大剂量照射后早期小鼠骨髓表达基因的初步研究
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摘要
目的 辐射能诱导细胞基因表达的改变，机体辐射损伤效应是大量基因表达变化的结果。骨髓型急性放射病以骨髓等造血组织损伤为基本病变，为深入理解骨髓辐射损伤的分子机制，有必要研究整体照射条件下，辐射前后骨髓基因表达的变化。方法 运用抑制消减杂交(SSH)和cDNA阵列杂交等分子生物学技术，以及序列比对等生物信息学分析方法研究大剂量照射后早期小鼠骨髓基因在mRNA水平的差异表达。结果成功构建C57BL／6J小鼠受7Gyγ射线照射后4hr的骨髓组织相对未受辐射正常对照的消减cDNA文库，经PCR鉴定后，保存480个含差异表达cDNA片段的单克隆。7Gyγ射线照射后4hr C57BL／6J小鼠骨髓表达可能升高的基因有一未命名蛋白、干扰素诱导蛋白(IFIT1)、嗜中性粒蛋白(NPG)、钙结合蛋白(S100a8)、珠蛋白α链(Hba1)、MPG(甲基嘌呤糖基化酶)、CDKN1A(周期蛋白依赖的蛋白激酶抑制蛋白)、CAT(过氧化氢酶)、HO1(血红素加氧酶)和CYP2E1(细胞色素P450家族成员)；表达可能降低的有CCT3(分子伴侣3)、NAT3(N-乙酰转移酶3)、NPM1(核仁蛋白)、PTGS1(前列腺素内过氧化物合酶)、UGT1A1(UDP葡糖甘酰转移酶)；同时分离了可能代表辐射相关新基因的EST序列。已验证CDKN1A与一新登录EST(BU101501)在辐射后表达升高，其余基因是否真正辐射后差异表达及差异表达机制仍有待进一步的实验研究。结论 辐射后差异表达基因的功能及表达调控规律反映骨髓作为造血免疫器官在辐射后功能的变化和辐射后骨髓神经内分泌调节系统的变化，也应证了辐射诱导基因对机体起保护作用的一般功能规律。本工作为进一步分离辐射相关差异表达基因、探索骨髓辐射损伤分子机制的研究奠定基础。
Aim Radiation can induce changes in gene expression in mammalian cells, and the radiation injury in an organism is also related to the expression changes of many genes. The injury of hematopoietic tissues including bone marrow is one of the major pathological changes of acute radiation syndrome (ARS). To explore the molecular mechanism of bone marrow injury caused by radiation, gene expression of bone marrow in mice after whole body irradiation was studied. Methods The molecular biology technology including suppression subtract!ve hybridization (SSH) and cDNA array hybridization, in addition to the bioinformatics analysis method such as sequence alignment, were used to study differential gene expression of mRNA level in mouse bone marrow early after high dose radiation in vivo. Results Taking cDNA from bone marrow in C57BL/6J mice at 4hr after whole-body irradiation with 7Gy 60 Co -rays as the tester, and the cDNA from the control mice as the driver, the subtracted cDNA library was constructed s
uccessfully. After PCR test, 480 clones which have inserted differential cDNA fragments were conserved. The possibly induced genes in bone marrow of C57BL/6J mice at 4hr after whole-body irradiation with 7Gy 60Co y-rays included an unnamed protein, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), neutrophilic granule protein (NPG) ,8100 calcium binding protein A8 (S100a8), Hemoglobin alpha (Hbal), N-methylpurine-DNA glycosylase (MPG), cyclin-dependent kinase inhibitor 1A (CDKN1A), heme oxygenase 1 (HOI) and cytochrome P450, family 2 subfamily e polypeptide 1 (CYP2E1); The possibly repressed genes included chaperonin subunit 3 (CCT3), nucleophosmin 1 (NPM1), N-acetyltransferase 3 (NAT3),prostaglandin-endoperoxide synthase (PTGS1), UDP-glucuronosyltransferase 1 family, member 1 (UGT1A1); Some expressed sequence tags (ESTs) which possibly represented the new radiation-related genes were also separated . CDKN1A and a new EST (BUI01501) were validated to be induced after radiation. Further wor
k need to do to decide whether mRNA level of the other genes were really changed or what was the mechanism of differential gene expression after radiation. Conclusion the biological function and the expression regulation of those differentially expressed genes illuminated that radiation influenced
the hematopoiesis and immune function and the neuroimmunoendocrine network in bone marrow. Some irradiation-induced genes protect organism from genotoxic injury. The work establishes a basis for further research of irradiation-related genes and the molecular mechanism of bone marrow injury caused by radiation.
