CN112535733A - tRFs在UV辐射致皮肤急性损伤的产品中的应用 - Google Patents
tRFs在UV辐射致皮肤急性损伤的产品中的应用 Download PDFInfo
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Abstract
本发明涉及生物医学领域，公开了tRFs在制备治疗或检测UV辐射致皮肤急性损伤的产品中的应用，所述tRFs为tRF‑Trp‑TCA‑001和tRF‑Gly‑CCC‑019。本发明为紫外线辐射致皮肤损伤的机制研究进一步奠定了基础，为诊治该方面的疾病提供了tRF‑Trp‑TCA‑001和tRF‑Gly‑CCC‑019新靶点，本发明表明tRF‑Trp‑TCA‑001和tRF‑Gly‑CCC‑019在紫外线辐射致皮肤损伤中起到了关键的调控作用，因此，靶点tRF‑Trp‑TCA‑001和tRF‑Gly‑CCC‑019在检测和治疗紫外线辐射致皮肤损伤及相关疾病中具有重要的应用价值。
Description
技术领域
本发明涉及生物医学领域，具体涉及tRFs在UV辐射致皮肤急性损伤的产品中的应用。
背景技术
紫外线辐射所致的晒伤、慢性累积损伤，如光老化和皮肤癌等，均对人类健康构成潜在危害。紫外线辐射可引起皮肤红斑、水肿、水疱、皮肤色素沉着、异常生长、光老化等不良反应，甚至是非黑素瘤皮肤癌。面对上述损伤，人体可通过各种保护措施来保护皮肤，如细胞凋亡、炎症、监测和对抗潜在癌细胞的增殖。
转运RNA(Transporter RNA，tRNA)是一种参与mRNA编码和蛋白质翻译的连接分子，最近的研究表明，tRNA也可以作为拥有特殊功能的小的非编码RNA(small non-codingRNA,sncRNA)的主要来源之一。这些tRNA衍生的ncRNA不是随机降解的产物，而是由精确的生物过程产生的。来自tRNA的ncRNA大致可分为两部分：tiRNA(tRNA衍生的应激诱导RNA)和tRFs(tRNA衍生片段)，它们有特定的分子大小、核苷酸组成、生理功能和生物发生。tRFs的长度约为16-28nt，来源于成熟tRNA或前体tRNA。tRFs和tiRNAs与多种病理状态甚至致病因素有关，如癌症、神经退行性疾病和遗传性代谢性疾病。
目前，tRFs&tiRNAs的研究受到了广泛关注，但还没有研究证明tRFs&tiRNAs与紫外线辐射致皮肤损伤之间的内在联系。因此，发明人研发了tRFs&tiRNAs在紫外线损伤中的作用机制，为诊治疾病提供了新靶点。
发明内容
基于以上问题，本发明提供tRFs在UV辐射致皮肤急性损伤的产品中的应用，本发明为诊治紫外线辐射致皮肤损伤提供了新的靶点。
为解决以上技术问题，本发明提供了tRFs在制备治疗或检测UV辐射致皮肤急性损伤的产品中的应用，所述tRFs为tRF-Trp-TCA-001和tRF-Gly-CCC-019。
进一步的，所述产品为检测tRF-Trp-TCA-001和tRF-Gly-CCC-019的表达量的试剂盒。
进一步的，所述试剂盒为生物标记诊断试剂盒，所述生物标记为tRF-Trp-TCA-001和tRF-Gly-CCC-019。
进一步的，所述产品为治疗用药物，所述药物的作用靶点为tRF-Trp-TCA-001和tRF-Gly-CCC-019。
进一步的，所述药物促进tRF-Trp-TCA-001和tRF-Gly-CCC-019调控Rac1基因，从而促进Rac1基因调节UV辐射导致的DNA损伤反应，从而保护皮肤免受紫外线诱导的角化细胞凋亡和皮肤癌变。
