CN113913436A - 逆转录转座基因l1-atp8b1及其作为肺鳞癌标志物的用途和治疗肺鳞癌的药物 - Google Patents
逆转录转座基因l1-atp8b1及其作为肺鳞癌标志物的用途和治疗肺鳞癌的药物 Download PDFInfo
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Abstract
本发明公开了一种逆转录转座基因L1‑ATP8B1及其作为肺鳞癌标志物的用途和治疗肺鳞癌的药物。所述逆转录转座基因L1‑ATP8B1的核酸序列为SEQ ID NO.1。逆转录转座基因L1‑ATP8B1的上、下游检测引物核酸序列如SEQ ID NO.2和SEQ ID NO.3所示。在小鼠模型中,本申请将N‑乙酰半胱氨酸与逆转录转座抑制剂奈韦拉平联合应用,可有效抑制肿瘤的生长。本发明逆转录转座基因L1‑ATP8B1可以作为一个新的肿瘤标志物,对L1‑ATP8B1的检测可用以进行肺鳞癌的早期诊断、分子分型以及预后评估,同时L1‑ATP8B1还可成为一个潜在的治疗靶点,应用于肺鳞癌的临床治疗。
Description
技术领域
本发明涉及生物技术领域,特别涉及一种逆转录转座基因L1-ATP8B1及其作为肺鳞癌标志物的用途和治疗肺鳞癌的药物。
背景技术
非小细胞肺癌约占所有肺癌的85%,在全球范围内发病率及病死率均居前列,5年生存率仅为19%。非小细胞肺癌主要包括肺腺癌,肺鳞癌和大细胞肺癌。肺鳞癌作为非小细胞肺癌的第二常见病理类型,具有进展快,预后差,化疗敏感性低等特点。相比肺腺癌,肺鳞癌基因变异虽多,却缺乏可靶向的变异位点,且容易发生免疫逃逸,故肺鳞癌患者从靶向治疗和免疫治疗单药治疗中获益有限,亟待寻找新的治疗手段。
长散布元件-1(long interspersed element-1,LINE-1)是人类基因组中唯一具有自主转座能力的逆转录转座子,约占基因组总量的17%。近年来,LINE-1插入形成的逆转录转座基因的促肿瘤作用一直是癌症研究的热点。全基因组泛癌分析表明,LINE-1逆转录转座基因可通过促进染色体上着丝粒和端粒丢失,导致抑癌基因的缺失;或启动“断裂-融合-桥接”循环触发癌基因扩增,引起复杂的基因易位和大规模基因重排,影响基因组稳定性,发挥致癌作用。已证实逆转录转座基因L1-APC,L1-PTEN,L1-MYC,L1-BRCA1与结直肠癌、卵巢癌、乳腺癌和肝癌的发生密切相关,但对于非小细胞肺癌而言,虽然已证实LINE-1插入是肺鳞癌的不良预后因素,但具体哪些LINE-1逆转录转座基因参与肺鳞癌的发生和发展过程,目前尚不清楚。
发明内容
本发明为了解决上述技术问题,提供了一种逆转录转座基因L1-ATP8B1及其作为肺鳞癌标志物的用途和治疗肺鳞癌的药物。
本发明是通过以下技术方案得以实现的。
一种逆转录转座基因L1-ATP8B1,所述逆转录转座基因L1-ATP8B1的核酸序列如SEQ ID NO.1所示。
一种上述逆转录转座基因L1-ATP8B1表达水平的检测试剂在制备诊断肺鳞癌产品中应用。
进一步的,所述检测试剂为逆转录转座基因L1-ATP8B1的上游检测引物和下游检测引物,所述上游检测引物的核酸序列如SEQ ID NO.2所示,所述下游检测引物的核酸序列如SEQ ID NO.3所示。
一种上述逆转录转座基因L1-ATP8B1表达水平的抑制剂在制备治疗肺鳞癌产品中应用。
进一步的,所述抑制剂选自逆转录转座抑制剂和抗氧化剂中的一种或两种。
进一步的,所述逆转录转座抑制剂为奈韦拉平;所述抗氧化剂为N-乙酰半胱氨酸。
一种上述逆转录转座基因L1-ATP8B1的检测引物,所述检测引物包括上游检测引物和下游检测引物,所述上游检测引物的核酸序列如SEQ ID NO.2所示,所述下游检测引物的核酸序列如SEQ ID NO.3所示。
