大剂量照射后造血功能变化规律的研究
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摘要
目的：探索大剂量照射后造血功能的变化规律，寻找判断大剂量照射后造血损伤程度的分子生物学指标，探索急性放射病的诊断新指标和治疗新靶点。方法：观察不同剂量照射后C57BL／6J小鼠的生存率、外周血象、CFU-GM系集落培养、细胞周期和细胞凋亡的变化规律，小鼠骨髓组织部分造血生长因子与受体的表达规律，应用基因芯片技术筛选大剂量照射后小鼠骨髓组织的差异表达基因。结果：经生存率观察，选定了4个照射剂量组：6.5Gy、7.5Gy、8.5Gy和10Gy。发现以下指标存在一定的剂量相关性：外周血白细胞数最低值和血小板数降低发生时相；骨髓细胞照射后6小时的G2／M期阻滞细胞百分比和脾细胞照射后6小时S期阻滞细胞百分比；骨髓照射后12小时凋亡细胞百分比和脾脏与胸腺照射后6小时凋亡细胞百分比。应用RT-PCR技术从小鼠骨髓组织中扩增出干细胞因子(SCF)、粒细胞集落刺激因子受体(G-CSFR)、促红细胞生成素受体(EPOR)、促血小板生成素受体(c-mp1)和干细胞因子受体(c-kit)的部分序列，并经测序证实。用RT-PCR与Southern杂交联用法初步探索了大剂量照射后部分造血生长因子与受体的表达规律，发现大剂量照射后早期，各基因的表达水平未见明显变化，随着照射后时间的延长，由于造血处于枯竭状态，不能得到足量的总RNA用于分析。经过两次基因芯片杂交，发现功能未知的新基因NM-025383与辐射相关，并从小鼠骨髓组织扩增出该基因。结论：从细胞水平上观察到的部分剂量相关性指标，对大剂量照射后造血损伤程度的判断有参考意义。从小鼠骨髓组织克隆出部分造血生长因子与受体的cDNA序列，为从整体水平上研究这些低拷贝基因在大剂量照射后的表达规律奠定了基础。RT-PCR与Southern杂交联用法研究基因表达水平具有较好的可行性。发现功能未知的新基因NM-025383与辐射相关，为此基因的存在提供了实验证据，为此基因的功能研究提供了线索。
Objective: To analyze the hematopoietic function after high dose irradiation. To seek molecular biology methods which can evaluate the severity of hematopoietic injury. To screen new biomarkers for the diagnosis and treatment of acute radiation sickness. Methods: To explore mortality rate, peripheral blood cell counts, CFU-GM counts, cell cycle retardation and apoptosis from mice strain C57BL/6J after y rays total body irradiation of different doses. To study the expression level of some hematopoietic growth factor and receptors of mouse bone marrow after high dose irradiation. To search differentially expressed genes from mouse bone marrow after high dose irradiation using DNA microarray. Results: 6.5Gy, 7.5Gy, 8.5Gy and lOGy were determined which represent the seventy of hematopoietic injury. The following criterions were found to be dose correlated: Nadir of white blood cell and reduction phase of platelet after high dose irradiation; Percentage of G2/M retardation cells in bone marrow and S retardation c
ells in spleen 6h post irradiation; Percentage of apoptosis cells 12h post irradiation in bone marrow and 6h post irradiation in spleen and thymus. RT-PCR was used to clone partial sequences of SCF, G-CSFR, EPOR, c-mpl and c-kit from mouse bone marrow. Combining RT-PCR and Southern hybridization, the post irradiation expression level of these hematopoietic growth factor and receptors was observed in mouse bone marrow. No significant changes were found within 24h after high dose irradiation. Since the severe depletion of hematopoietic cells, no sufficient amount of total RNA can be obtained for investigation 48h after high dose irradiation. New gene NM-025383 was found to be radiation associated through DNA microarray screening and was cloned from mouse bone marrow. Conclusion: The 7 dose correlated criterions may help evaluate the severity of hematopoietic injury. The successful cloning of 5 low copy genes make it possible to investigate their expression level in vivo after high dose irradiation. Combining R
T-PCR and Southern hybridization is a practical method to analyze expression level of low copy genes in vivo. Functional research of radiation associated NM-025383 is feasible.
