CN111621559A - 关联转录组和蛋白组数据筛选低剂量电离辐射效应基因 - Google Patents
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Abstract
本发明涉及关联转录组和蛋白组数据筛选低剂量电离辐射效应基因，所述基因包括ORM1、ORM2、LYZ、FBXO7、SNCA、HBZ和HIST1H4J中的一种或多种，该组基因的转录和蛋白水平变化相关性较好，能够为探讨低剂量辐射效应机制提供新的靶标；并基于所筛选基因有望检测受试者是否受低剂量辐射效应影响的非诊断方法和提供相应的检测试剂盒、芯片，其有助于及时发现受试者是否遭受低剂量电离辐射效应影响，对于充分保护职业人群免受辐射暴露增加的潜在影响具有重要意义。
Description
技术领域
本发明涉及电离辐射领域，具体而言，涉及关联转录组和蛋白组数据筛选低剂量电离辐射效应基因。
背景技术
人类每天都有暴露在低剂量自然和人为电离辐射下的风险，特别是计算机断层扫描使用和放射治疗的增加使得现代医疗照射成为电离辐射非本底照射的主要来源，同时增加辐射暴露。研究表明，长期职业暴露低剂量电离辐射可引起医疗职业人群细胞遗传学损伤和甲状腺机能损伤；此外，暴露在外的正常组织有在长期存活中发展辐射诱导毒性和继发性疾病的风险。了解暴露于低剂量电离辐射的生物效应，及时检测受试者遭受低剂量电离辐射效应的影响水平，对于充分保护职业人群免受辐射暴露增加的潜在影响显得至关重要。
但目前本领域对低剂量电离辐射的生物效应研究较少，低剂量电离辐射的风险评估是从高剂量暴露数据推断而来，少有能够准确反映低剂量电离辐射生物效应的相关基因及其筛选技术，导致在确定低剂量区域的影响程度以及剂量反应关系等方面存在很大不确定性。有鉴于此，特提出本发明。
发明内容
本发明的第一目的，提供关联转录组和蛋白组数据筛选低剂量电离辐射效应基因。
本发明的第二目的，提供检测受试者是否受到低剂量电离辐射效应影响的非诊断方法。
本发明的第三目的和第四目的，提供检测前述低剂量电离辐射效应基因的试剂盒和芯片。
本发明的第五目的，提供一种筛选低剂量电离辐射效应基因的方法。
为实现上述目的，本发明特提供以下技术方案：
低剂量电离辐射效应基因，所述效应基因包括ORM1、ORM2、LYZ、FBXO7、SNCA、HBZ和HIST1H4J中的一种或多种。
在一些具体的实施方案中，所述效应基因包括：ORM1、ORM2、LYZ、FBXO7、SNCA、HBZ和HIST1H4J。
一种检测受试者是否受到低剂量电离辐射效应影响的非诊断方法，所述方法包括以下步骤：
a.从受试者处收集待测样本；
b.检测所述样本中前述低剂量电离辐射效应基因的转录水平和对应编码蛋白的表达水平；
c.与未受到低剂量电离辐射的对照样品进行比较，如果基因FBXO7和SNCA的转录水平和对应编码蛋白的表达水平均上调，基因ORM1、ORM2、HIST1H4J、HBZ和LYZ的转录水平和对应编码蛋白的表达水平均下调，则判断所述受试者受到低剂量电离辐射效应的影响。
在一些具体的实施方式中，所述低剂量电离辐射是指剂量低于200mGy的X或γ-射线外照射。
在一些具体的实施方式中，所述低剂量电离辐射是指是指剂量率低于0.1mGy/min的X或γ-射线外照射。
在一些具体的实施方式中，所述受试者具有低剂量电离辐射的职业暴露，或者，所述受试者疑似遭受低剂量电离辐射。
在一些具体的实施方式中，所述待测样本为血液样本。
在一些具体的实施方式中，所述血液样本为全血样本、血浆样本或血清样本。
在一些具体的实施方式中，所述蛋白水平通过ELISA、免疫层析或WB的方式检测。
在一些具体的实施方式中，所述转录水平通过PCR反应、测序或杂交探针的方式检测。
一种试剂盒，所述试剂盒包括检测前述效应基因的转录水平和编码蛋白表达水平的试剂。
优选地，所述转录水平检测试剂为PCR试剂或测序试剂；所述蛋白水平的检测试剂为ELISA检测试剂、免疫层析检测试剂和/或WB检测试剂。
一种芯片，所述芯片上包被有特异性结合权利要求1或2所述基因标记物的蛋白质和mRNA的探针。
一种筛选低剂量电离辐射效应基因的方法，所述方法包括：a.获得未受电离辐射的受试者血液样本和对照血液样本；b.之后，对所述受试者血液样本进行低剂量电离辐射处理，对照血液样本不进行低剂量电离辐射处理；c.检测受低剂量电离辐射处理的受试者血液样本和未经低剂量电离辐射处理的对照血液样本的mRNA表达水平和蛋白质表达水平；d.通过与所述对照血液样本进行比较，获得所述受试血液样本在转录组和蛋白组水平的变化信息；e.将所述受试者在转录组和蛋白组表达水平进行关联性分析，根据关联性筛选低剂量电离辐射效应基因。
在一些具体的实施方案中，所述转录组学信息通过测序或芯片杂交技术获得。