一种治疗肺鳞癌的药物,所述药物以逆转录转座基因L1-ATP8B1为治疗靶点,所述逆转录转座基因L1-ATP8B1的核酸序列如SEQ ID NO.1所示。
进一步的,所述药物选自逆转录转座抑制剂和抗氧化剂中的一种或两种。
进一步的,所述逆转录转座抑制剂为奈韦拉平;所述抗氧化剂为N-乙酰半胱氨酸。
本申请具有以下有益效果。
本发明L1-ATP8B1的高表达能够增加肺鳞癌细胞增殖和侵袭的能力,伴随有细胞内活性氧升高,促进小鼠肺鳞癌的发生发展。同时,本申请利用抗氧化剂联合逆转录转座抑制剂,以逆转录转座基因L1-ATP8B1为靶点对肺鳞癌进行治疗,获得了很好的治疗效果。本发明的逆转录转座基因L1-ATP8B1可以作为一个新的肿瘤标志物,对L1-ATP8B1的检测可以用以进行肺鳞癌的早期诊断、分子分型,以及预后评估,同时L1-ATP8B1还可能成为一个潜在的治疗靶点,应用于肺鳞癌的临床治疗。
附图说明
图1是本发明定量检测逆转录转座基因L1-ATP8B1在肺鳞癌和癌旁组织中的表达图;
图2是本发明在细胞水平上验证逆转录转座基因L1-ATP8B1的存在及其与肺鳞癌发生的相关性图(其中,A为逆转录转座基因L1-ATP8B1在不同细胞中表达的琼脂糖电泳图;B为逆转录转座基因L1-ATP8B1在不同细胞中的相对表达量对比图;C为逆转录转座基因L1-ATP8B1的测序结果图);
图3是本发明逆转录转座基因L1-ATP8B1的表达量与患者总生存以及TNM分期之间的关系图(其中,A为逆转录转座基因L1-ATP8B1与患者总生存的关系图;B为逆转录转座基因L1-ATP8B1与TNM分期的关系图);
图4是本发明逆转录转座基因L1-ATP8B1的表达量与肺鳞癌细胞增殖能力、侵袭潜能、凋亡数量的关系,以及奈韦拉平对L1-ATP8B1表达水平、肺鳞癌细胞的增殖能力,细胞侵袭数量的影响图(其中,A为不同细胞以及奈韦拉平处理前后L1-ATP8B1表达量的对比图;B为不同细胞以及奈韦拉平处理前后细胞增殖能力对比图;C为不同细胞以及奈韦拉平处理前后细胞凋亡数量对比图;D为不同细胞以及奈韦拉平处理前后细胞迁移能力对比图;E为不同细胞以及奈韦拉平处理前后细胞侵袭能力对比图);
图5是本发明逆转录转座基因L1-ATP8B1促进细胞产生高水平活性氧,以及N-乙酰半胱氨酸对肺鳞癌细胞的增殖、侵袭能力的影响图(其中,A为不同细胞以及奈韦拉平处理前后活性氧含量对比图;B为不同细胞以及N-乙酰半胱氨酸处理前后细胞增殖能力对比图;C为不同细胞以及N-乙酰半胱氨酸处理前后细胞凋亡数量对比图;D为不同细胞以及N-乙酰半胱氨酸处理前后细胞迁移能力对比图;E为不同细胞以及N-乙酰半胱氨酸处理前后细胞侵袭能力对比图);
图6是本发明小鼠体内逆转录转座基因L1-ATP8B1的表达对肺鳞癌发生发展的影响,以及奈韦拉平单独治疗和与不同浓度N-乙酰半胱氨酸联合治疗的抗肿瘤效果图(其中,A为对照组和不同处理组肿瘤大小实物图;B为对照组和不同处理组肿瘤大小对比图;C为对照组和处理组小鼠体重对比图)。
具体实施方式
下面结合附图和实施例对本发明进行进一步的说明。
一、核酸序列
逆转录转座基因L1-ATP8B1序列(SEQ ID NO.1):
GTTGCCGCCTTGCAGTTTGATCTCAGACTGCTGTGCTAGCAATCAGCGAGATTCCGTGGGCGTAGGACCCTCTGAGCCAGGTGTGGGATATAGTCTCGTGGTGCGCCGTTTCTTAAGCCGGTCTGAAAAGCGCAATATTCGGATGGGAGTGACCCGATTTTCCAGAATGTACATGGCAAGTCAAAGCAAACGATCGCAAGTACCACGAACAACCTCACTTTATGAACACAAAATTCTTGTGTATTAAGGAGAGTAAATATGCGAATAATGCAATTAAAACATACAAGTACAACGCATTTACCTTTATACCAATGAATCTGTTTGAGCAGTTTAAGAGAGCAGCCAATTTATATTTCCTGGCTCTTCTTATCTTACAGGCAGTTCCTCAAATCTCTACCCTGGCTTGGTACACCACACTAGTGCCCCTG
