,HIF-1低氧反应通路基因表达下调;高氧亲和力血红蛋白组份(胚胎和胎儿期Hb基因)表达上调;凋亡抑制基因上调,促凋亡基因与凋亡蛋白基因表达下调;多数有氧代谢、无氧代谢酶类、电子传递链多种复合体、细胞周期相关蛋白以及RBC网架结构蛋白中的基因表达下调;多种细胞因子以及自由基清除酶类基因表达上调。
初步推测,HIF—1低氧反应通路基因的下调可能是藏族人低氧适应的一个特征,通过HIF-1通路基因的下调和抗凋亡能力的提高,产生细胞保护机制;藏族人通过上调高亲和力Hb基因表达,提高摄氧与供氧能力,通过降低代谢水平和增殖能力,减少氧与能量消耗,产生一种低氧条件下合理补充与利用能源的良性模式;藏族人通过下调AQP1、CA2表达,上调自由基清除酶类和一些细胞因子表达,保持细胞内环境的稳定。这种多环节、多基因的综合调控,有利于藏族人对高原低氧环境的适应。
Tibetan is unique ethnic group which exhibit best adaptation to the hypoxic environment at high altitude. Investigating differential expressed genes in bone marrow between Tibetan and Han ethnic group would be helpful to explore the mechanism of Tibetan adaptation to hypoxic environment and to develop the measurements in promoting hypoxia acclimatization of sea-level residents.In order to screen the specific genes to high altitude hypoxic adaptation , the SSH cDNA library of differential expression genes in bone marrow tissue between Tibetan and Han ethnic group was constructed and sequenced, and at mean time, the expression profile of differential expression genes between Tibetan and Han was analyzed by the gene chip method by using the bone marrow tissue samples from high altitude Tibetan natives and sea-level Han ethnic group. Some differential genes were reexamined by quantity real-time RT-PCR method.The reexamined result of real-time RT-PCR suggested that Hbε, Hbζ, Hbγ , BCL2, CCL5, IL28RA, IGFBPland TGFBR3 genes were upregulated , and TP53, BAX, EPOR, AQP1, Caspase2, Caspase8 和 CA2 genes were downregulated in the Tibetan bone marrow tissue compared with Han's.Compared with Han bone marrow myeloid tissue, genes encoded enzymes concerned with glycolysis, aerobic metabolism , genes encoded Complex I, IV, V of electron transport chain were downregulated in the Tibetan's, furthermore cell cycle-related genes, and RBC framework structure protein genes in Tibetan's were almost downregulated too. On the contrast, the hemoglobin genes, anti-apoptosis genes, some cytokine and chemokine genes are upregulated in the Tibetan's.These findings indicated that the effects of high altitude hypoxia on Tibetan bone marrow may be the result of multicultural genes' regulation. And the long-term
引文
1.吕永达,李开兴,尹昭云主编.高原医学与生理学.天津:天津科技出版社1995.
2.吕永达,霍仲厚主编.特殊环境生理学.北京:军事医学科学院出版社,2003:10—12.
3. Ramirez G, Bittle PA, Rosen R, et al. High altitude living: Genetic and environmental adaptation. Aviat Space Environ Med, 1999, 70:73-81.
4. Beall CM. An Ethiopian pattern of human adaptation to high altitude hypoxia. PNAS, 2002, 99(26): 17215-17218.
5. Lorna G. Moore. Human genetic adaptation to high altitude. High Aft Med Biol 2001, 2(2): 257-279.
6. Wu Tianyi. The Qinghai-Tibetan Plateau: how high altitude do Tibetan live? High Alt Med Biol 2001, 2(4): 489-499.
7. Wu Tianyi. Life on the High Tibetan Plateau. High Alt Med Biol, 2004, 5(1): 1-2.
8. Beall CM. High-Altitude Adaptations. The Lancet. Dec. 2003, 362: 14-15.
9.周兆年,吴秀凤,蒋海燕,等。急性低氧对藏族移居海平4年后藏族个体氧传递的影响.科学通报。1996,41(8):735-737.
10. Beall CM. Tibetan and Andean patterns of adaptation to high-altitude hypoxia. Hum Biol. 2000 Feb; 72(1): 201-28.
11. Beall CM, Song K, Elston RC, et al. Higher offspring survival among Tibetan women with high oxygen saturation genotypes residing at 4,000 m. Proc Natl Acad Sci U S A. 2004; 101(39): 14300-14304.
