人肝癌SMMC-7721细胞受低线性能量传递射线照射后线粒体DNA和核DNA断裂状况的比较
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摘要
目前肿瘤一直是导致人类死亡的主要原因之一,其发病率呈逐年上升的趋势。放射治疗作为肿瘤的三大主要治疗手段(放疗、化疗、外科手术)之一有着不可替代的重要地位。人们发现射线之所以能够杀死肿瘤细胞主要在于它能够损伤肿瘤细胞的DNA分子,引起DNA分子双链断裂,导致细胞失活和染色体畸变,最终使细胞凋亡。目前人们越来越关注肿瘤细胞DNA的变化与其凋亡的关系,获得了有关肿瘤细胞核DNA(nDNA)改变的许多知识,但对肿瘤细胞线粒体DNA(mtDNA)的研究相对较少。很多报道发现mtDNA与肿瘤之间存在着密切的关系,无论是肿瘤细胞的发生、发展,还是肿瘤细胞的死亡都和mtDNA密切相关。
过去的文献大多报道机体遭辐照后nDNA的损伤情况,对mtDNA的损伤研究较少。目前有文献报道高线性能量传递(Liner energy transmission,LET)射线引起的核DNA双链断裂是非随机分布的,而低线性能量传递射线中X射线引起的核DNA双链断裂位点是随机分布的,我们希望通过本试验研究了解肿瘤细胞经辐照后其mtDNA与nDNA损伤的具体情况,检测在肿瘤细胞mtDNA与nDNA上是否存在对射线敏感的位点,这些位点的分布是否随机,并对mtDNA和nDNA经射线照射后的受损情况作出比较,希望能为肿瘤的临床治疗提供一定的指导,并对细胞的辐射敏感性研究提供一个新的参考指标。
国内目前在临床上放疗使用的主要还是低LET射线,即γ和X射线。本实验对肿瘤细胞照射不同剂量的γ和X射线,对其mtDNA和nDNA的断裂敏感位点的分布进行检测,并作出mtDNA和nDNA的损伤程度比较,同时对mtDNA的编码区和D-loop区、重链和轻链,nDNA的两条链之间的断裂损伤情况作了比较。
射线引起细胞DNA断裂的途径有直接损伤和间接损伤。直接损伤是射线直接作用于DNA分子引起DNA断裂;间接损伤主要是由射线照射导致细胞内的水分子发生电离,产生氧自由基(主要是·OH),这些自由基再和生物大分子发生作用,导致不可逆损伤。两种效应有同等的重要性。本实验选取人肝癌细胞SMMC-7721细胞作为研究对象,对其采用由小到大的辐射剂量照射,用10Gy、20Gy、30Gy、50Gy四种不同剂量的γ和X射线来照射细胞,并测定了细胞存活度,检测其mtDNA和nDNA的断裂状况。由于mtDNA是多拷贝的,一个线粒体含有2到10个mtDNA[3],1个细胞含有几百到几千个线粒体,要比较mtDNA和nDNA的损伤程度需测定mtDNA的相对拷贝数,我们建立了用PCR检测细胞mtDNA拷贝数的方法。我们在辐照细胞后即刻提取对照组和各实验组细胞的基因组DNA,然后用接头介导PCR(LM-PCR)方法进行检测,并结合碱性凝胶电泳实验观察其大致损伤程度,再通过基因扫描得出其具体损伤位点和频率。结果显示:①在SMMC-7721细胞mtDNA与nDNA中确实存在着对射线敏感的断裂位点,断裂损伤频率随辐射剂量的增加呈上升趋势,并随剂量增加出现了新的断裂位点;②mtDNA与nDNA对γ射线敏感的断裂位点和对X射线敏感的断裂位点位置均相同;③射线照射后SMMC-7721细胞mtDNA的损伤程度要高于nDNA;④mtDNA的重链损伤程度高于轻链,编码区(选取细胞色素b基因作为研究对象)的损伤程度要高于D-loop区;⑤nDNA(选取18SrRNA基因作为研究对象)的模板链的损伤程度高于编码链,这种结果很可能与蛋白保护或DNA的空间结构有一定的关系;⑥从碱基组成来看,胸腺嘧啶(T)和鸟嘌呤(G)的损伤占到损伤碱基的82%,推测这两种碱基是·OH的主要攻击对象,这也从侧面说明了辐照对DNA的损伤是以间接损伤为主。mtDNA损伤重于nDNA,mtDNA编码区损伤重于D-loop区的的主要原因可能是nDNA和mtDNA D-loop区受蛋白保护所致。nDNA模板链损伤程度重于编码链,mtDNA重链的损伤程度重于轻链的主要原因可能是由于模板链和重链在转录时暴露较多,更易受辐射损伤之故。
Now a days,, tumor is still the major reason which causes death of people, and the attack rate is increasing every year. As one of the three main treatments of cancer (chemiotherapy, radiotherapy and surgery), Radiotherapy is too important to be substituted. People find that the radioactive ray can break the DNA molecular of cancer cell. Then the damaged cell loses vigor and chromosome undergoes aberration. At last, apoptosis is irreversible. There have been many studies about the relationship between DNA change and apoptosis in malignant cells. Thus we have obtained a vast number of information about nuclear DNA (nDNA). Compared with nDNA, there are less knowledge about mitochondrial DNA(mtDNA)in the cancer cells. However, there are many evidences indicating mtDNA is tightly connected with the happen、development and death of cancer cell.