Aim Radiation can induce changes in gene expression in mammalian cells, and the radiation injury in an organism is also related to the expression changes of many genes. The injury of hematopoietic tissues including bone marrow is one of the major pathological changes of acute radiation syndrome (ARS). To explore the molecular mechanism of bone marrow injury caused by radiation, gene expression of bone marrow in mice after whole body irradiation was studied. Methods The molecular biology technology including suppression subtract!ve hybridization (SSH) and cDNA array hybridization, in addition to the bioinformatics analysis method such as sequence alignment, were used to study differential gene expression of mRNA level in mouse bone marrow early after high dose radiation in vivo. Results Taking cDNA from bone marrow in C57BL/6J mice at 4hr after whole-body irradiation with 7Gy 60 Co -rays as the tester, and the cDNA from the control mice as the driver, the subtracted cDNA library was constructed s
uccessfully. After PCR test, 480 clones which have inserted differential cDNA fragments were conserved. The possibly induced genes in bone marrow of C57BL/6J mice at 4hr after whole-body irradiation with 7Gy 60Co y-rays included an unnamed protein, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), neutrophilic granule protein (NPG) ,8100 calcium binding protein A8 (S100a8), Hemoglobin alpha (Hbal), N-methylpurine-DNA glycosylase (MPG), cyclin-dependent kinase inhibitor 1A (CDKN1A), heme oxygenase 1 (HOI) and cytochrome P450, family 2 subfamily e polypeptide 1 (CYP2E1); The possibly repressed genes included chaperonin subunit 3 (CCT3), nucleophosmin 1 (NPM1), N-acetyltransferase 3 (NAT3),prostaglandin-endoperoxide synthase (PTGS1), UDP-glucuronosyltransferase 1 family, member 1 (UGT1A1); Some expressed sequence tags (ESTs) which possibly represented the new radiation-related genes were also separated . CDKN1A and a new EST (BUI01501) were validated to be induced after radiation. Further wor
k need to do to decide whether mRNA level of the other genes were really changed or what was the mechanism of differential gene expression after radiation. Conclusion the biological function and the expression regulation of those differentially expressed genes illuminated that radiation influenced
the hematopoiesis and immune function and the neuroimmunoendocrine network in bone marrow. Some irradiation-induced genes protect organism from genotoxic injury. The work establishes a basis for further research of irradiation-related genes and the molecular mechanism of bone marrow injury caused by radiation.