二、实验方法
1.临床样本收集
本发明共收集来自于肺部肿瘤科就诊并接受部分肺切除手术治疗的肺鳞癌患者样本190例,其中包括153名男性和37名女性,中位年龄为61岁(35-84岁)。这些患者都被确诊为肺鳞癌,临床分期为I期76例,II期37例,III期50例,和IV期27例。在进行肺切除手术前,未进行包括化疗或放疗在内的治疗。术后随访时间67-96个月。
2.PCR检测
(1)Trizol法提取总RNA
①收集的组织样本,加入适量的Trizol,吹打后移入无酶Eppendorf管中。确保细胞完全裂解,液体基本澄清;
②将上述Eppendorf管室温静置5min,加入氯仿(200μl/1ml Trizol),上下颠倒混匀,室温静置10min,4℃,12000g,离心15min;
③小心吸取上层水相置于另一个新的无酶Eppendorf管中,加入等体积异丙醇,上下颠倒混匀,室温静置10min,4℃,12000g,离心10min。弃上清;
④用75%乙醇(1ml/1mlTrizol)洗沉淀,4℃,7500g,离心5min,弃上清,室温静置数分钟,使沉淀自然干燥。加入适量DEPC处理的DDW使其溶解,-80℃保存备用;
⑤紫外分光光度法测定RNA的浓度和纯度;1%琼脂糖凝胶电泳检测RNA的完整性。
(2)反转录实验(20μl体系)
①配制如下反应体系,70℃,5min,然后立即置于冰上。体系如下:
Oligo-(dT)15primer(500μg/ml) 1μl
total RNA sample 1μg
Sterile water 补足到10μl
②在反应体系中加入以下试剂,置于PCR仪中,42℃,1h。
(3)普通PCR反应
PCR扩增反应体系:
上游检测引物:5’-CGC CTT GCA GTT TGA TCT CA-3’,下游检测引物:5’-CGTGGTACTTGCGATCGTTT-3’。
扩增条件:预变性94℃,5min,变性94℃,30s,退火54℃,30s,延伸72℃,30s,35个循环,72℃延伸7min。根据预测的序列,通过Primer Premier 5.0设计检测逆转录转座基因的引物,并由Santa Cruz公司合成。用β-actin做为内参。PCR产物进行1.5%琼脂糖电泳。为了确认在PCR反应中观察和检测的条带确实是最初预测的基因,本发明纯化了PCR产物并对其进行测序验证。
(4)实时荧光定量PCR反应
反应体系(20μl)如下:
反应条件:95℃,30sec;95℃,5sec;64℃,34sec;重复40个循环。每个反应设3个复孔。ABI PRISM 7500仪器自动生成CT值,△CT=CT目的基因-CTactin,mRNA的相对表达量为2-△CT。
表1
(5)高通量定量PCR
收集所有肺鳞癌样本,并提取总RNA,然后反转录成cDNA。然后,将纯化的cDNA送至上海微分基因技术有限公司,利用Smartchip芯片进行高通量定量PCR检测。
3.构建高表达L1-ATP8B1的细胞系
纯化PCR扩增得到的条带,作为模板,并重新设计带有酶切位点序列的引物(以BamHI和EcoRI为酶切位点设计克隆引物。上游引物:
5’-CGCGGATCCGTTGCCGCCTTGCAGTTTGAT-3’;下游引物:5’-CCGGAATTCTTACAGGGGCACTAGTGTGGTGT-3’),进行二次PCR。将得到的PCR产物与克隆载体(带有GFP-Puro标签的克隆载体)使用相同的内切酶进行酶切反应。酶切产物经过纯化后,使用T4连接酶进行连接。连接产物转化进DH5α感受态细胞中进行扩增,在平板上挑取克隆,筛选鉴定阳性克隆。将构建好的阳性核心质粒送至上海汉恒公司进行慢病毒包装。
