CN107208130A - 对低水平放射线反应的dna修复相关基因的检测方法 - Google Patents
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Abstract
本发明涉及对低水平放射线反应的DNA修复相关基因的检测方法。根据如上所述的本发明提供包括以下步骤的对低水平放射线反应的DNA修复相关基因的检测方法:(1)在低水平放射线环境中饲养胸腺癌模型AKR/J小鼠和正常ICR小鼠;(2)采集所述步骤(1)中饲养的胸腺癌模型AKR/J小鼠和正常ICR小鼠的胸腺;(3)对在所述步骤(2)中采集的胸腺的基因进行分析;(4)在所述步骤(3)中分析的基因中检测胸腺癌模型AKR/J小鼠和正常ICR小鼠中共同或单独表达的DNA修复相关基因;以及(5)对所述步骤(4)中检测出的基因进行扩增并测定表达量。通过提供上述方法能够带来以下效果:能够用作评价产业及医疗从事人员的放射线暴露与致癌之间的相关性的低水平放射线DNA修复指标基因,并且不仅能够用作评价癌症患者的癌症进展及治疗程度的低水平放射线DNA修复基因,而且还能够用作评价放射线和致癌的因果关系的低水平DNA修复指标。
Description
技术领域
本发明涉及对低水平放射线反应的DNA修复相关基因的检测方法,更详细而言,涉及对胸腺癌模型AKR/J小鼠和正常ICR小鼠照射低水平放射线,然后采集胸腺并检测共同或单独表达的DNA修复相关基因,并进行扩增,然后测定表达量的方法。
背景技术
现有技术中,为了找出对低水平(0.7mGy/小时)放射线敏感的基因而利用了多种方法,但是没有确认到分子性的蛋白质水平,对于本发明中提出的基因,不仅确认了RNA,还确认了蛋白质水平的表达差异。并且,现有技术中,对于DNA损伤和修复相关的基因,没有对照射低水平放射线的健康的小鼠和发生癌症的小鼠进行比较来确认,本发明中发掘出的基因并未在现有技术中被已知为对低水平放射线敏感的基因。对低水平放射线敏感的基因表达谱的发掘中的现有技术如下所示。
i)对人骨髓性白血病细胞株照射2~50cGy放射线,然后确认CDKN1A、GADD45A、MDM2、BTG2、PHLDA3等基因的表达(Amundson等,2003)
ii)为了用于放射线事故时的剂量评价,对人血液照射0.5、2、8Gy,然后发现根据剂量反应的FDXR、CDKN1A、PHPT1、BBC3、SESN1基因(Paul和Amundson,2008)
iii)对C57BL/6J小鼠以0.032~13μGy/分钟照射放射线485天,然后采集肾脏和睾丸,并通过微阵列(microarray)发现了对低水平放射线敏感的基因(Taki等,2009)
iv)对C57BL/6J雄性小鼠以17~20mGy/天、0.86~1.0mGy/天照射放射线401~485天(8Gy、0.4Gy或0.02Gy),然后采集肝并利用微阵列和逆转录核酸扩增法发现了对低水平放射线敏感的基因(Uehara等,2010)
v)利用微阵列和定量核酸扩增法在照射低水平放射线的黑腹果蝇中发现39个对低水平放射线敏感的基因(Seong等,2011)
vi)对人血液照射0、0.02、0.1、0.5、1、2、4Gy放射线,然后通过微阵列发掘出共9个对低水平放射线敏感的基因,并且为了用于生物学剂量评估,按剂量进行照射后按时间选取表达量具有差异的基因组(Knops等,2012)。
vii)对斑马鱼(zebra danio)胚胎和成体照射0.1或1Gy的放射线,然后利用肝的mRNA实施微阵列和定量核酸扩增法,并发掘出对放射线敏感的基因(Jaafar等,2013)
viii)通过微阵列在照射5~500mGy的放射线的人血液中发掘出对低水平放射线敏感的基因(Nosel等,2013)
发明内容
要解决的技术问题
本发明人在研究因放射线引起的癌症时,通常利用细胞株的研究结果难以直接适用于人或动物来解释,在实验动物的情况下,一般利用最多的是小鼠,但由于癌发病率低,并且低水平放射线的影响直到表现为人体影响方面有很多限制,因此为了弥补这种缺点,利用胸腺癌模型AKR/J小鼠和通常用在癌症研究中的正常ICR小鼠,并通过以下方法分析个体之间的差异,从而完成了本发明。
1)对胸腺癌模型AKR/J小鼠和正常ICR小鼠照射低水平(0.7mGy/小时)放射线,然后在胸腺中发掘出对低水平放射线敏感的基因表达谱(profile),然后,2)以DNA修复相关基因为中心进行鉴定后对其功能进行分析。