12. Lundby C, Pilegaard H, Andersen JL, et al. Acclimatization to 4100 m does not change capillary density or mRNA expression of potential angiogenesis regulatory factors in human skeletal muscle. J Exp Biol. 2004; 207(Pt 22): 3865-3871.
13. Guyton AC, Hall JE. Red blood cells, Anemia, and Polycythemia. From Textbook of Medical Physiology.. New York: W. B. Saunders Company, 2000. 498-500.
14.金伯泉.细胞和分子免疫学.世界图书出版社,2000年(第二版).
15. Lundby C, Pilegaard H, Andersen JL, et al. Acclimatization to 4100 m does not change capillary density or mRNA expression of potential angiogenesis regulatory factors in human skeletal muscle. J Exp Biol. 2004; 207(Pt 22): 3865-3871.
16.孙志新,冯建明,王建国等.高原红细胞增多症的骨髓细胞形态和活检病理组织的系列观察,现代临床医学生物工程学杂志 1999;5(04):245—247.
17. Hayashida K, Fujita J, Miyake Y, et al. Bone marrow-derived cells contribute to pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension. Chest. 2005 May; 127(5): 1793-1798.
18. Greijer A, van der Groep P, Kemming D, et al. Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1). J Pathol. 2005 19(5); [Epub ahead of print]
19.杨曦明 谢印芝 张东祥.血管内皮细胞生长因子与高原肺水肿发病的关系.中华医学杂志2000;80(12):931-935.
20. Moore LG, Shriver M, Bemis L, et al. Maternal adaptation to high-altitude pregnancy: an experiment of nature—a review. Placenta. 2004 Apr; 25 Suppl A:S60-71.
21. Lockhart DJ, Winzeler EA. Genomics, gene expression and DNA arrays. Nature. 2000 Jun 15; 405(6788): 827-836.
22. Diatchenko L, Lau YC, Campbell AP, et al. Supression Subtractive hybridization: A method for generating differential regulated or tissue specificial cDNA probe and libraries.[J] Proc Natl Acad Sci, 1996, 93(12): 6025-6030.
23. Kuang WW, Thompson DA, Hoch RV, et al. Differential screening and suppression subtractive hybridization identified genes differentially expressed in an estrogen receptor-positive breast carcinoma cell line. Nucleic Acids Res, 1998, 26(4): 1116-1123
24. Yang GP, Ross DT, Kuang WW, et al. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Res. 1999 15; 27(6): 1517-1523.
25. Cao W, Epstein C, Liu H, et al. Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study. BMC Genomics. 2004 Apr 27; 5(1): 26.
26. Ruchuang D, Baogui L, Thangamani M, et al. CD103 mRNA levels in urinary cells rejection of renal allografts. Transplantation. 2003; 2003; 75(8): 1307-1312.
27. Razeghi P, Essop MF, Huss JM, et al. Hypoxia-induced switches of myosin heavy chain iso-gene expression in rat heart. Biochem Biophys Res Commun. 2003 18;303(4): 1024-1027.
28. Richardson RS, Wagner H, Mudaliar SR, et al. Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise. Am J Physiol. 1999; 277(6 Pt 2): H2247-2252.
29.曾溢滔主编.人类血红蛋白[M].北京:科学出版社.2002.49-51.
30. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual[M]. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989.49-59.
31. Zhao W, Wilson Jb, Webber BB, et al. A second observation of Hb Abruzzo [alpha 2 beta 2 (143)(H21)His->Arg] in an Italian family[J], Hemoglobin, 1990; 14(4): 463-466.
32. Mosca A, Paleari R, Rubino FM, et al. Hb Abruzzo [beta143(H21)His->Arg] identified by mass spectrometry and DNA analysis[J]. Hemoglobin, 1993,17(3):261-268.
33.格日力.中国高原医学研究回顾.青海医学院学报,2005,26(1):1—2.
34.吕国蔚.低氧适应的进化 首都医科大学学报 2002,23(2):185-189
35. Fink T, Ebbesen P, Zachar V. Quantitative gene expression profiles of human liver derived cell lines exposed to moderate hypoxia. Cell Physiol Biochem 2001; 11(2): 105-114.