Objective: To analyze the hematopoietic function after high dose irradiation. To seek molecular biology methods which can evaluate the severity of hematopoietic injury. To screen new biomarkers for the diagnosis and treatment of acute radiation sickness. Methods: To explore mortality rate, peripheral blood cell counts, CFU-GM counts, cell cycle retardation and apoptosis from mice strain C57BL/6J after y rays total body irradiation of different doses. To study the expression level of some hematopoietic growth factor and receptors of mouse bone marrow after high dose irradiation. To search differentially expressed genes from mouse bone marrow after high dose irradiation using DNA microarray. Results: 6.5Gy, 7.5Gy, 8.5Gy and lOGy were determined which represent the seventy of hematopoietic injury. The following criterions were found to be dose correlated: Nadir of white blood cell and reduction phase of platelet after high dose irradiation; Percentage of G2/M retardation cells in bone marrow and S retardation c
ells in spleen 6h post irradiation; Percentage of apoptosis cells 12h post irradiation in bone marrow and 6h post irradiation in spleen and thymus. RT-PCR was used to clone partial sequences of SCF, G-CSFR, EPOR, c-mpl and c-kit from mouse bone marrow. Combining RT-PCR and Southern hybridization, the post irradiation expression level of these hematopoietic growth factor and receptors was observed in mouse bone marrow. No significant changes were found within 24h after high dose irradiation. Since the severe depletion of hematopoietic cells, no sufficient amount of total RNA can be obtained for investigation 48h after high dose irradiation. New gene NM-025383 was found to be radiation associated through DNA microarray screening and was cloned from mouse bone marrow. Conclusion: The 7 dose correlated criterions may help evaluate the severity of hematopoietic injury. The successful cloning of 5 low copy genes make it possible to investigate their expression level in vivo after high dose irradiation. Combining R
T-PCR and Southern hybridization is a practical method to analyze expression level of low copy genes in vivo. Functional research of radiation associated NM-025383 is feasible.
引文
1 毛秉智．切尔诺贝利核事故后十年：事故后果概述．解放军医学情报．1996；10(6)：305-10．
2 毛秉智．急性放射病防治研究进展．中华放射医学与防护杂志．1998；18(5)：322-5．
3 毛秉智．放射生物剂量估计．军事医学科学出版社．2002；140-65．
4 毛秉智．辐射血液学—基础与临床．军事医学科学出版社．2002；186-203．
5 毛秉智，陈肖华．辐射血液学—基础与临床．军事医学科学出版社．2002；204-54．
6 毛秉智．急性放射病基础与临床．军事医学科学出版社．2002；152-62．
7 Baranov AE, Selidovkin GD, Butturini A, et al.Hematopoietic recovery after 10-Gy acute total body radiation. Blood. 1994 Jan 15;83 (2):596-9.
8 Bertho JM, Demarquay C, Frick J, et al.Level of Flt3-ligand in plasma: a possible new bio-indicator for radiation-induced aplasia. Int J Radiat Biol. 2001 Jun;77(6):703-12.
9 叶飞，刘树铮．不同剂量χ射线全身照射对小鼠脾细胞周期进程的影响．辐射研究与辐射工艺学报．1999；17(2)：111-4．
10 马淑梅，付士波，鞠桂芝．电离辐射对小鼠骨髓造血细胞周期进程的影响．辐射研究与辐射工艺学报．2000；18(2)：104-7．
11 彭瑞云，王德文，雄呈琦，等．辐射诱导骨髓细胞凋亡及相关基因表达研究．中华放射医学与防护杂志．2001；21(1)：19-20．
12 Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162:156-9.
