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电离辐射能够引起造血系统损伤,包括急性骨髓抑制和持久性骨髓损伤,前者主要是快速增殖的造血祖细胞(hematopoietic progenitor cell,HPC)损伤所致,后者主要是由于电离辐射引起的造血干细胞(hematopoietic stem cell,HSC)损伤所致。已有的研究显示,电离辐射引起HSC和HPC损伤的机制主要是细胞内活性氧升高激活下游一系列信号通路,最终引起造血系统急性损伤和HSC、HPC衰老[1]。应用系列抗氧化剂能够部分缓解电离辐射引起的造血干祖细胞损伤。细胞内活性氧水平升高同样引起细胞代谢发生变化[2],细胞代谢是近年研究的热点,也是细胞最基本、最重要的活动之一。代谢通路在肿瘤的发生发展、糖尿病、衰老等病理生理过程中发挥着重要作用[3-5],然而在电离辐射引起造血系统损伤中的作用却少有研究。c-kit又称为CD117,是正常表达于干祖细胞表面的细胞因子受体。c-kit阳性细胞是一群经过富集的富含HPC和HSC的一类细胞,利用磁珠分选系统能在短时间内快速分选出大量的c-kit阳性细胞,对c-kit阳性细胞电离辐射损伤的研究能够初步反映HPC和HSC的损伤情况。因此,本研究以c-kit阳性细胞为对象,利用基因表达谱芯片技术筛选出经4 Gy γ射线照射后在代谢过程中表达发生变化的基因,并分析这些基因的性质和功能,为寻找电离辐射引起造血系统损伤的分子机制奠定基础。
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在基因表达芯片散点图上,每个点代表 1个基因探针,X轴表示对照组c-kit阳性细胞信号强度,Y轴表示照射组c-kit阳性细胞信号强度。散点图中共有3条斜线,最上端的斜线代表上调超过2倍的差异基因,最下端的斜线代表下调超过2倍的差异基因,由图 1可见,电离辐射引起c-kit阳性细胞的许多基因的表达发生显著改变。
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GO聚类分析结果显示,与对照组比较,照射组经4 Gy γ射线照射后参与代谢过程(GO通路编号:0008152)的差异表达基因中,62个基因表达上调2倍以上,其中13个基因表达上调3倍以上(表 1)。
基因名称 中文全称 英文全称 基因序列号 变化倍数 Pld5 磷脂酶D5 phospholipase D5 AK082725 5.045 Neu2 细胞质唾液酸酶 neuraminidase2 AF139059 4.683 Bpgm 2,3-二磷酸甘油酸变位酶 2,3-bisphosphoglycerate mutase BC004589 4.527 Alas2 红细胞氨基乙酰丙酸合成酶2 aminolevulinic acid synthase 2,erythroid NM_001102446 3.888 Satl1 精胺N1乙酰转移酶样蛋白1 spermine N1-acetyl transferase-like 1 XM_136025 3.343 Rdh16 视黄醇脱氢酶16 retinol dehydrogenase 16 BC089612 3.325 Mccc1 甲基丁烯酰辅酶A羧化酶1 methylcrotonoyl-Coenzyme A carboxylase 1 AK160184 3.313 Sat2 精胺N1乙酰转移酶2 spermine N1-acetyl transferase 2 BC061227 3.254 Smug1 单链选择性单功能的尿嘧啶DNA糖基化酶 Single-strand selective monofunctional uracil DNA glycosylase AK204061 3.240 Cml5 - camello-like 5 BC024605 3.224 Adhfe1 含铁乙酸脱氢酶1 alcohol dehydrogenase,iron containing,1 AK050178 3.084 Idh3α 异柠檬酸脱氢酶3α isocitrate dehydrogenase 3(NAD+)alpha AK146717 3.034 Slc27a5 溶质运载蛋白27号家族(脂肪酸运输)成员5 solute carrier family 27(fatty acid transporter),member 5 BC145823 3.010 表中,“-”表示未查到此项。 表 1 4 Gy y射线照射后与代谢过程相关的表达上调3倍以上的基因
Table 1. Metabolic process related genes up-regulated more than 3 times after 4 Gyy-ray irradiation
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GO聚类分析结果显示,与对照组比较,照射组经4 Gy γ射线照射后参与代谢过程(GO通路编号:0008152)的差异表达基因中,89个基因表达下调2倍以上,其中22个基因表达下调3倍以上(表 2)。
基因名称 中文全称 英文全称 基因序列号 变化倍数 Acss2 乙酰辅酶A合成酶短链家族成员2 acyl-CoA synthetase short-chain family member 2 BC051432 5.076 Phgdh 3-磷酸甘油脱氢酶 3-phosphoglycerate dehydrogenase BC086668 4.661 Psat1 磷酸丝氨酸转氨酶1 phosphoserine aminotransferase 1 AK153093 4.525 Nat10 N-乙酰转移酶10 N-acetyltransferase 10 BC034516 4.494 Glb1 β-半乳糖苷酶1 galactosidase, beta 1 AK171760 4.295 Gpam 线粒体甘油三磷酸酰基转移酶 glycerol-3-phosphate acyltransferase, mitochondrial BC019201 4.280 Dus3l 二氢尿嘧啶合成酶3样 dihydrouridine synthase 3-like BC023942 4.187 Fuca2 岩藻糖苷酶类2 fucosidase, alpha-L-2 