总结而言,本发明的目的在于提供对低水平放射线反应的DNA修复相关基因的检测方法,其包括以下步骤:(1)在低水平放射线环境中饲养胸腺癌模型AKR/J小鼠和正常ICR小鼠;(2)采集所述步骤(1)中饲养的胸腺癌模型AKR/J小鼠和正常ICR小鼠的胸腺;(3)对在所述步骤(2)中采集的胸腺的基因进行分析;(4)在所述步骤(3)中分析的基因中检测胸腺癌模型AKR/J小鼠和正常ICR小鼠中共同或单独表达的DNA修复相关基因;以及(5)对所述步骤(4)中检测出的基因进行扩增并测定表达量。
技术方案
为了实现上述目的,本发明提供对低水平放射线反应的DNA修复相关基因的检测方法,其包括以下步骤:(1)在低水平放射线环境中饲养胸腺癌模型AKR/J小鼠和正常ICR小鼠;(2)采集所述步骤(1)中饲养的胸腺癌模型AKR/J小鼠和正常ICR小鼠的胸腺;(3)对在所述步骤(2)中采集的胸腺的基因进行分析;(4)在所述步骤(3)中分析的基因中检测胸腺癌模型AKR/J小鼠和正常ICR小鼠中共同或单独表达的DNA修复相关基因;以及(5)对所述步骤(4)中检测出的基因进行扩增并测定表达量。
所述步骤(1)的特征在于,低水平放射线剂量为0.7mGy/小时。
所述步骤(1)的特征在于,使在0.7mGy/小时的低水平放射线环境中的累计剂量达到1.7Gy来进行饲养。
所述步骤(5)的特征在于,用选自Cyp11a1、Ptgs2、Rnd3、Plxncl及cyp2el中的引物对DNA修复相关基因进行扩增。
所述步骤(5)的特征在于,通过维恩图、定量核酸扩增法、特殊蛋白质检测检查及统计程序SAS测定表达量。
发明效果
根据如上所述的本发明提供包括以下步骤的对低水平放射线反应的DNA修复相关基因的检测方法:(1)在低水平放射线环境中饲养胸腺癌模型AKR/J小鼠和正常ICR小鼠;(2)采集所述步骤(1)中饲养的胸腺癌模型AKR/J小鼠和正常ICR小鼠的胸腺;(3)对在所述步骤(2)中采集的胸腺的基因进行分析;(4)在所述步骤(3)中分析的基因中检测胸腺癌模型AKR/J小鼠和正常ICR小鼠中共同或单独表达的DNA修复相关基因;以及(5)对所述步骤(4)中检测出的基因进行扩增并测定表达量。通过提供上述方法能够带来以下效果:能够用作评价产业及医疗从事人员的放射线暴露与致癌之间的相关性的低水平放射线DNA修复指标基因,并且不仅能够用作评价癌症患者的癌症进展及治疗程度的低水平放射线DNA修复基因,而且还能够用作评价放射线和致癌的因果关系的低水平DNA修复指标。
附图说明
图1为对于在实验例1的DNA损伤/修复相关基因中对低水平放射线显示种类特异性反应的基因,利用核酸扩增法确认相对表达量的图表。
图2为示出实验例2的照射低水平放射线的AKR/J和JCR小鼠的胸腺蛋白质的分析结果的图表。((1)低水平放射线DNA修复基因(Cyp11a、Rnd3、Plxnc1)蛋白质量,(2)加样对照(loading control),(3)特殊蛋白质检测法的定量分析,测定各条带(band)的密度后用β-微管蛋白(tubulin)密度值来定量化)
图3为图示化了低水平放射线照射所引起的小鼠胸腺的分子变化。
具体实施方式
下面,将详细说明本发明。
通常而言,已知高剂量放射线会引起DNA损伤、产生癌症,然而,另一方面,已知低剂量放射线会修复受损的DNA、诱导细胞死亡、促进免疫活性和抗氧化功能,从而抑制癌症。然而,未满足联合国原子辐射效应科学委员会(UNSCEAR,2000)提出的低剂量率放射线(6≥mGy/h)标准的情况较多。此外,利用细胞株而确保的研究结果难以直接适用于人体来解释,即使是在利用实验动物的情况下,对于低水平放射线对DNA修复带来的影响,在现有技术中也没有确认到蛋白质水平。本发明中,在低水平(0.7mGy/小时)放射线环境下饲养AKR/J小鼠和正常小鼠(ICR),并在达到开始死亡的时期(100天)时,采集胸腺并分析了基因。此外,采集了在相同的条件下饲养的正常小鼠(ICR)的胸腺,并分析了基因。最终,对两种小鼠中共同或单独表达的DNA修复相关基因进行扩增,并通过蛋白质检测最终发掘出基因组(genome)、蛋白质组(proteome)。
下面,通过实施例对本发明进行更详细的说明。这些实施例仅用于例示本发明,本发明的范围并不解释为被限定于这些实施例,对于本领域技术人员来说是显而易见的。
实施例1
本发明中,从日本静冈县实验室(Shizuoka laboratory)中心购入雌性(7周龄)AKR/J和ICR小鼠,并在附有特定病原菌的设施中饲养1周而使其稳定化。
各组利用5只小鼠并在低水平(0.7mGy/小时)放射线环境下饲养100天(累计剂量为共1.7Gy),然后采集胸腺并在液体氮中急速冷冻后实施了基因分析。