与现有技术相比，本发明的有益效果是：本发明为紫外线辐射致皮肤损伤的机制研究进一步奠定了基础，为诊治该方面的疾病提供了tRF-Trp-TCA-001和tRF-Gly-CCC-019新靶点，tRF-Trp-TCA-001和tRF-Gly-CCC-019在UV辐射致损伤小鼠中的表达水平明显降低，与正常小鼠中的tRF-Trp-TCA-001和tRF-Gly-CCC-019的表达量存在明显差异，且tRF-Trp-TCA-001和tRF-Gly-CCC-019可通过调控Rac1基因来保护皮肤免受紫外线诱导的角化细胞凋亡和皮肤癌变。本发明表明tRF-Trp-TCA-001和tRF-Gly-CCC-019在紫外线辐射致皮肤损伤中起到了关键的调控作用，因此，靶点tRF-Trp-TCA-001和tRF-Gly-CCC-019在检测和治疗紫外线辐射致皮肤损伤及相关疾病中具有重要的应用价值。
附图说明
图1为本发明的实施例的HE染色(分别为光学显微镜下放大10倍和20倍)结果图；
图2为本发明的实施例的各样本的质量评分图；
图3为本发明的实施例中的散点图；
图4为本发明的实施例中的热点图；
图5为本发明的实施例的tRF-Gly-CCC-019与Rac1基因的结合位点图；
图6为本发明的实施例的tRF-Trp-TCA-001与Rac1基因的结合位点图；
图7为本发明的实施例的研究步骤；
图8为本发明的实施例的U6、tRF-Gly-CCC-019和tRF-Trp-TCA-001的标准曲线图；
图9为本发明的实施例的RT-qPCR验证tRF-Gly-CCC-019和tRF-Trp-TCA-001的相对表达量结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例：
本实施例首先构建UV辐射致皮肤急性损伤动物模型，研究动物对象为C57BL/6小鼠，共12只，成年雄性(SCXK 2016-0002，2018年3月29号，中南大学湘雅三医院中心动物房提供)，鼠龄6-8周，体重18±2g左右。小鼠被安置在恒温22-25℃和潮湿的环境中，小鼠可以自由获取食物和水；小鼠接受12小时的光/暗循环。实验流程均按照实验室要求进行，并得到了中南大学生物伦理委员会的批准。
实验前，用1％戊巴比妥钠对C57BL/6小鼠进行术前麻醉，用脱毛膏及电推刀剔除小鼠背部毛发约2×2cm2。
之后应用上海西格玛高技术有限公司SS-03B型紫外线光疗仪对实验组小鼠皮肤进行照射，除了对照组不予照射外，其余各组照射剂量根据预实验结果分别定为90mJ/cm2、180mJ/cm2、360mJ/cm2。具体操作为：将小鼠固定在工作台面上，预热SS-03B型紫外线辐射仪，调节灯管高度至距离工作台50cm处，连续照射8天。
实验分为了对照组A组、UV照射组B组(辐射强度为90mJ/cm2)、UV照射组C组(辐射强度为180mJ/cm2)和UV照射组D组(辐射强度为360mJ/cm2)，UV照射组B组、UV照射组C组和UV照射组D组均为实验组。
每日定时使用同一台日本尼康数码相机采集图片，记录小鼠每天照射前及照射后的改变及特殊情况，第9天处死小鼠，用无菌消毒剪刀收集照射组和对照组小鼠的背部皮肤，每只小鼠收集四块约1×1cm2大小的皮肤组织。
所切除的皮肤组织标本平均分为四部分用于保存，第一部分装于冻存管，储存于液氮中，制作成冰冻切块，供日后备用；第二部分和第三部分用甲醛溶液固定24h后，分别用于HE和Masson三色染色，第四部分置于EP管中，由干冰保存，用于进行分子检测，每份皮肤样本均作相应的标号标记。
皮肤组织标本HE染色：按照固定、脱水、透明、浸蜡、包埋、切片、烤片的顺序依次操作，制作石蜡切片；脱蜡入水：用二甲苯I、二甲苯II溶液各浸泡10min，晾干后加入无水乙醇，脱去二甲苯；用乙醇水化后甩干；染色：苏木素染色，分化，冲洗，甩干；氨水中返蓝，冲洗、甩干；再用伊红溶液染色，甩干；脱水透明：乙醇脱水；二甲苯透明。中性树胶封片，镜下观察，使用普通光学显微镜，观察皮肤表皮的厚度、完整性，基底膜有无降解，真皮乳头层是否明显，有无炎症细胞浸润，是否可见其他病变等。实验结果见附图1，说明动物模型构建成功。