AK163042 4.053 Impdh1 肌苷单磷酸脱氢酶1 inosine 5′-monophosphate dehydrogenase 1 AK171139 3.898 Dlat 二氢硫辛酰胺S乙酰转移酶(丙酮 酸脱氢酶E2组分) dihydrolipoamide S-acetyltransferase(E2 component of pyruvate dehydrogenase complex) BC031495 3.708 Enpp4 外核苷酸焦磷酸酶/磷酸二酯酶4 ectonucleotide pyrophosphatase/phosphodiesterase 4 AK157622 3.677 Prim2 DNA引物酶,p58亚基 DNA primase, p58 subunit BC019500 3.649 Mettl10 甲基转移酶样10 methyltransferase like 10 BC116375 3.638 Slc27a2 溶质运载蛋白27号家族成员2 solute carrier family 27, member 2 BC013442 3.515 Dera 2-脱氧核糖-5-磷酸醛缩酶 2-deoxyribose-5-phosphate aldolase BC016218 3.393 Qdpr 醌型二氢喋呤还原酶 quinoid dihydropteridine reductase BC002107 3.385 Dus1l 二氢尿嘧啶核苷合成酶1样 dihydrouridine synthase 1-like AK147967 3.251 Cdyl2 染色体区域蛋白,Y染色体2 chromodomain protein, Y chromosome-like 2 NM_029441 3.192 Dhodh 二氢乳清酸脱氢酶 dihydroorotate dehydrogenase BC019542 3.155 Srr 丝氨酸消旋酶 serine racemase BC011164 3.055 Spr 墨蝶呤还原酶 sepiapterin reductase AK004941 3.046 Mical2 微管相关单氧酶,钙调蛋白和LIM 区域2 microtubule associated monooxygenase, calponin and LIM domain containing 2 AK220353 3.035 表 2 4 Gy y酌射线照射后与代谢过程相关的表达下调3倍以上的基因
Table 2. Metabolic process related genes down-regulated more than 3 times after 4 Gyy-ray irradiation
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将上述差异表达上调及下调3倍以上的参与代谢过程的基因带入KEGG数据库进行信号通路分析(Fisher精确检验,P < 0.05),涉及8条信号通路(表 3)。
通路名称 涉及基因 嘌呤代谢 Impdh1,Prim2 嘧啶代谢 Prim2,Dhodh 初级胆汁酸生物合成 Slc27a5 真核生物核糖体合成 Nat10 柠檬酸循环 (三羧酸循环) Dlat DNA 复制 Prim2 RNA 转运 Dhodh mRNA 监测通路 Dhodh 表中,KEGG:Kyoto Encyclopedia of Genes and Genomes。 表 3 差异表达基因的KEGG通路富集分析
Table 3. KEGG pathway enrichment analysis of differen-tially expressed genes
基因芯片表达分析4 Gy γ射线对小鼠骨髓c-kit阳性细胞影响
Expression changes of genes in c-kit positive cells after 4 Gy γ-ray irradiation:a gene chip analysis
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摘要:
目的 利用基因芯片技术分析经4 Gy γ射线照射后c-kit(CD117,正常表达于干祖细胞表面的细胞因子受体)阳性细胞与代谢过程相关的基因及信号通路表达变化。 方法 利用磁珠分选系统分选出骨髓c-kit阳性细胞。实验分为对照组、4 Gy照射组。取1×106个c-kit阳性细胞进行照射,照射剂量率为0.99 Gy/min。照射后将细胞培养18 h后取出,进行全基因组的高通量基因芯片检测,并进行Gene Ontology聚类分析和Kyoto Encyclopedia of Genes and Genomes信号通路分析。 结果 4 Gy γ射线照射引起c-kit阳性细胞包括Pld5、Neu2、Bpgm、Alas2、Satl1、Rdh16、Mccc1、Sat2、Smug1、Cml5、Adhfe1、Idh3a、Slc27a5在内的13个基因表达上调3倍以上,包括Acss2、Phgdh、Psat1、Glb1、Gpam、Dus3l、Fuca2、Impdh1、Dlat、Glb1、Enpp4、Prim2、Mettl10、Slc27a2、Dera、Qdpr、Dus1l、Cdyl2、Dhodh、Srr、Spr、Mical2在内的22个基因表达下调3倍以上,这些基因主要参与嘌呤代谢、嘧啶代谢、三羧酸循环等信号通路。 结论 4 Gy γ射线照射能引起c-kit阳性细胞中与代谢过程相关的许多基因表达发生变化,这些变化的基因有待进一步的实验验证。 -
关键词:
- 原癌基因蛋白质c-kit /
- 寡核苷酸序列分析 /
- 辐射,电离 /
- 细胞代谢