引文
1．陈家佩，毛秉智．辐射血液学—基础与临床．军事医学科学出版社，2002
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7. Limanni A, Baker WH, Chang CM, etc. c-kit ligand gene expression in normal and sublethally irradiated mice. Blood, 1995, 85(9): 2377-84
8. Lockhart DJ,Winzeler E.Genomics, gene, expression and DNA arrays.Nature,2000,405:827-836
9. Amundson SA, Do KT, Shahab S, etc. Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation. Radiat Res, 2000,154(3): 342-6
10．孙志贤，王怀宾，夏寿萱．γ射线对哺乳动物细胞核DNA转录活性的影响及其机理研究。细胞生物学杂志，1983，5：18—21
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13. Smith ML, Fornace AJ Jr. Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis. Mutat Res, 1996, 340(2-3): 109-24
14．邹仲敏，罗成基．细胞凋亡和细胞增殖抑制在射线损伤造血功能中的作用．第三军医大学学报，1997，2：136，141
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19. Verlaet M, Deregowski V, Denis G, etc. Genetic imbalances in preleukemic thymuses. Biochem Biophys Res Commun, 2001,283(1):12-8
20. Bole-Feysot C, Perret E, Roustan P etc. Analysis of prolactin-modulated gene expression profiles during the Nb2 cell cycle using differential screening
techniques. Genome Biol 2000,1(4) : research0007． 1-0007． 15
21． 李衍达，孙之荣等译．生物信息学基因和蛋白质分析的实用指南．清华大学出版 社，2000，133-156
22． 贺林．解码生命．科学出版社，2000，333-351
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2．金伯泉．细胞和分子免疫学．世界图书出版社，1995，1-13
3．毕爱华．医学免疫学．人民军医出版社，1995，1-4
4. Dainiak N. Hematologic consequences of exposure to ionizing radiation. Experimental Hematology, 2002,30:513-528
5. Chang CM,Limanni A, Baker WH,etc.Sublethal gamma irradiation increases IL-lalpha, IL-6,and TNF-alpha mRNA levels in murine hematopoietic tissues. J Interferon Cytokine Res, 1997,17(9): 567-72
6. Chang CM, Elliott TB, Dobson ME, etc. Ionizing radiation and bacterial challenge alter splenic cytokine gene expression. J Radiat Res (Tokyo), 2000,41(3): 259-77
7. Limanni A, Baker WH, Chang CM, etc. c-kit ligand gene expression in normal and sublethally irradiated mice. Blood, 1995, 85(9): 2377-84
8. Lockhart DJ,Winzeler E.Genomics, gene, expression and DNA arrays.Nature,2000,405:827-836
9. Amundson SA, Do KT, Shahab S, etc. Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation. Radiat Res, 2000,154(3): 342-6
10．孙志贤，王怀宾，夏寿萱．γ射线对哺乳动物细胞核DNA转录活性的影响及其机理研究。细胞生物学杂志，1983，5：18—21
11．左连富．流式细胞术与生物医学．辽宁科学技术出版社，1996，129—135
12．夏寿萱．放射生物学．军事医学科学出版社，1998
13. Smith ML, Fornace AJ Jr. Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis. Mutat Res, 1996, 340(2-3): 109-24
14．邹仲敏，罗成基．细胞凋亡和细胞增殖抑制在射线损伤造血功能中的作用．第三军医大学学报，1997，2：136，141
15. Zanders ED, GouldenMG, Kennedy TC, etc. Analysis of immune gene expression in small rheumatoid arthritis biopsies using a comnination of subtractive hybridization and high-density cDNA arrays.Journal of Immunological Methods,2000, 233:131-140
16. Terskikh A, Lykyanov S, Diatchenko L, etc. Hunting hematopoietic stem-specific genes using PCR-select cDNA subtraction, http://www.clontech.com
17. Zhou R, Vander Heiden MG, Rudin CM. Genotoxic exposure is associated with alterations in glucose uptake and metabolism. Cancer Res, 2002,62(12):3515-20
18. Rimner A, Wischhusen J, Naumann U, etc. Identification by suppression subtractive hybridization of p21 as a radio-inducible gene in human glioma cells. Anticancer Res, 2001, 21(5):3505-8
19. Verlaet M, Deregowski V, Denis G, etc. Genetic imbalances in preleukemic thymuses. Biochem Biophys Res Commun, 2001,283(1):12-8