在一些具体的实施方案中，所述蛋白组学信息通过相对和绝对定量同位素标志(isobaric tags for relative and absolute quantitation,iTRAQ)技术联合二维液相色谱-串联质谱(two-dimensional liquid chromatography/tandem mass spectrometry,2D LC-MS/MS)。
与现有技术相比，本发明的有益效果为：
现有技术尚不能够准确全面地筛选低剂量辐射效应相关的基因，本发明提供了一组筛选所得的效应基因(即关联转录组和蛋白组数据筛选低剂量电离辐射效应基因)，该组基因的转录和蛋白表达水平变化关联性较好，能够较为准确地反映低剂量辐射效应变化；
本发明还依据该组基因，建立检测受试者受到低剂量电离辐射影响水平的非诊断方法，其有助于及时发现受试者是否遭受低剂量电离辐射影响，充分保护职业人群免受辐射暴露增加的潜在影响；
本发明还提供一种试剂盒或芯片，为前述效应基因的检测提供便利；
另外本发明还建立一种筛选低剂量电离辐射效应基因的方法(即一种筛选分析方法)，该方法运用高通量多组学数据关联技术，通过基因转录变化和蛋白质丰度变化确定单体组学技术不能明确阐明的低剂量辐射效应相关基因。
附图说明
图1为定量蛋白和基因的表达关联；
图2为变化趋势相同的差异蛋白和基因表达关联；
图3为变化趋势相反的差异蛋白和基因的表达关联。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1经低剂量电离辐射的血液样本和对照样本的制备
在签署知情同意书的前提下，选择非放射工作者、无急慢性疾病、半年内无射线和化学毒物接触史的6名健康志愿者，男3名，女3名，年龄25-48岁，平均年龄为(33±9.03)岁。
每名志愿者采集静脉血10ml，分装BD管和肝素锂抗凝管，分别用于转录组与蛋白组数据库构建。将已分装的血样于中国疾病预防控制中心辐射防护与核安全医学所在室温下用137Csγ射线进行照射。6个样本分为对照组和照射组，每组各3个生物学重复。吸收剂量为58.4cGy/h，源靶距为70cm，平均照射野为30cmx30cm，总照射剂量为150mGy。本次研究获北京市职业病防治研究院伦理委员会审核。
实施例2总RNA和蛋白的提取
提取实施例1所述血液样本的总RNA和蛋白：
1.使用TRIzol Reagent试剂盒(Invitrogen，Life Technologies，USA)提取总RNA，然后通过DNase I(Invitrogen，Life Technologies，USA)处理。利用NanoDrop紫外分光光度计(LabTech，USA)和Agilent 2100Bioanalyzer(Agilent，USA)进行RNA质检。质检合格再使用BGISEQ-500平台进行测序。
2.使用Proteominer试剂盒去高峰度；采用丙酮沉淀法提取照射组与对照组蛋白溶液；对提取后的蛋白样品进行还原烷基化处理；用Brandford法进行蛋白的浓度测定，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测。
实施例3转录组数据分析
将测序获得的原始序列数据(raw data)进行过滤，得到有效数据(clean data)，比对参考人类基因组(HG19,Accession Number:PRJNA31257)，运用RPKM(reads per kbper million reads)法计算基因表达量。差异表达基因筛选标准为差异表达倍数(foldchange)>＝1.5，错赔率(false discover rate,FDR)<0.05以及P<＝0.01。
经BGISEQ-500深度测序后，对照组和照射组经去除杂质处理分别得到24114683和24115342个reads的净序列数，比对到基因组的序列均占90％以上，见表1。根据差异表达基因筛选标准，发现差异表达基因486个，其中上调基因202个，下调基因284个。
表1 RNA-seq序列与参考基因组的比对
实施例4蛋白组数据分析
采用相对和绝对定量同位素标志(isobaric tags for relative and absolutequantitation,iTRAQ)技术联合二维液相色谱-串联质谱(two-dimensional liquidchromatography/tandem mass spectrometry,2D LC-MS/MS)分析和鉴定组间差异表达蛋白。差异表达蛋白的挑选标准是P<＝0.05，且差异倍数>1.20(上调)或<0.83(下调)的则视为差异表达蛋白。
蛋白质ITRAQ定量分析中，共有19508条肽段和3807个蛋白被鉴定到。3次重复实验之间的蛋白定量比值平均值都在1附近，表明3组蛋白样品之间重复性较好，检测出的蛋白质可信度较高。利用iTRAQ技术鉴定蛋白表达，根据差异标准，共筛选到266个差异蛋白，其中上调147个，下调119个。