将H520细胞铺于6孔板中,每孔约3×105细胞,铺板时细胞的融合率为50%左右,培养24小时。将冻存在-80℃的病毒需要先在冰上融化后使用,备用。感染目的细胞:准备好病毒后,取出6孔板,观察细胞状态,细胞的融合率为70%,取其中1孔细胞用于细胞计数,计数结果为每孔内含5×105细胞。选生长状态最好的2个孔,吸取其内培养液。取慢病毒100μl,用完全培养基稀释10倍(MOI=30),分别加入这2孔中,每孔再加入ploybrene 8μg,轻摇混匀,培养24小时后换液。48小时后,将培液完全更换为加有2μg/ml的puromycin的培液,约2天更换一次培液,待细胞生长稳定之后,可以进行细胞传代,2代之后无需加puromycin培养,建系完成。细胞进行验证后用于下一步实验或-80℃保存。
4.细胞培养与处理
细胞于含有10%FBS和1%青霉素/链霉素的RPMI1640培养基中,在37℃、5%CO2条件下培养。对于逆转录转座抑制剂处理实验,将奈韦拉平溶解在二甲基亚砜(DMSO)中,制成储液,工作浓度为450μM。对于抗氧化剂处理实验,N-乙酰半胱氨酸溶解在PBS中,制成储液,工作浓度为5mM。
5.细胞增殖
分别取生长状态良好的对数生长期的细胞,每孔按4×103个接种至96孔板中,每组设置3个复孔,置于细胞培养箱中培养,待细胞贴壁后,弃上层培养基,每孔加入100μl新鲜配制的含10μl增殖检测液CCK-8的培养液,置于培养箱中继续培养2h后,用酶标仪测波长为450nm的OD值。实验重复3次,取实验结果的平均值作为最终实验结果。按公式计算生长抑制率=[(对照组OD-实验组OD)/对照组OD]×100%,以分组为横坐标,生长抑制率为纵坐标,绘制细胞生长抑制率柱状图。
6.细胞凋亡
使用Annexin-V-FITC检测所构建的不同细胞的细胞凋亡。使用Annexin-V-FITC凋亡检测试剂盒来测量细胞凋亡。收集细胞后,用PBS洗涤细胞,并以1×106个细胞/ml的浓度重悬于结合缓冲液中。随后,将5μl Annexin-V和10μl PI加入到100μl细胞悬液中,并将混合物在黑暗中温育15分钟,使用流式细胞仪进行分析。实验至少重复3次。
7.细胞迁移
将所构建的不同细胞接种于6孔板中,24h后达到80-90%的密度。在单层细胞上用10μl移液枪头以直线划线以形成“划痕”。用PBS除去细胞碎片,并用新鲜培养基培养划完线的细胞。在0小时和划线48小时后拍照,以测量划痕的距离。细胞迁移率=(0小时划痕距离-48小时划痕距离)/0小时划痕距离×100%。实验至少重复3次。
8.细胞侵袭
使用Matrigel胶和Trans-well板检测所构建的不同细胞的侵袭能力。将细胞以1×105个细胞的密度接种在Matrigel和100μl无血清RPMI-1640中,接种到具有8μm孔径聚碳酸酯滤膜的24孔板Trans-well系统的小室中,下室是含有10%FBS的培养基。细胞孵育48小时后,将膜下表面的细胞用甲醇固定,并用1%甲苯胺蓝染色。通过显微镜拍摄染色的膜并计数发生侵袭的细胞。实验至少重复3次。
9.细胞内活性氧的测定
使用活性氧检测试剂盒测定细胞内活性氧。收集细胞,用PBS洗涤,与DCFH-DA在37℃孵育30分钟,然后用无血清培养基洗涤3次。流式细胞仪检测1×107/mL细胞在488nm激发波长和525nm发射波长下的DCF荧光。
10.动物实验
将处于对数生长期的不同细胞按5×105细胞/100μl/只,皮下接种NOD/SCID小鼠(6-8周龄,18-22g,每组5只)腹股沟部位,构建不同动物模型。每3天观察肿瘤生长情况,连续观察4-6周,比较对照组和实验组小鼠的成瘤率和成瘤时间,成瘤后每天测量肿瘤长短径,按照公式:肿瘤体积=πab2/6计算肿瘤大小(a为长径,b位短径),绘制小鼠肿瘤生长曲线。对于药物治疗实验,动物每天接受奈韦拉平(100mg/kg/天)单独治疗,或与不同浓度N-乙酰半胱氨酸(分别为75mg/kg/天,150mg/kg/天和300mg/kg/天)联合治疗。