Abstract:Objective To analyze the metabolic process-related genes and pathway variations in the c-kit(CD117, a cytokine receptor expressed on the surface of hematopoietic stem cells)positive cells after 4 Gy irradiation by using gene chip technology. Methods c-kit positive cells were sorted by microbead separation system. Two groups were involved:control group and 4 Gy irradiation group. A total of 1×106 c-kit positive cells received 4 Gy irradiation at a dose rate of 0.99 Gy/min. After the two groups were cultured for 18 h, all the cells were collected and detected by gene chip arrays. Data were processed and analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Results After 4 Gy γ-ray irradiation, 13 metabolic process-related genes were up-regulated more than 3 times:Pld5, Neu2, Bpgm, Alas2, Satl1, Rdh16, Mccc1, Sat2, Smug1, Cml5, Adhfe1, Idh3a, and Slc27a5. Additionally, 22 genes were down-regulated more than 3 times:Acss2, Phgdh, Psat1, Glb1, Gpam, Dus3l, Fuca2, Impdh1, Dlat, Glb1, Enpp4, Prim2, Mettl10, Slc27a2, Dera, Qdpr, Dus1l, Cdyl2, Dhodh, Srr, Spr, and Mical2. These genes were involved in pyrimidine metabolism, purine metabolism, and citrate cycle(TCA cycle) pathways. Conclusion Many variations of genes and pathways in c-kit positive cells existed after 4 Gy irradiation. However, further laboratory technology is required to confirm these variations. -
表 1 4 Gy y射线照射后与代谢过程相关的表达上调3倍以上的基因
Table 1. Metabolic process related genes up-regulated more than 3 times after 4 Gyy-ray irradiation
基因名称 中文全称 英文全称 基因序列号 变化倍数 Pld5 磷脂酶D5 phospholipase D5 AK082725 5.045 Neu2 细胞质唾液酸酶 neuraminidase2 AF139059 4.683 Bpgm 2,3-二磷酸甘油酸变位酶 2,3-bisphosphoglycerate mutase BC004589 4.527 Alas2 红细胞氨基乙酰丙酸合成酶2 aminolevulinic acid synthase 2,erythroid NM_001102446 3.888 Satl1 精胺N1乙酰转移酶样蛋白1 spermine N1-acetyl transferase-like 1 XM_136025 3.343 Rdh16 视黄醇脱氢酶16 retinol dehydrogenase 16 BC089612 3.325 Mccc1 甲基丁烯酰辅酶A羧化酶1 methylcrotonoyl-Coenzyme A carboxylase 1 AK160184 3.313 Sat2 精胺N1乙酰转移酶2 spermine N1-acetyl transferase 2 BC061227 3.254 Smug1 单链选择性单功能的尿嘧啶DNA糖基化酶 Single-strand selective monofunctional uracil DNA glycosylase AK204061 3.240 Cml5 - camello-like 5 BC024605 3.224 Adhfe1 含铁乙酸脱氢酶1 alcohol dehydrogenase,iron containing,1 AK050178 3.084 Idh3α 异柠檬酸脱氢酶3α isocitrate dehydrogenase 3(NAD+)alpha AK146717 3.034 Slc27a5 溶质运载蛋白27号家族(脂肪酸运输)成员5 solute carrier family 27(fatty acid transporter),member 5 BC145823 3.010 表中,“-”表示未查到此项。 表 2 4 Gy y酌射线照射后与代谢过程相关的表达下调3倍以上的基因
Table 2. Metabolic process related genes down-regulated more than 3 times after 4 Gyy-ray irradiation
基因名称 中文全称 英文全称 基因序列号 变化倍数 Acss2 乙酰辅酶A合成酶短链家族成员2 acyl-CoA synthetase short-chain family member 2 BC051432 5.076 Phgdh 3-磷酸甘油脱氢酶 3-phosphoglycerate dehydrogenase BC086668 4.661 Psat1 磷酸丝氨酸转氨酶1 phosphoserine aminotransferase 1 AK153093 4.525 Nat10 N-乙酰转移酶10 N-acetyltransferase 10 BC034516 4.494 Glb1 β-半乳糖苷酶1 galactosidase, beta 1 AK171760 4.295 Gpam 线粒体甘油三磷酸酰基转移酶 glycerol-3-phosphate acyltransferase, mitochondrial BC019201 4.280 Dus3l 二氢尿嘧啶合成酶3样 dihydrouridine synthase 