比较照射低水平放射线的AKR小鼠和ICR小鼠的胸腺,并发掘出对低水平(0.7mGy/小时)放射线反应的DNA修复相关基因并解释了功能。
利用维恩图、定量核酸扩增法、特殊蛋白质检测检查及统计程序SAS(方差分析(ANOVA),t-检验(t-test))进行了分析。
另外,为了对于在照射低水平(0.7mGy/小时)放射线的小鼠胸腺中产生敏感反应的DNA修复相关基因的表达量进行测量,所使用的引物碱基序列如下述表1所示。
表1
基因号 | 基因名称 | 正向(5'→3') | 反向(5'→3') |
NM_019779 | Cyp11a1 | CCTGGAAGAAAGACCGAATC | TGCTTGATGCGTCTGTGTAA |
NM_011198 | Ptgs2 | AGAACCTGCAGTTTGCTGTG | GCTCCTGCTTGAGTATGTCG |
NM_028810 | Pnd3 | TATGACAACGTCCGTCCACT | CCTGGATTTCACCTTTCCAC |
NM_018797 | Plxnc1 | TCCTCATCCCATGAAGAACA | CGCTGCTAAGCACTCTGAAC |
NM_021282 | Cyp2el | TCTCTTCAACAAACGCTTCG | CCAGGGAGTACTCAGCAGGT |
实验例1.DNA损伤/修复相关基因的表达量测定
对AKR/J和ICR小鼠照射低水平(0.7mGy/小时)放射线,然后在癌症发生初期阶段(100天)采集胸腺,然后利用核酸扩增法确认了在DNA损伤/修复相关基因中对低水平放射线显示种类特异性反应的基因的相对表达量。
其结果如图1所示,在照射低水平放射线的AKR/J小鼠中,Cyp11a1、Ptgs2、Rnd3、Plxnc1等对低水平放射线产生了种类特异性的敏感反应,在ICR小鼠中,Cyp2e1等对低水平放射线产生了种类特异性的敏感反应。
实施例2.照射低水平放射线的AKR/J和ICR小鼠胸腺的蛋白质分析
对于实施例1中的照射低水平放射线的情况下的蛋白质和作为对照组的照射高水平放射线(0.8Gy/分钟,总剂量:4.5Gy)的小鼠胸腺的蛋白质进行了分析。
其结果如图2所示,鉴定了在照射低水平放射线的AKR/J小鼠中Cyp11a1、Rnd3、Plxnc1等对低水平放射线产生了种类特异性的敏感反应。
以上,对本发明内容的特定部分进行了详细记述,这些具体的记述只是优选的实施方式,本发明的范围并不限定于此,这对具有本领域常规知识的技术人员来说是明确的。因此,本发明的实质范围定义为附上的权利要求及其等价物。
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Claims (5)
1.对低水平放射线反应的DNA修复相关基因的检测方法,其包括以下步骤:(1)在低水平放射线环境中饲养胸腺癌模型AKR/J小鼠和正常ICR小鼠;(2)采集所述步骤(1)中饲养的胸腺癌模型AKR/J小鼠和正常ICR小鼠的胸腺;(3)对在所述步骤(2)中采集的胸腺的基因进行分析;(4)在所述步骤(3)中分析的基因中检测胸腺癌模型AKR/J小鼠和正常ICR小鼠中共同或单独表达的DNA修复相关基因;以及(5)对所述步骤(4)中检测出的基因进行扩增并测定表达量。
2.根据权利要求1所述的对低水平放射线反应的DNA修复相关基因的检测方法,其特征在于,所述步骤(1)中低水平放射线剂量为0.7mGy/小时。
3.根据权利要求1所述的对低水平放射线反应的DNA修复相关基因的检测方法,其特征在于,在所述步骤(1)中使在0.7mGy/小时的低水平放射线环境中的总累计剂量达到1.7Gy来进行饲养。
4.根据权利要求1所述的对低水平放射线反应的DNA修复相关基因的检测方法,其特征在于,在所述步骤(5)中用选自Cyp11a1、Ptgs2、Rnd3、Plxncl及cyp2el中的引物对DNA修复相关基因进行扩增。
5.根据权利要求1所述的对低水平放射线反应的DNA修复相关基因的检测方法,其特征在于,在所述步骤(5)中通过维恩图、定量核酸扩增法、特殊蛋白质检测检查及统计程序SAS来测定表达量。
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CN111148848B (zh) * | 2017-09-26 | 2023-08-29 | 韩国水力原子力株式会社 | 被照射低剂量率低水平放射线的小鼠胸腺淋巴瘤细胞中的端粒调节基因家族及其检测方法 |
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