Compared with our limited knowledge about damage of mtDNA after radiation, previous literature mainly focused on the damage of nDNA. Some documents have reported that high Liner energy transmission(LET) can cause nonrandom break of the double DNA strand, and low LET cause random break. Now, we hope to get some information about the damage of mtDNA and nDNA of cancer cell after radiation.We want to know whether there exist radioactive sensitive sites in the mtDNA and nDNA,whether these sites are distributed randomly and what the damage state of mtDNA and nDNA. The results of the work may provide some guidance for the clinical therapy of cancer, and offer a new reference index for the study of radiation sensitiveness of cell.
In the present, the most frequently used ray is low LET, namely in the aspect ofγand X treatment. In the present study, through exposure to different dose ofγand X ray, we detected the distribution of sensitive site of mtDNA and nDNA; And we compare the degree of damage between mtDNA and nDNA, coding region and D-loop region,heavy strand and light strand, the two strands of nDNA.
Radiation can cause direct and indirect damage of DNA. The former is free radical produced due to the direct damage of polar molecule by the radiation; The latter is mainly the radiation leads to ionization of H20 in the cells, and produces free radicals(most of them are·OH ).There is interaction of free radicals and biomacromolecule, and results in irreversible damage. The two ways are tantamount important. In this study, SMMC-7721 cell are radiated by different dose, namely 10Gy、20Gy、30Gy and 50Gy, and the survival rate of cell, the break of mtDNA and nDNA are detected respectively. The mtDNA is multi-copy molecular, one mitochondria contains 2-10 mtDNA,thus one cell contains hundreds to thousands of mtDNA. To compare the damage degree of mtDNA and nDNA, we established methods based on PCR through which the copy number of mtDNA can be determined. Immediately after radiation, the genome DNA of cells was extracted from experimental and control group. Ligation mediated PCR (LM-PCR) and alkaline gel electrophoresis were used to detect the break site and observe the damage degree. At last, the concrete damage site and frequency can be analyzed by the result of gene-scan technique. We found that there exists sensitive break site of mtDNA and nDNA, moreover,the degree of damage showed an increasing tendency with the increase of irradiation doses, and new break points appeared with the increase of irradiation doses. The sensitive sites ofγray and X ray are identical; The damage degree of mtDNA is more serious than nDNA in SMMC-77221 cell after radiation. We observed that the damage degree of heavy strands(H-strands) is higher than that of light strands(L-stRanDs), coding region(cytochrome b gene)higher than D-loop region ,and the damage degree of the template strands of nDNA(18SrRNA gene) is higher than coding region. From the point of composition of base, damaged thymine(T)and guanine(G)accounts for 81% of the whole damaged base. The reason may be this two types of bases are major attacking target of·OH, this also indicates that radiation mainly cause indirect damage to DNA. We think that protein has definite function of protecting DNA from the damage of radioactive ray, this can explain why the damage degree of mtDNA is higher than that of nDNA and the damage degree of coding region is higher than that of D-loop region. The damage degree of template strand is higher than the coding strand for nDNA and the damage degree of the heavy strand is greater than light strand for mtDNA. We proposed that template strand and heavy strand are exposed more frequently during transcription and thus are more susceptible to radioactive damage.