13 Chirgwin JJ, Przbyla AE, MacDonald RJ,et al.Isolation of biologically active ribonucleic acid from souces enriched in ribonuclease. Biochemistry. 1979; 18:5294.
14 Fukunaga R, Ishizaka-Ikeda E, Seto Y, et al. Expression cloning of a receptor for murine granulocyte colony-stimulating factor. Cell. 1990;61 (2):341-50.
15 D'Andrea AD, Lodish HF, Wong GG. Expression cloning of the murine erythropoietin receptor. Cell. 1989;57(2):277-85.
16 Skoda RC, Seldin DC, Chiang MK, et al. Murine c-mpl:a member of the
hematopoietic growth factor receptor superfamily that transduces a proliferative signal. EMBOJ. 1993; 12(7) :2645-53．
17 Qiu FH, Ray P, Brown K, et al. Primary structure of c-kit:relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus. EMBO J.1988;7(4) :1003-11．
18 Marchuk D,Drumm M,Saulino A,et al.Construction of T-vecters,a rapid and general system for direct cloning of unmodified PCR products.Nucl Acid Res. 1991; 19:1154．
19 Birnboim HC. A rapid alkaline extraction for the isolation of plasmid DNA. Methods Enzymol.l983;100:243-55．
20 Holmes DS,Quigley M.A rapid boiling method for the preparation of bacterial plasmids.Anal Biochem. 1981;114:193-7．
21 Southern EM.Detection of specific sequences among DNA fragments separated by gel electrophoresis.J Mol Biol.l975;98:503-17．
22 Wang AM,Doyle MV,Mark DF.Quantitation of mRNA by the polymerase chain reaction.Proc Natl Acad Sci USA. 1989;86:9717-21．
23 Gilliland G,Perrin S,Blanchard K,et al.Analysis of cytokine mRNA and DNA:Detection and Quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA.1990;87:2725-9．
24 Nedelman J,Heagerty P,Lawrence F. Quantitative PCR with internal controls.CABIOS.1992;8:65-70．
25 Peng R, Wang D, Xiong C, et al.Interleukin-3 gene expression in irradiated mouse bone marrow.J Environ Pathol Toxicol Oncol. 1998;17(2) : 135-9．
26 Peterson VM, Adamovicz JJ, Elliott TB, et al.Gene expression of hematoregulatory cytokines is elevated endogenously after sublethal gamma irradiation and is differentially enhanced by therapeutic administration of biologic response modifiers.J Immunol. 1994 Sep 1;153(5) :2321-30．
27 Cong YW, Chen JP, Shao Y, et al. Studies on the responsiveness of hematopoietic cells to hematopoietic growth factors after irradiation. 45th Annual Meeting of Radiation Research Society. 1997;21:425．
28 Chang CM, Limanni A, Baker WH, et al. Bone marrow and splenic granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta mRNA levels in irradiated mice.Blood. 1995 Sep 15;86(6) :2130-6．
29 Limanni A, Baker WH, Chang CM, et al.c-kit ligand gene expression in normal and sublethally irradiated mice. Blood. 1995 May 1;85(9) :2377-84．
30 Peng R, Wang D, Xiong C, et al.The changes and significance of c-kit expression in the irradiated bone marrow of mice.J Environ Pathol Toxicol Oncol. 1998;17(2) : 129-33．
31 Chang CM, Limanni A, Baker WH, et al. Sublethal gamma irradiation increases IL-1 alpha, IL-6, and TNF-alpha mRNA levels in murine hematopoietic tissues.J Interferon Cytokine Res. 1997 Sep;17(9) :567-72．
32 Chang CM, Elliott TB, Dobson ME, et al.Ionizing radiation and bacterial challenge alter splenic cytokine gene expression. J Radiat Res (Tokyo). 2000 Sep;41(3) :259-77．