小鼠皮肤组织总RNA的提取：
(1)组织匀浆：每50-100mg组织样品，加入1ml的TRI Reagent，用电动匀浆器进行匀浆；所加样品体积不能超过匀浆此样品所使用的TRI REAGENT试剂体积的10％；
(2)两相分离：匀浆后样品于15到30℃孵育5分钟，以便核酸蛋白复合体完全解离；每1ml的TRI Reagent试剂匀浆的样品中加入0.2ml的氯仿，盖紧管盖；手动剧烈振荡管体15秒后，15到30℃孵育2到3分钟，4℃下12000×g离心15分钟；离心后混合液体将分为下层的红色酚氯仿相，中间层核上层的无色的水相；RNA全部被分配于水相中，水相的体积大约使匀浆时加入的TRI Reagent试剂的60％；
(3)RNA沉淀：将水相转移到新离心管中，水相与异丙醇混合以沉淀其中的RNA，加入异丙醇的量为每个样品匀浆时加入1ml TRI Reagent试剂的此时加0.5ml的异丙醇；混匀后15到30℃孵育10分钟后，于4℃ 12000g离心10分钟，此时离心前不可见的RNA沉淀将在管底部和侧壁上形成胶状沉淀块；
(4)RNA清洗：移去上清液，每1ml TRI Reagent试剂匀浆的样品中加入至少1ml的75％乙醇，清洗RNA沉淀，振荡后，4℃ 7500g离心5分钟；
(5)重新溶解RNA沉淀：去除乙醇溶液，空气中干燥RNA沉淀5-10分钟，切勿真空离心干燥；注意RNA沉淀不要完全干燥，否则将大大降低RNA的可溶性；部分溶解的RNA样品A260/280比值将小于1.6，溶解RNA时，先加入无RNA酶的水用枪反复吹打几次，然后55到60℃孵育10分钟，获得的RNA溶液保存于-70℃；
(6)小量RNA的抽提：从少量的组织(1到10mg)或者细胞(102到104)样品中抽提RNA：加入800μl的TRI Reagent试剂至样品中，样品裂解后，按步骤2加氯仿进行两相分离；在加异丙醇沉淀RNA前，加5-10μg的无RNA酶的糖原作为水相载体；为降低溶液粘度，在加氯仿前先将样品两次过26号针头以剪切基因组DNA；两相分离后糖原留在水相中并和RNA共沉淀，只要糖原浓度不高于4mg/ml，不会影响cDNA的合成，同样也不会对PCR过程产生影响。
RNA质量检测：紫外吸收测定法：使用
ND-1000测定RNA浓度和纯度，测量前先用溶解RNA用的DEPC水调零，操作方法如下：a)滴加1ul DEPC水或者RNA样品至测量基座的表面；b)液滴会自动在上下基座之间形成液柱并自动完成测定，RNA浓度及质量的各种参数将在电脑中自动生成文件；c)一次测定完成后，用柔软的擦镜纸擦去上下基座表面上的样本液，便可进行下一个样品的测量；d)测定结果(电脑自动生成的EXCEL和JPEG文件附于实验报告文件夹中)：浓度测定：260nm处读值为1表示40ng RNA/ul。样品RNA浓度计算公式为：A260 X 40ng/ul；具体计算如下：RNA溶于20μl DEPC水中，取1ul用于测定，测得A260＝65.003；RNA浓度＝65.003X 40ng/ul＝2600.12ng/ul；取1ul用来测量以后，剩余样品RNA为19μl，剩余RNA总量为：19μl X2600.12ng/ul＝49.4μg；纯度检测：RNA溶液的A260/A280的比值是一种RNA纯度检测方法，比值范围1.8到2.1；即使比值超出这个范围，RNA样品也一样可以用于一些普通实验中如Northern杂交，RT-PCR和RNA酶保护实验。