过去的文献大多报道机体遭辐照后nDNA的损伤情况,对mtDNA的损伤研究较少。目前有文献报道高线性能量传递(Liner energy transmission,LET)射线引起的核DNA双链断裂是非随机分布的,而低线性能量传递射线中X射线引起的核DNA双链断裂位点是随机分布的,我们希望通过本试验研究了解肿瘤细胞经辐照后其mtDNA与nDNA损伤的具体情况,检测在肿瘤细胞mtDNA与nDNA上是否存在对射线敏感的位点,这些位点的分布是否随机,并对mtDNA和nDNA经射线照射后的受损情况作出比较,希望能为肿瘤的临床治疗提供一定的指导,并对细胞的辐射敏感性研究提供一个新的参考指标。
国内目前在临床上放疗使用的主要还是低LET射线,即γ和X射线。本实验对肿瘤细胞照射不同剂量的γ和X射线,对其mtDNA和nDNA的断裂敏感位点的分布进行检测,并作出mtDNA和nDNA的损伤程度比较,同时对mtDNA的编码区和D-loop区、重链和轻链,nDNA的两条链之间的断裂损伤情况作了比较。
射线引起细胞DNA断裂的途径有直接损伤和间接损伤。直接损伤是射线直接作用于DNA分子引起DNA断裂;间接损伤主要是由射线照射导致细胞内的水分子发生电离,产生氧自由基(主要是·OH),这些自由基再和生物大分子发生作用,导致不可逆损伤。两种效应有同等的重要性。本实验选取人肝癌细胞SMMC-7721细胞作为研究对象,对其采用由小到大的辐射剂量照射,用10Gy、20Gy、30Gy、50Gy四种不同剂量的γ和X射线来照射细胞,并测定了细胞存活度,检测其mtDNA和nDNA的断裂状况。由于mtDNA是多拷贝的,一个线粒体含有2到10个mtDNA[3],1个细胞含有几百到几千个线粒体,要比较mtDNA和nDNA的损伤程度需测定mtDNA的相对拷贝数,我们建立了用PCR检测细胞mtDNA拷贝数的方法。我们在辐照细胞后即刻提取对照组和各实验组细胞的基因组DNA,然后用接头介导PCR(LM-PCR)方法进行检测,并结合碱性凝胶电泳实验观察其大致损伤程度,再通过基因扫描得出其具体损伤位点和频率。结果显示:①在SMMC-7721细胞mtDNA与nDNA中确实存在着对射线敏感的断裂位点,断裂损伤频率随辐射剂量的增加呈上升趋势,并随剂量增加出现了新的断裂位点;②mtDNA与nDNA对γ射线敏感的断裂位点和对X射线敏感的断裂位点位置均相同;③射线照射后SMMC-7721细胞mtDNA的损伤程度要高于nDNA;④mtDNA的重链损伤程度高于轻链,编码区(选取细胞色素b基因作为研究对象)的损伤程度要高于D-loop区;⑤nDNA(选取18SrRNA基因作为研究对象)的模板链的损伤程度高于编码链,这种结果很可能与蛋白保护或DNA的空间结构有一定的关系;⑥从碱基组成来看,胸腺嘧啶(T)和鸟嘌呤(G)的损伤占到损伤碱基的82%,推测这两种碱基是·OH的主要攻击对象,这也从侧面说明了辐照对DNA的损伤是以间接损伤为主。mtDNA损伤重于nDNA,mtDNA编码区损伤重于D-loop区的的主要原因可能是nDNA和mtDNA D-loop区受蛋白保护所致。nDNA模板链损伤程度重于编码链,mtDNA重链的损伤程度重于轻链的主要原因可能是由于模板链和重链在转录时暴露较多,更易受辐射损伤之故。
Now a days,, tumor is still the major reason which causes death of people, and the attack rate is increasing every year. As one of the three main treatments of cancer (chemiotherapy, radiotherapy and surgery), Radiotherapy is too important to be substituted. People find that the radioactive ray can break the DNA molecular of cancer cell. Then the damaged cell loses vigor and chromosome undergoes aberration. At last, apoptosis is irreversible. There have been many studies about the relationship between DNA change and apoptosis in malignant cells. Thus we have obtained a vast number of information about nuclear DNA (nDNA). Compared with nDNA, there are less knowledge about mitochondrial DNA(mtDNA)in the cancer cells. However, there are many evidences indicating mtDNA is tightly connected with the happen、development and death of cancer cell.