33 Hieter P, Boguski M. Functional genomics: it's all how you read it. Science. 1997; 278:601-2．
34 Collins FS, Patrinos A. Jordan E, et al. New goals for the US human genome project: 1998-2003． Science. 1998; 282:682-9．
35 Persidis A.Biochips.Nature Biotechnol. 1998; 16:981-3．
36 Duggan DJ,Bittner M,Chen Y,et al.Expression profiling using cDNA microarrays.Nat Genet. 1999;21 (suppl): 10-14．
37 Brown PO,Botstein D.Exploring the new world of the genome with DNA microarrays. Nat Genet.l999;21(suppl):48-50．
38 Pietu G,Alibert O,Guichard V,et al.Novel gene transcripts preferentially expressed in human muscles revealed by quantitative hybridization of a high density cDNA array.Genome Res.1996;6:492-503．
39 Hacia JG,Resequencing and mutational analysis using oligonucleotide microarrays. Nat Genet. 1999;21 (suppl):42-47．
40 Schena M,Shalon D,Heller R,et al.Parallel human genome analysis:microarray-based expression monitoring of 1000 genes.Proc Natl Acad Sci USA.1996;93:10614-9．
41 Park WY, Hwang CI, Im CN, et al.Identification of radiation-specific responses from
gene expression profile.Oncogene. 2002 Dec 5;21(55) :8521-8．
42 Fornace AJ, Amundson SA, Bittner M, et al.The complexity of radiation stress responses: analysis by informatics and functional genomics approaches. Gene Expr. 1999;7(4-6) :387-400．
43 Amundson SA, Bittner M, Chen Y, et al.Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses. Oncogene. 1999 Jun 17;18(24) :3666-72．
44 Amundson SA, Bittner M, Meltzer P, et al.Induction of gene expression as a monitor of exposure to ionizing radiation.Radiat Res. 2001 Nov;156(5 Pt 2) :657-61．
45 Sebastian JL, Rigberg DA, Shrivatsan E, et al.The use of large-scale cDNA analysis to profile differential gene expression in KYSE 410 human esophageal cancer cells after irradiation.Bull Exp Biol Med. 2000 Sep;130(9) :882-5．
46 Tusher VG, Tibshirani R, Chu G.Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci USA. 2001 Apr 24;98(9) :5116-21．
47 Kitahara O, Katagiri T, Tsunoda T, et al.Classification of sensitivity or resistance of cervical cancers to ionizing radiation according to expression profiles of 62 genes selected by cDNA microarray analysis.Neoplasia. 2002 Jul-Aug;4(4) :295-303．
48 Amundson SA, Do KT, Shahab S, et al.Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation. Radiat Res. 2000 Sep;154(3) :342-6．
49 Achary MP, Jaggernauth W, Gross E, et al. Cell lines from the same cervical carcinoma but with different radiosensitivities exhibit different cDNA microarray patterns of gene expression.Cytogenet Cell Genet. 2000;91(1-4) :39-43．
50 Amundson SA, Fornace AJ. Gene expression profiles for monitoring radiation exposure. Radiat Prot Dosimetry. 2001;97(1) :11-6．
2 毛秉智．急性放射病防治研究进展．中华放射医学与防护杂志．1998；18(5)：322-5．
3 毛秉智．放射生物剂量估计．军事医学科学出版社．2002；140-65．
4 毛秉智．辐射血液学—基础与临床．军事医学科学出版社．2002；186-203．
5 毛秉智，陈肖华．辐射血液学—基础与临床．军事医学科学出版社．2002；204-54．
6 毛秉智．急性放射病基础与临床．军事医学科学出版社．2002；152-62．
7 Baranov AE, Selidovkin GD, Butturini A, et al.Hematopoietic recovery after 10-Gy acute total body radiation. Blood. 1994 Jan 15;83 (2):596-9.