3-like BC023942 4.187 Fuca2 岩藻糖苷酶类2 fucosidase, alpha-L-2 AK163042 4.053 Impdh1 肌苷单磷酸脱氢酶1 inosine 5′-monophosphate dehydrogenase 1 AK171139 3.898 Dlat 二氢硫辛酰胺S乙酰转移酶(丙酮 酸脱氢酶E2组分) dihydrolipoamide S-acetyltransferase(E2 component of pyruvate dehydrogenase complex) BC031495 3.708 Enpp4 外核苷酸焦磷酸酶/磷酸二酯酶4 ectonucleotide pyrophosphatase/phosphodiesterase 4 AK157622 3.677 Prim2 DNA引物酶,p58亚基 DNA primase, p58 subunit BC019500 3.649 Mettl10 甲基转移酶样10 methyltransferase like 10 BC116375 3.638 Slc27a2 溶质运载蛋白27号家族成员2 solute carrier family 27, member 2 BC013442 3.515 Dera 2-脱氧核糖-5-磷酸醛缩酶 2-deoxyribose-5-phosphate aldolase BC016218 3.393 Qdpr 醌型二氢喋呤还原酶 quinoid dihydropteridine reductase BC002107 3.385 Dus1l 二氢尿嘧啶核苷合成酶1样 dihydrouridine synthase 1-like AK147967 3.251 Cdyl2 染色体区域蛋白,Y染色体2 chromodomain protein, Y chromosome-like 2 NM_029441 3.192 Dhodh 二氢乳清酸脱氢酶 dihydroorotate dehydrogenase BC019542 3.155 Srr 丝氨酸消旋酶 serine racemase BC011164 3.055 Spr 墨蝶呤还原酶 sepiapterin reductase AK004941 3.046 Mical2 微管相关单氧酶,钙调蛋白和LIM 区域2 microtubule associated monooxygenase, calponin and LIM domain containing 2 AK220353 3.035 表 3 差异表达基因的KEGG通路富集分析
Table 3. KEGG pathway enrichment analysis of differen-tially expressed genes
通路名称 涉及基因 嘌呤代谢 Impdh1,Prim2 嘧啶代谢 Prim2,Dhodh 初级胆汁酸生物合成 Slc27a5 真核生物核糖体合成 Nat10 柠檬酸循环 (三羧酸循环) Dlat DNA 复制 Prim2 RNA 转运 Dhodh mRNA 监测通路 Dhodh 表中,KEGG:Kyoto Encyclopedia of Genes and Genomes。 -
[1] Zhang H, Zhai Z, Wang Y, et al. Resveratrol ameliorates ionizing irradiation-induced long-term hematopoietic stem cell injury in mice[J]. Free Radic Biol Med, 2013, 54:40-50. doi: 10.1016/j.freeradbiomed.2012.10.530 [2] Ghesquiere B, Wong BW, Kuchnio A, et al. Metabolism of stromal and immune cells in health and disease[J]. Nature, 2014, 511(758):167-176. [3] De Mas IM, Aguilar E, Jayaraman A, et al. Cancer cell metabolism as new targets for novel designed therapies[J]. Future Med Chem, 2014, 6(16):1791-1810. doi: 10.4155/fmc.14.119 [4] Yun JS, Ko SH. Avoiding or coping with severe hypoglycemia in patients with type 2 diabetes[J]. Korean J Intern Med, 2015, 30(1):6-16. doi: 10.3904/kjim.2015.30.1.6 [5] Kushiyama A, Tanaka K, Hara S, et al. Linking uric acid metabolism to diabetic complications[J]. World J Diabetes, 2014, 5(6):787-795. doi: 10.4239/wjd.v5.i6.787 [6] Ema H, Morita Y, Yamazaki S, et al. Adult mouse hematopoietic stem cells:purification and single-cell assays[J]. Nat Protoc, 2006, 1(6):2979-2987. doi: 10.1038/nprot.2006.447 [7] Shao L, Luo Y, Zhou D. Hematopoietic stem cell injury induced by ionizing radiation[J]. Antioxid Redox Signal, 2014, 20(9):1447-1462. doi: 10.1089/ars.2013.5635 [8] Vacanti NM, Metallo CM. Exploring metabolic pathways that contribute to the stem cell phenotype[J]. Biochim Biophys Acta, 2013, 1830(2):2361-2369.