实施例5转录组结果与蛋白质鉴定结果的关联分析
关联分析：基于参考基因得到的转录组结果和蛋白质鉴定结果进行关联，当某一个蛋白质在转录组水平表达量时，被认为关联到。对转录组与蛋白组关联上的mRNA和蛋白的表达量相关性进行分析，确定mRNA和蛋白的相关性强弱。
转录组与蛋白质组关联数量关系：对相同基因在mRNA和蛋白质表达情况进行关联，当某一个基因的mRNA水平和蛋白质水平均可以检测到表达时，认为这个基因mRNA和蛋白质是相关的。在鉴定、定量、显著差异3个层面，能关联到的蛋白质和基因数量见表2。结果表明在mRNA和蛋白质水平均鉴定到的基因有3619个，其中3614个基因在mRNA和蛋白质水平均有表达。266个差异蛋白中有12个同基因显著关联(P<0.05)，即12个基因在转录水平和蛋白水平均差异表达且达到显著水平。可见有254个蛋白在蛋白质水平显著差异表达，在mRNA水平表达差异不显著；474个基因在mRNA水平显著差异表达，在蛋白质水平表达差异不显著。
表2关联转录组和蛋白质组数量
| 类型 | 基因数量 | 蛋白质数量 | 关联数量 |
| 鉴定 | 16158 | 3807 | 3619 |
| 定量 | 16158 | 3801 | 3614 |
| 差异表达 | 486 | 266 | 12 |
蛋白质组与转录组相关性分析：由于转录组与蛋白组水平之间存在复杂的非线性关系，本研究通过定量蛋白和基因、差异蛋白和基因变化趋势相同及相反3种类型进行关联分析。由图1可知，人类外周血通过低剂量辐射后，发现定量蛋白和基因呈现正相关，其间的相关性并不高，相关系数为0.0034；变化趋势相反的差异蛋白和基因相关系数为-0.1，表达变化趋势相同的差异蛋白和基因相关系数为0.6786，相关性较高。即上述图2为变化趋势相同的差异蛋白和基因表达关联；图3为变化趋势相反的差异蛋白和基因的表达关联。
关联到差异基因：关联到12个差异基因和相应蛋白如表3，涉及免疫系统调节、信号转导、酶活性调节、跨膜运输、防御、转录、DNA修复等方面功能。其中上调表达7个，主要与信号转导、跨膜转运、免疫调节作用相关；下调表达5个，主要参与了转录调控、DNA修复、免疫系统调节等。
表3关联到的差异基因和蛋白
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (10)
1.关联转录组和蛋白组数据筛选低剂量电离辐射效应基因，其特征在于，所述效应基因包括ORM1、ORM2、LYZ、FBXO7、SNCA、HBZ和HIST1H4J中的一种或多种。
2.根据权利要求1所述的关联转录组和蛋白组数据筛选低剂量电离辐射效应基因，其特征在于，所述效应基因包括：ORM1、ORM2、LYZ、FBXO7、SNCA、HBZ和HIST1H4J。
3.一种检测受试者是否受到低剂量电离辐射效应影响的非诊断方法，其特征在于，所述方法包括以下步骤：
a.从受试者处收集待测样本；
b.检测所述样本中权利要求1或2所述低剂量电离辐射效应基因的转录水平或对应编码蛋白的表达水平；
c.与未受到低剂量电离辐射的对照样品进行比较，如果基因FBXO7和SNCA的转录水平和对应编码蛋白的表达水平均发生上调，基因ORM1、ORM2、HIST1H4J、HBZ和LYZ的转录水平和对应编码蛋白的表达水平均发生下调，则判断所述受试者受到低剂量电离辐射的效应影响。
4.根据权利要求3所述的方法，其特征在于，所述低剂量电离辐射是指剂量低于200mGy的X或γ-射线外照射；或，所述低剂量电离辐射是指剂量率低于0.1mGy/min的X或γ-射线外照射。
5.根据权利要求3所述的方法，其特征在于，所述受试者具有低剂量电离辐射的职业暴露，或者，所述受试者疑似遭受低剂量电离辐射。
6.根据权利要求3-5任一项所述的方法，其特征在于，所述待测样本为血液样本。
7.根据权利要求6所述的方法，其特征在于，所述血液样本为全血样本、血浆样本或血清样本。
8.根据权利要求4-5任一项所述的方法，其特征在于，所述蛋白水平通过ELISA、免疫层析或WB的方式检测；所述转录水平通过PCR反应、测序或杂交探针的方式检测。
9.一种试剂盒，其特征在于，所述试剂盒包括检测权利要求1或2所述基因的转录水平和其编码蛋白表达水平的试剂；优选地，所述转录水平检测试剂为PCR试剂或测序试剂；所述蛋白表达水平的检测试剂为ELISA检测试剂、免疫层析检测试剂和/或WB检测试剂。
10.一种芯片，其特征在于，所述芯片上包被有特异性结合权利要求1或2所述基因的蛋白质和mRNA的探针。
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