8 Bertho JM, Demarquay C, Frick J, et al.Level of Flt3-ligand in plasma: a possible new bio-indicator for radiation-induced aplasia. Int J Radiat Biol. 2001 Jun;77(6):703-12.
9 叶飞，刘树铮．不同剂量χ射线全身照射对小鼠脾细胞周期进程的影响．辐射研究与辐射工艺学报．1999；17(2)：111-4．
10 马淑梅，付士波，鞠桂芝．电离辐射对小鼠骨髓造血细胞周期进程的影响．辐射研究与辐射工艺学报．2000；18(2)：104-7．
11 彭瑞云，王德文，雄呈琦，等．辐射诱导骨髓细胞凋亡及相关基因表达研究．中华放射医学与防护杂志．2001；21(1)：19-20．
12 Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162:156-9.
13 Chirgwin JJ, Przbyla AE, MacDonald RJ,et al.Isolation of biologically active ribonucleic acid from souces enriched in ribonuclease. Biochemistry. 1979; 18:5294.
14 Fukunaga R, Ishizaka-Ikeda E, Seto Y, et al. Expression cloning of a receptor for murine granulocyte colony-stimulating factor. Cell. 1990;61 (2):341-50.
15 D'Andrea AD, Lodish HF, Wong GG. Expression cloning of the murine erythropoietin receptor. Cell. 1989;57(2):277-85.
16 Skoda RC, Seldin DC, Chiang MK, et al. Murine c-mpl:a member of the
hematopoietic growth factor receptor superfamily that transduces a proliferative signal. EMBOJ. 1993; 12(7) :2645-53．
17 Qiu FH, Ray P, Brown K, et al. Primary structure of c-kit:relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus. EMBO J.1988;7(4) :1003-11．
18 Marchuk D,Drumm M,Saulino A,et al.Construction of T-vecters,a rapid and general system for direct cloning of unmodified PCR products.Nucl Acid Res. 1991; 19:1154．
19 Birnboim HC. A rapid alkaline extraction for the isolation of plasmid DNA. Methods Enzymol.l983;100:243-55．
20 Holmes DS,Quigley M.A rapid boiling method for the preparation of bacterial plasmids.Anal Biochem. 1981;114:193-7．
21 Southern EM.Detection of specific sequences among DNA fragments separated by gel electrophoresis.J Mol Biol.l975;98:503-17．
22 Wang AM,Doyle MV,Mark DF.Quantitation of mRNA by the polymerase chain reaction.Proc Natl Acad Sci USA. 1989;86:9717-21．
23 Gilliland G,Perrin S,Blanchard K,et al.Analysis of cytokine mRNA and DNA:Detection and Quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA.1990;87:2725-9．
24 Nedelman J,Heagerty P,Lawrence F. Quantitative PCR with internal controls.CABIOS.1992;8:65-70．
25 Peng R, Wang D, Xiong C, et al.Interleukin-3 gene expression in irradiated mouse bone marrow.J Environ Pathol Toxicol Oncol. 1998;17(2) : 135-9．
26 Peterson VM, Adamovicz JJ, Elliott TB, et al.Gene expression of hematoregulatory cytokines is elevated endogenously after sublethal gamma irradiation and is differentially enhanced by therapeutic administration of biologic response modifiers.J Immunol. 1994 Sep 1;153(5) :2321-30．
27 Cong YW, Chen JP, Shao Y, et al. Studies on the responsiveness of hematopoietic cells to hematopoietic growth factors after irradiation. 45th Annual Meeting of Radiation Research Society. 1997;21:425．
28 Chang CM, Limanni A, Baker WH, et al. Bone marrow and splenic granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta mRNA levels in irradiated mice.Blood. 1995 Sep 15;86(6) :2130-6．
29 Limanni A, Baker WH, Chang CM, et al.c-kit ligand gene expression in normal and sublethally irradiated mice. Blood. 1995 May 1;85(9) :2377-84．