Compared with our limited knowledge about damage of mtDNA after radiation, previous literature mainly focused on the damage of nDNA. Some documents have reported that high Liner energy transmission(LET) can cause nonrandom break of the double DNA strand, and low LET cause random break. Now, we hope to get some information about the damage of mtDNA and nDNA of cancer cell after radiation.We want to know whether there exist radioactive sensitive sites in the mtDNA and nDNA,whether these sites are distributed randomly and what the damage state of mtDNA and nDNA. The results of the work may provide some guidance for the clinical therapy of cancer, and offer a new reference index for the study of radiation sensitiveness of cell.
In the present, the most frequently used ray is low LET, namely in the aspect ofγand X treatment. In the present study, through exposure to different dose ofγand X ray, we detected the distribution of sensitive site of mtDNA and nDNA; And we compare the degree of damage between mtDNA and nDNA, coding region and D-loop region,heavy strand and light strand, the two strands of nDNA.
Radiation can cause direct and indirect damage of DNA. The former is free radical produced due to the direct damage of polar molecule by the radiation; The latter is mainly the radiation leads to ionization of H20 in the cells, and produces free radicals(most of them are·OH ).There is interaction of free radicals and biomacromolecule, and results in irreversible damage. The two ways are tantamount important. In this study, SMMC-7721 cell are radiated by different dose, namely 10Gy、20Gy、30Gy and 50Gy, and the survival rate of cell, the break of mtDNA and nDNA are detected respectively. The mtDNA is multi-copy molecular, one mitochondria contains 2-10 mtDNA,thus one cell contains hundreds to thousands of mtDNA. To compare the damage degree of mtDNA and nDNA, we established methods based on PCR through which the copy number of mtDNA can be determined. Immediately after radiation, the genome DNA of cells was extracted from experimental and control group. Ligation mediated PCR (LM-PCR) and alkaline gel electrophoresis were used to detect the break site and observe the damage degree. At last, the concrete damage site and frequency can be analyzed by the result of gene-scan technique. We found that there exists sensitive break site of mtDNA and nDNA, moreover,the degree of damage showed an increasing tendency with the increase of irradiation doses, and new break points appeared with the increase of irradiation doses. The sensitive sites ofγray and X ray are identical; The damage degree of mtDNA is more serious than nDNA in SMMC-77221 cell after radiation. We observed that the damage degree of heavy strands(H-strands) is higher than that of light strands(L-stRanDs), coding region(cytochrome b gene)higher than D-loop region ,and the damage degree of the template strands of nDNA(18SrRNA gene) is higher than coding region. From the point of composition of base, damaged thymine(T)and guanine(G)accounts for 81% of the whole damaged base. The reason may be this two types of bases are major attacking target of·OH, this also indicates that radiation mainly cause indirect damage to DNA. We think that protein has definite function of protecting DNA from the damage of radioactive ray, this can explain why the damage degree of mtDNA is higher than that of nDNA and the damage degree of coding region is higher than that of D-loop region. The damage degree of template strand is higher than the coding strand for nDNA and the damage degree of the heavy strand is greater than light strand for mtDNA. We proposed that template strand and heavy strand are exposed more frequently during transcription and thus are more susceptible to radioactive damage.
引文
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[1] 孙乃恩,孙东旭,朱德煦主编.(1999)分子遗传学. 南京:南京大学出版社,64-67.
[2] Wei YH,Lee CF,Lee HC,et al.Increases of mitochondrial mass and mitochondrial genome in association with enhanced oxidative stress in human cells harboring 4977BP-deleted mitochondrial DNA.Ann N Y Acad Sci,2001,928:97-112.
[3] Kang D,Hamasaki N.Mitochondrial oxidative and mitochondrial DNA.Clin Chem Lab Med.2003,41(10):1281-1288.
[4] Zischler H.Nuclear integrations of mitochondrial DNA in primates:inference of associated mutational events.2000,21(3):531-536.
[5] Polyak K,Li Y,Zhu H,et al.Somatic mutation of the mitochondrial genome in human colorectal tumors.Nature Genet.1998,20(3):291-293.
[6] Nishikawa M,Nishiguchi S,Shiomi S,et al.Somatic mutation of the mitochondrial DNA in cancerous and noncancerous liver tissue with hepatocelluar carcinoma.Cancer Res.2001,61(5):1843-1845.