36.王金惠,善亚君,从玉文等.人体肝癌细胞急性低氧及低氧习服差异表达基因分析生理学报,2003,55(3):324-330
37. Greijer A, van der Groep P, Kemming D, et al. Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (H/F-1). J Pathol. 2005 May 19;206(3):291-304 [Epub ahead of print]
38. Semenza GL. Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochem Pharmacol. 2000 Jan 1; 59(1): 47-53. Review.
39. Semenza G. Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol. 2002; 64(5-6): 993-998.
40.赵惠卿,朱玲玲,赵彤等.低氧对胚胎干细胞增殖的影响.中国应用生理学杂志,2004,2(3):209—213.
41. Martin G, Andriamanalijaona R, Grassel S, et al. Effect of hypoxia and reoxygenation on gene expression and response to interleukin-1 in cultured articular chondrocytes. Arthritis Rheum. 2004 Nov; 50(11): 3549-3560.
42. Narayan AD, Ersek A, Campbell TA, et al.. The effect of hypoxia and stem cell source on haemoglobin switching.Br J Haematol. 2005 Feb; 128(4):562-570.
43. Bichet S, Wenger RH, Camenisch Get al. Oxygen tension modulates beta-globin switching in embryoid bodies. FASEB J. 1999 Feb; 13(2):285-295.
44. Sumin MN, Rezaikin AV, Yushkov BG. Hemoglobin heterogeneity under conditions of abnormal erythropoiesis. Bull Exp Biol Med. 2003 Jun; 135(6): 563-565.
45. Kunz M, Ibrahim SM. Molecular responses to hypoxia in tumor cells. Mol Cancer. 2003; 2: 23.
46. Foo RS, Mani K, Kitsis RN. Death begets failure in the heart. J Clin Invest. 2005 Mar 1; 115(3): 565-571.
47. Gomez-Navarro J, Arafat W, Xiang J. Gene therapy for carcinoma of the breast: Pro-apoptotic gene therapy. Breast Cancer Res. 2000; 2(1): 32-44.
48. Lassus P. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science 2002; 297: 1352-1354.
49.刘刊,聂鸿靖,马子敏等。复方党参对高原低氧大鼠脑细胞凋亡相关基因表达谱影响的分析中国现代应用药学杂志2004,21(5):345—349。
50. Dong JW, Zhu HF, Zhu WZ, Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression. Cell Res. 2003 Oct; 13(5): 385-391.
51. Yu HJ, Chien CT, Lai YJ, Hypoxia preconditioning attenuates bladder overdistension—induced oxidative injury by up-regulation of Bcl-2 in the rat. J Physiol. 2004 Feb 1; 554(Pt 3): 815-828.
52. Karsten Ruscher, Nikolaj Isaev, George Trendelenburg, et al. Induction of hypoxia inducible factor 1 by oxygen glucose deprivation is attenuated by hypoxic preconditioning in rat cultured neurons. Neuroscience Letters, 1998;254:117-120.
53. Semenza GL. Hypoxia-inducible factor 1: control of oxygen homeostasis in health and disease. Pediatr Res. 2001; 49(5): 614-617.
54.高继东 高原地区藏、汉男性血糖血脂的比较研究,高原医学杂志2001;11(1):19—22.
55. Hochachka PW, Stanley C, Matheson GO, et al. Metabolic and work efficiencies during exercise in Andean natives. J Appl physiol, 1991, 70: 1720～1730.
56. Lundby C, Saltin B, Hall VG. The 'lactate paradox', evidence for a transient change in the course of acclimatization to severe hypoxia in lowlanders. Acta Physiol Scand, 2000; 170: 265-269.
57. LaManna JC, Kutina-Nelson KL, Hritz MA, et all Decreased rat brain cytochrome oxidase activity after prolonged hypoxial. Brain Res, 1996,720(1-2): 1-61
58. Abdel-Aleem S, St Louis JD, Hughes GC et al. Metabolic changes in the normal and hypoxic neonatal myocardium. Ann N Y Acad Sci. 1999 Jun 30; 874: 254-261.
59.韩骀仁主编,分子细胞生物学,北京:科学出版社.2001(第二版).
60. Gamboa J, Caceda R, Gamboa A. Carbonic anhydrase activity in the red blood cells of sea level and high altitude natives. Biol Res. 2000;33(3-4):207-208.
61. Yang H, Hewett-Emmett D, Tashian RE. Absence or reduction of carbonic anhydrase Ⅱ in the red cells of the beluga whale and llama: implications for adaptation to hypoxia. Biochem Genet. 2000 Aug; 38(7-8): 241-252.