30 Peng R, Wang D, Xiong C, et al.The changes and significance of c-kit expression in the irradiated bone marrow of mice.J Environ Pathol Toxicol Oncol. 1998;17(2) : 129-33．
31 Chang CM, Limanni A, Baker WH, et al. Sublethal gamma irradiation increases IL-1 alpha, IL-6, and TNF-alpha mRNA levels in murine hematopoietic tissues.J Interferon Cytokine Res. 1997 Sep;17(9) :567-72．
32 Chang CM, Elliott TB, Dobson ME, et al.Ionizing radiation and bacterial challenge alter splenic cytokine gene expression. J Radiat Res (Tokyo). 2000 Sep;41(3) :259-77．
33 Hieter P, Boguski M. Functional genomics: it's all how you read it. Science. 1997; 278:601-2．
34 Collins FS, Patrinos A. Jordan E, et al. New goals for the US human genome project: 1998-2003． Science. 1998; 282:682-9．
35 Persidis A.Biochips.Nature Biotechnol. 1998; 16:981-3．
36 Duggan DJ,Bittner M,Chen Y,et al.Expression profiling using cDNA microarrays.Nat Genet. 1999;21 (suppl): 10-14．
37 Brown PO,Botstein D.Exploring the new world of the genome with DNA microarrays. Nat Genet.l999;21(suppl):48-50．
38 Pietu G,Alibert O,Guichard V,et al.Novel gene transcripts preferentially expressed in human muscles revealed by quantitative hybridization of a high density cDNA array.Genome Res.1996;6:492-503．
39 Hacia JG,Resequencing and mutational analysis using oligonucleotide microarrays. Nat Genet. 1999;21 (suppl):42-47．
40 Schena M,Shalon D,Heller R,et al.Parallel human genome analysis:microarray-based expression monitoring of 1000 genes.Proc Natl Acad Sci USA.1996;93:10614-9．
41 Park WY, Hwang CI, Im CN, et al.Identification of radiation-specific responses from
gene expression profile.Oncogene. 2002 Dec 5;21(55) :8521-8．
42 Fornace AJ, Amundson SA, Bittner M, et al.The complexity of radiation stress responses: analysis by informatics and functional genomics approaches. Gene Expr. 1999;7(4-6) :387-400．
43 Amundson SA, Bittner M, Chen Y, et al.Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses. Oncogene. 1999 Jun 17;18(24) :3666-72．
44 Amundson SA, Bittner M, Meltzer P, et al.Induction of gene expression as a monitor of exposure to ionizing radiation.Radiat Res. 2001 Nov;156(5 Pt 2) :657-61．
45 Sebastian JL, Rigberg DA, Shrivatsan E, et al.The use of large-scale cDNA analysis to profile differential gene expression in KYSE 410 human esophageal cancer cells after irradiation.Bull Exp Biol Med. 2000 Sep;130(9) :882-5．
46 Tusher VG, Tibshirani R, Chu G.Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci USA. 2001 Apr 24;98(9) :5116-21．
47 Kitahara O, Katagiri T, Tsunoda T, et al.Classification of sensitivity or resistance of cervical cancers to ionizing radiation according to expression profiles of 62 genes selected by cDNA microarray analysis.Neoplasia. 2002 Jul-Aug;4(4) :295-303．
48 Amundson SA, Do KT, Shahab S, et al.Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation. Radiat Res. 2000 Sep;154(3) :342-6．
49 Achary MP, Jaggernauth W, Gross E, et al. Cell lines from the same cervical carcinoma but with different radiosensitivities exhibit different cDNA microarray patterns of gene expression.Cytogenet Cell Genet. 2000;91(1-4) :39-43．
50 Amundson SA, Fornace AJ. Gene expression profiles for monitoring radiation exposure. Radiat Prot Dosimetry. 2001;97(1) :11-6．