变性琼脂糖凝胶电泳：制胶，1g琼脂糖溶于72ml水中，冷却至60℃，10ml的10×MOPS电泳缓冲液和18ml的37％甲醛溶液(12.3M)。灌制凝胶板，预留加样孔至少可以加入25μl溶液。胶凝后取下梳子，将凝胶板放入电泳槽内，加足量的1×MOPS电泳缓冲液至覆盖胶面几个毫米。准备RNA样品，取3μg RNA，加3倍体积的甲醛上样染液，加GoldView于甲醛上样染液中至终浓度为10μg/ml。加热至70℃孵育5分钟使样品变性。电泳：上样于胶孔中，5-6V/cm电压下电泳至溴酚兰指示剂进胶至少2-3cm。紫外透射光下观察并拍照：28S和18S核糖体RNA的带非常亮而浓(其大小决定于用于抽提RNA的物种类型)，上面一条带的密度大约是下面一条带的2倍。还有可能观察到一个更小稍微扩散的带，它由低分子量的RNA(tRNA和5S核糖体RNA)组成。在18S和28S核糖体带之间一般可以看到一片弥散的EB染色物质，可能是由mRNA和其它异型RNA组成。RNA制备过程中如果出现DNA污染，将会在28S核糖体RNA带的上面出现，即更高分子量的弥散迁移物质或者带。RNA的降解表现为核糖体RNA带的弥散。
随后进行高通量测序，首先对总RNA样本进行质量检测，合格后进行以下预处理，以去除一些干扰小型RNA-seq文库构建的RNA修饰:3'-氨基酰基(带电)脱酰至3'-OH进行3'-配对连接，3'–cP(2'，3'-环磷酸盐)去除至3'-OH进行3'-配对连接，5'–OH(羟基)磷酸化至5'-P进行5'-配对连接，m1A和m3C去甲基化进行高效逆转录；使用预处理后的总RNA制备测序文库，步骤如下：3’-端配对连接，5’-端配对连接；互补脱氧核糖核酸(cDNA)的合成；PCR扩增，PCR扩增片段大小选择134～160bp(对应14～40nt小RNA大小范围)；这些文库被变性为单链DNA分子，在Illumina流动池上捕获，原位扩增为测序簇，按照的说明书在IlluminaNextSeq 500系统上进行50个周期的测序；使用Solexa pipeline v1.8(Off-lineBase Caller软件)进行图像分析和基本调用；测序质量由FastQC进行检测，剪切读数(通过Illumina quality filter，cut adapt剪切5'，3'-adaptorbase)对齐，只允许与miRDeep2miRNA参考序列不匹配1次；miRNA的丰度是通过测序计数来评估的，并以每百万总比对读数(CPM)来标准化；根据R包edgeR的计数值，在R或perl环境中进行筛选差异表达的miRNA分析。
本实施例在对tRF&tiRNA表达谱进行筛选时，使用Agilengt 2100生物芯片分析仪和FASTQC对RNA序列数据进行质量控制，FASTQ格式的原始数据文件是由Illumina测序器生成的。为了检验测序质量，绘制了每个样本的质量评分图，见附图2，读取的位置绘制在X轴上，Q值绘制在Y轴上；红线(图2中最下方的区域)是Q得分的中值，蓝线(图2中最上方的区域)是Q得分的平均值，箱形图表示四分位之间的范围，而触须线表示10％和90％的点；Q值高于30(>99.9％正确)被认为是高质量的数据，质量分数Q与基本调用错误概率(P)对数相关:Q＝-10log10(P)。
见附图3中的散点图，其中X轴和Y轴均为两组之间的log2(平均CMP值)，黑/灰点(必要时可提供相应的彩色图片，彩色图片中为红/绿点)显示显著差异表达miRNA与倍数变化≧1.5，P≦0.05(红色:上调；绿色:下调)；两条虚线之间的点表示无差异表达的miRNA，FC或P值没有达到阈值；结果显示差异表达的tRFs&tiRNAs有31个，其中上调8个，下调23个。
分析tRFs&tiRNAs表达数据最广泛使用的是热点图见附图4，层次聚类分析将样本按照tRFs&tiRNAs表达水平(CPM值)进行分组，从而使我们可以了解样本之间的关系。树状图显示了样品tRFs&tiRNAs表达模式之间的关系，使用差异表达的tRFs&tiRNAs(如果数量超过两个)进行分层聚类，每一行表示一个tRFs&tiRNAs，每列代表一个样本，分层聚类结果表明不同样本间tRFs&tiRNAs表达差异显著。根据筛选条件(log2FC≥0.585,P-value≤0.05,Q-value≤1.00and CMP>3)再次筛选出10个差异表达的tRFs&tiRNAs。