62. Leikauf GD, McDowell SA, Wesselkamper SC. Pathogenomic mechanisms for particulate matter induction of acute lung injury and inflammation in mice. Res Rep Health Eff Inst. 2001; (105): 5-58; 59-71.
63. Bai C, Fukuda N, Song Y, et al. Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice[J]. J Clin Invest, 1999, 103 (4): 555.
64. Jones JI, Clemmons D R. Insulin2 like growth factors and their binding proteins, biological actions. Endocr Rev, 1995; 16(1): 3.
65. Kajimura S, Aida K, Duan C. Insulin-like growth factor-binding protein-1 (IGFBP-1) mediates hypoxia-induced embryonic growth and developmental retardation. Proc Natl Acad Sci. 2005: 25; 102(4): 1240-1245.
66. Huang ST, Vo KC, Lyell DJ, et al. Developmental response to hypoxia. FASEB J. 2004 Sep; 18(12): 1348-1365.
67. Krampl E, Kametas NA, McAuliffe F, et al. Maternal serum insulin-like growth factor binding protein-1 in pregnancy at high altitude. Obstet Gynecol. 2002; 99(4): 594-598.
68. Dhandapani KM, Brann DW. Transforming growth factor-beta: a neuroprotective factor in cerebral ischemia. Cell Biochem Biophys. 2003; 39(1): 13-22.
69. Zhou S, Lechpammer S, Greenberger J, et al. Hypoxia inhibition of adipocytogenesis in human bone marrow stromal cells requires TGFbeta/Smad3 signaling. J Biol Chem. 2005 Apr 20; [Epub ahead of print]
70. Kempuraj D, Donelan J, Frydas S, et al. Interleukin-28 and 29 (IL-28 and IL-29): new cytokines with anti-viral activities. Int J Immunopathol Pharmacol. 2004; 17(2): 103-106.
71. Sheppard P, Kindsvogel W, Xu W, et al. IL-28, IL-29 and their class Ⅱ cytokine receptor IL-28R. Nat Immunol. 2003; 4(1): 63-68.
72. Laure Dumoutier, Amel Tounsi, Thomas Michiels, et al. Role of the Interleukin (IL)-28 Receptor Tyrosine Residues for Antiviral and Antiproliferative Activity of IL-29/Interferon-λ1: similarities with type Ⅰ interferon signaling. J Biol Chem. 2004; 279(31): 32269-32274.
73. Meager A, Visvalingam K, Dilger P, et al. Biological activity of interleukins-28 and-29: Comparison with type Ⅰ interferons. Cytokine. 2005 May 14; [Epub ahead of print]
74. Langer JA, Cutrone EC, Kotenko S. The Class Ⅱ cytokine receptor (CRF2) family: overview and patterns of receptor-ligand interactions. Cytokine Growth Factor Rev. 2004; 15(1): 33-48.
75. Gao JL et al Structure and functional expression of the human macrophage inflammatory protein 1 alpha/RANTES receptor. Journal of Experimental Medicine (1993); 177: 1421-1427.
76. Lim JK, Bums JM, Lu W, et al. Multiple pathways of amino terminal processing produce two truncated variants of RANTES/CCLS. J Leukoc Biol. 2005 May 27; [Epub ahead of print].
77. Saccani A, Saccani S, Orlando S, et al. Redox regulation of chemokine receptor expression. Proc Natl Acad Sci. 2000 14; 97(6): 2761-2766.
78. Bona E, Andersson AL, Blomgren K, et al. Chemokine and inflammatory cell response to hypoxia-ischemia in immature rats. Pediatr Res. 1999; 45(4 Pt 1): 500-509.
79. Kobusiak-PM, Orzeszko J, Mazur G, et al. Kinetics of chemokines in acute myocardial infarction. Kardiol Pol. 2005 Apt; 62(4): 301-316.
80. Kremlev SG, Palmer C. Interleukin-10 inhibits endotoxin-induced pro-inflammatory cytokines in microglial cell cultures. 63 J Neuroimmunol. 2005 May; 162(1-2): 71-80.
81. Meyer UA, Zanger UM. Molecular mechanisms of genetic polymorphisms of drug metabolism[J]. Annu Rev Pharmacol Toxicol, 1997; 37: 269-296
82.张鑫生,刘丽萍,郭雪微,等.高原居民SOD和LPO的变化.中华老年医学杂志,1993,12(6):365.
83.张鑫生,籍元放,刘丽萍,等.平原人进驻高原后体内自由基代谢变化和药物干预作