[7] Fliss Ms,Usadel H,Caballero OL,et al.Facile detection of mitochondrial DNA mutation in tumors and bodily fluids.Science.2000,287(5460):2017-2019.
[8] Hibi K,Nakayama H,Yamazaki T,et al.Detection of mitochondrial DNA alterations in primary tumors and corresponding serum o colorectal cancer patients.Int J Cancer,2001, 94(3):429-431.
[9] Penta JS,Johnson FM,Wachsman JT,et al.Mitochondrial DNA in huamn malignancy. Mutat Res,2001,488:119-133.
[10] Han CB,Li F,Yang XF,et al.Alterations of mtDNA copy number in gastric carcinoma.Shijie Huaren Xiaohua Zazhi,2004,12:258-261.
[11] Luciakova K,Kuzala S.Increased steady-state levels of several mitochondrial DNA in human malignancy.Eur J Biochem,1992,205:1187-1193.
[12] Han CB,Zhao YJ,Li F,et al.Quantitation and detection of deletion in tumor mitochondrial DNA by microarray technique.Zhonghua Zhongjiu Zazhi,2004, 26:10-13.
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[1] 孙乃恩,孙东旭,朱德煦主编.(1999)分子遗传学. 南京:南京大学出版社,64-67.
[2] Wei YH,Lee CF,Lee HC,et al.Increases of mitochondrial mass and mitochondrial genome in association with enhanced oxidative stress in human cells harboring 4977BP-deleted mitochondrial DNA.Ann N Y Acad Sci,2001,928:97-112.
[3] Kang D,Hamasaki N.Mitochondrial oxidative and mitochondrial DNA.Clin Chem Lab Med.2003,41(10):1281-1288.
[4] Zischler H.Nuclear integrations of mitochondrial DNA in primates:inference of associated mutational events.2000,21(3):531-536.
[5] Polyak K,Li Y,Zhu H,et al.Somatic mutation of the mitochondrial genome in human colorectal tumors.Nature Genet.1998,20(3):291-293.
[6] Nishikawa M,Nishiguchi S,Shiomi S,et al.Somatic mutation of the mitochondrial DNA in cancerous and noncancerous liver tissue with hepatocelluar carcinoma.Cancer Res.2001,61(5):1843-1845.
[7] Fliss Ms,Usadel H,Caballero OL,et al.Facile detection of mitochondrial DNA mutation in tumors and bodily fluids.Science.2000,287(5460):2017-2019.
[8] Hibi K,Nakayama H,Yamazaki T,et al.Detection of mitochondrial DNA alterations in primary tumors and corresponding serum o colorectal cancer patients.Int J Cancer,2001, 94(3):429-431.
[9] Penta JS,Johnson FM,Wachsman JT,et al.Mitochondrial DNA in huamn malignancy. Mutat Res,2001,488:119-133.
[10] Han CB,Li F,Yang XF,et al.Alterations of mtDNA copy number in gastric carcinoma.Shijie Huaren Xiaohua Zazhi,2004,12:258-261.
[11] Luciakova K,Kuzala S.Increased steady-state levels of several mitochondrial DNA in human malignancy.Eur J Biochem,1992,205:1187-1193.
[12] Han CB,Zhao YJ,Li F,et al.Quantitation and detection of deletion in tumor mitochondrial DNA by microarray technique.Zhonghua Zhongjiu Zazhi,2004, 26:10-13.
[13] Wang J,Silva JP,Gustafasson CM,et al.Increased in vivo apoptosis in cells lacking mitochondrial DNA gene expression.Proc Natl Acad Sci U S A,2001,98:4038-4043.
[14] Savre-Train I,Piatyszek MA,Shay JW,et al.Transcription of deleted mitochondrial DNA in human colon adenocarcinoma cells.Hum Mol Genet,1992,1:203-204.
[15]Ikebe S,Tanaka M,Ohno K,et al.Increase of deleted mitochondrial DNA in the striatum in Parkinson`s disease and senescence.Biochem Biophys Res Commun.1990,170(3):1044-1048.
[16]Celia H,et al.Biochem et Biophys Acta,2002,1588:65-70.
[17]Wang Y,et al.Proc Natl Acad Sci USA,2001,98:4022-4027.
[18]Del Bo R,et al.Ann Neurol,2001,49:137-138.
[19]Rberto Del Bo,et al.J Neuro Sci,2002,202:85-91.