将上述10个差异表的tRFs&tiRNAs进行靶基因预测，其中tRF-Trp-TCA-001和tRF-Gly-CCC-019的靶基因为Rac1，tRF-Gly-CCC-019与Rac1基因的结合位点见附图5，tRF-Trp-TCA-001与Rac1基因的结合位点见附图6。Rac1基因可调节DNA损伤反应，保护皮肤免受紫外线诱导的角化细胞凋亡和皮肤癌变。研究人员发现，暴露在阳光下的黑色素瘤患者中，有9.2％的患者在接触紫外线信号后出现Rac1激活突变，这些与我们预测结果一致。我们推测tRF-Trp-TCA-001和tRF-Gly-CCC-019可调控Rac1基因，从而在紫外线辐射引起的皮肤损伤中发挥重要作用。
RT-qPCR验证，RNA质量检测及提取同前所述。RNA前处理与cDNA合成：1)3’末端去乙酰化处理，配置去乙酰化反应液，涡旋混合，37℃孵育40分钟，依次加入19μLDeacylation Stop Buffer，涡旋混合，室温孵育5分钟，终止去乙酰化反应；2)去除3’-cP与添加5’-P；3)去甲基化处理；4)连接3′接头；5)反转录引物(Reverse TranscriptionPrimer)杂交；6)连接5′接头；7)反转录反应。
制备用于绘制标准曲线的梯度稀释DNA模板，针对每一个需要测量的基因和管家基因，选择并确定表达该基因的cDNA模板进行PCR反应；轻弹管底将溶液混合，5000rpm短暂离心，设置PCR反应：95℃，10min；40个PCR循环(95℃，10秒；60℃，60秒(收集荧光))。PCR产物与100bp DNA Ladder在2％琼脂糖凝胶电泳，溴化乙锭染色，检测PCR产物是否为单一特异性扩增条带。将PCR产物进行10倍梯度稀释：设定PCR产物浓度为1，分别稀释梯度浓度的DNA。
进行Realtime PCR反应：将所有cDNA样品分别配置Realtime PCR反应体系。加样后，将上述384-PCR板置于Realtime PCR仪上进行PCR反应。所有的指标均按以下程序进行：95℃，10min；40个PCR循环(95℃，10秒；60℃，60秒(收集荧光))。为了建立PCR产物的熔解曲线，扩增反应结束后，按(95℃，10秒；60℃，60秒；95℃，15秒)；并从60℃缓慢加热到95℃(仪器自动进行-Ramp Rate为0.075℃/秒)。各样品的目的基因和管家基因分别进行RealtimePCR反应。根据绘制的梯度稀释DNA标准曲线，各样品目的基因和管家基因的浓度结果直接由机器生成。每个样品的目的基因浓度除以其管家基因的浓度，即为此样品此基因的校正后的相对含量。U6、tRF-Gly-CCC-019和tRF-Trp-TCA-001的标准曲线见附图8。
实时定量PCR使用引物列表：
见附图9，RT-qPCR验证结果为tRF-Trp-TCA-001和tRF-Gly-CCC-019表达下降，且上述RT-qPCR实验结果进一步验证了tRF-Trp-TCA-001和tRF-Gly-CCC-019在各实验组中的表达量下降。
附图7为本发明的实施例的实验步骤图。
上述结果说明tRF-Trp-TCA-001和tRF-Gly-CCC-019可作为靶点在紫外线辐射致皮肤损伤中起调控作用，tRF-Trp-TCA-001的序列为AAGTTTAACTTCTGCCA，tRF-Gly-CCC-019的序列为GCATTGGTGGTTCAGT，具体见SEQ ID NO：1和SEQ ID NO：2。因此上述tRF-Trp-TCA-001和tRF-Gly-CCC-019可应用于制备治疗或检测UV辐射致皮肤急性损伤的产品中，所述产品可以是检测tRF-Trp-TCA-001和tRF-Gly-CCC-019的表达量的试剂盒，所述试剂盒可以是生物标记诊断试剂盒，其中的生物标记为tRF-Trp-TCA-001和tRF-Gly-CCC-019。