20. Bole-Feysot C, Perret E, Roustan P etc. Analysis of prolactin-modulated gene expression profiles during the Nb2 cell cycle using differential screening
techniques. Genome Biol 2000,1(4) : research0007． 1-0007． 15
21． 李衍达，孙之荣等译．生物信息学基因和蛋白质分析的实用指南．清华大学出版 社，2000，133-156
22． 贺林．解码生命．科学出版社，2000，333-351
23． Mao M, Fu G, Wu JS, ect. Identification of genes expressed in human CD34+ hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning. Proc Natl Acad Sci USA, 1998, 98:8175-8180
24． Nishiguchi S, Sakuma R, Nomura M, etc. A catalogue of genes in mouse embryonal carcinoma F9 cells identified with expressed sequence tags. J Biochem, 1996,119:749-767
25． Kawai J, Shinagawa A, Shibata K, etc. Functional annotation of a full-length mouse cDNA collection. Nature, 2001, 409 (6821) : 685-690
26． TolstonogGV, Wang X, Shoeman R, etc. Intermediate filaments reconstituted from vimentin, desmin, and glial fibrillary acidic protein selectively bind repetitive and mobile DNA sequences from a mixture of mouse genomic DNA fragments. DNA Cell Biol, 2000,19(11) : 647-77
27． Fomenko L, Sirota N, Ravin V,etc. Interferon alpha reduces the level of radiation-induced micronuclei in mouse bone marrow cells. Arch Immunol Ther Exp (Warsz), 1997,45 (5-6) :475-8
28． Smith JB, Herschman HR. The glucocorticoid attenuated response genes GARG-16, GARG-39, and GARG-49/IRG2 encode inducible proteins containing multiple tetratricopeptide repeat domains. Arch Biochem Biophys, 1996,330(2) : 290-300
29． Bluyssen HA, Vlietstra RJ, Faber PW, etc. Structure, chromosome localization, and regulation of expression of the interferon-regulated mouse Ifi54/Ifi56 gene family. Genomics, 1994, 24(1) : 137-48
30． Moscinski LC, Hill B. Molecular cloning of a novel myeloid granule protein. J Cell Biochem, 1995, 59(4) : 431-42
31． Scott LM, Mueller L, Collins SJ. E3, a hematopoietic-specific transcript directly regulated by the retinoic acid receptor alpha. Blood, 1996, 88(7) : 2517-30
32． Gebhardt C, Breitenbach U, Tuckermann JP, etc. Calgranulins S100A8 and S100A9 are negatively regulated by glucocorticoids in a c-Fos-dependent manner and overexpressed throughout skin carcinogenesis. Oncogene, 2002, 21(27) : 4266-76
33． Nacken W, Manitz MP, Sorg C. Molecular characterisation of the genomic locus of the mouse MRP8 gene. Biochim Biophys Acta, 1996, 1315(1) : 1-5
34． Thomas Brittain. Molecular aspects of embryonic hemoglobin function. Molecular Aspects of Medicine, 2002, 23 :293-342
35． Hanson KP, Zhivotovsky BD. Effects of X-irradiation on the hybridization of rat thymus nuclear RNA with repeated and unique DNA sequences. Int J Radiat Biol, 1976,30: 129-139
36． O' Neill J. Patrick, Rogan Peter K, Cariello Neal, etc. Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum. Mutation Research, 1998, 411: 179-214
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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi(NCBI PubMed)
http://www.mlpla.org.cn/(军事医学图书馆)
http://www.freemedical journals.com/htm/index.htm(部分西文期刊全文)
http://elsevier.lib.tsinghua.edu.cn/(部分西文期刊全文)
http://www.ncbi.nlm.nih.gov/(NCBI主页常用联结有：PubMed、BLAST、LocusLink、UniGene、NCBIHandbook、dbEST等)
http://fantom.gsc.riken.go.jp/(RIKEN数据库)
http://bioinf.bmi.ac.cn/postnuke/services.php(PCR引物设计软件(primer3)，基于JAVA的分子生物学分析工具(JaMBW))
http://rsb.info.nih.gov/ij/index.html(ImageJ软件)
http://www.roche-applied-science.com/no cache/molecular.cfm(Roche试剂使用)
http://www.clontech.com/techinfo/manuals/index.shtml(Clontech试剂使用)