所述产品也可以是治疗用药物，所述药物的作用靶点为tRF-Trp-TCA-001和tRF-Gly-CCC-019，所述药物的作用机制可以为：促进tRF-Trp-TCA-001和tRF-Gly-CCC-019调控Rac1基因，从而促进Rac1基因调节UV辐射导致的DNA损伤反应，从而保护皮肤免受紫外线诱导的角化细胞凋亡和皮肤癌变。靶点tRF-Trp-TCA-001和tRF-Gly-CCC-019在预防紫外线辐射致损伤的产品中也具有一定的应用价值，tRF-Trp-TCA-001和tRF-Gly-CCC-019在紫外线辐射致损伤的产品中的应用价值并不仅限于本实施例中所述的范围。
如上即为本发明的实施例。上述实施例以及实施例中的具体参数仅是为了清楚表述发明验证过程，并非用以限制本发明的专利保护范围，本发明的专利保护范围仍然以其权利要求书为准，凡是运用本发明的说明书及附图内容所作的等同结构变化，同理均应包含在本发明的保护范围内。
序列表
<110> 中南大学湘雅三医院
<120> tRFs在UV辐射致皮肤急性损伤的产品中的应用
<130> 2020
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> aagtttaact tctgcca
<400> 1
aagtttaact tctgcca 17
<210> 2
<211> 16
<212> DNA
<213> GCATTGGTGGTTCAGT
<400> 2
gcattggtgg ttcagt 16
Claims (5)
1.tRFs在制备治疗或检测UV辐射致皮肤急性损伤的产品中的应用，其特征在于，所述tRFs为tRF-Trp-TCA-001和tRF-Gly-CCC-019。
2.根据权利要求1所述的应用，其特征在于，所述产品为检测tRF-Trp-TCA-001和tRF-Gly-CCC-019的表达量的试剂盒。
3.根据权利要求2所述的应用，其特征在于，所述试剂盒为生物标记诊断试剂盒，所述生物标记为tRF-Trp-TCA-001和tRF-Gly-CCC-019。
4.根据权利要求1所述的产品，其特征在于，所述产品为治疗用药物，所述药物的作用靶点为tRF-Trp-TCA-001和tRF-Gly-CCC-019。
5.根据权利要求4所述的产品，其特征在于，所述药物促进tRF-Trp-TCA-001和tRF-Gly-CCC-019调控Rac1基因，从而促进Rac1基因调节UV辐射导致的DNA损伤反应，从而保护皮肤免受紫外线诱导的角化细胞凋亡和皮肤癌变。
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| KATERINA GRAFANAKI: "Translation regulation in skin cancer from a tRNA point of view", 《EPIGENOMICS》 * |
| LINWEN ZHU: "tRNA-derived fragments and tRNA halves: The new players in cancers", 《CANCER LETTERS》 * |
| YUAN FANG: "Differential Expression Profiles and Function Predictions for tRFs & tiRNAs in Skin Injury Induced by Ultraviolet Irradiation", 《FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY》 * |
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