三、实验结果
1.在前期研究中,本申请利用deFuse工具发现TCGA数据库中90%肺鳞癌样本存在LINE-1插入。通过整合公共大数据和本地临床样本资料,筛选出13种肺鳞癌相关高频LINE-1逆转录转座基因,其中L1-ATP8B1不仅在肺鳞癌中高表达,还与患者预后显著相关,提示高致病性。进一步的,本发明收取了52对肺鳞癌组织及其癌旁正常组织作为对照,抽提RNA后进行反转录,合成的cDNA通过定量qPCR,定量检测逆转录转座基因L1-ATP8B1在肺鳞癌和癌旁组织中的表达。实验结果参见图1,结果显示,逆转录转座基因L1-ATP8B1在肺鳞癌中的表达显著高于其癌旁对照组织(PCR扩增后获得长度为180bp的片段即为目的基因),提示逆转录转座基因L1-ATP8B1参与调控肺鳞癌的发生。
2.本发明又选取肺癌细胞系(肺腺癌细胞系A549、H1299、肺鳞癌细胞系H520、大细胞肺癌细胞系H460、小细胞肺癌细胞系H446),以及正常细胞系(人正常肺上皮细胞系BEAS-2B和人上皮细胞系HEK293T)作为对照,利用RT-PCR和定量real-time qPCR分别检测逆转录转座基因L1-ATP8B1在肺癌细胞系和正常细胞系中的表达。结果显示,逆转录转座基因L1-ATP8B1主要在H520细胞中呈现出高表达(图2A-B)。在其他肺癌细胞系,包括2个正常细胞系中的表达几乎未检测到,这提示逆转录转座基因L1-ATP8B1的表达与肺鳞癌的发生有关。为了验证在PCR实验中观察的条带确实是最初所预测的基因序列,本发明纯化了PCR产物并对其进行测序。结果显示,逆转录转座基因L1-ATP8B1的测序结果与预测的基因序列完全匹配(图2C)。这些结果在细胞水平上验证了逆转录转座基因L1-ATP8B1的存在,并表明其存在与肺鳞癌的发生相关。
3.本发明还进行了逆转录转座基因L1-ATP8B1的表达与肺鳞癌患者性别、年龄、生存时间、TNM分期,和吸烟指数等临床病理指标相关性的研究。本发明收集了190例肺鳞癌标本,检测了其逆转录转座基因L1-ATP8B1的表达。根据高通量定量qPCR的结果,依据逆转录转座基因L1-ATP8B1表达量的中位数(2-ΔCt值大于中位数的样本被定义为高表达,2-ΔCt的值小于中位数的样本被定义为低表达),将样本分成2组,分析其表达量与患者总生存(OS)之间的关系。结果显示,逆转录转座基因L1-ATP8B1表达低的患者生存率较高,而L1-ATP8B1表达高的患者生存率较低(图3A),显示出了显著性的差异。其他临床参数的分析结果显示,逆转录转座基因L1-ATP8B1的表达量与患者TNM分期显著相关,逆转录转座基因L1-ATP8B1表达低的患者肿瘤体积较小,不容易发生淋巴转移和远处转移,而L1-ATP8B1表达高的患者肿瘤体积较大,容易发生淋巴转移和远处转移(p<0.05,图3B)。此外,L1-ATP8B1表达高的患者中吸烟人数显著多于L1-ATP8B1表达低的患者(p<0.05)。而L1-ATP8B1的表达量与性别、年龄等临床指标无明显相关性(p>0.05)。以上数据表明,逆转录转座基因L1-ATP8B1可能是肺鳞癌的一个潜在肿瘤标志物,与肺鳞癌的发生和发展相关,并影响肺鳞癌病人预后。
4.为了进一步研究逆转录转座基因L1-ATP8B1对于肺鳞癌发生发展的影响,本发明构建了高表达L1-ATP8B1的H520细胞(H520OV-L1-ATP8B1),通过定量PCR检测H520OV-L1-ATP8B1细胞中L1-ATP8B1的表达明显升高(图4A),细胞增殖能力也明显增强(图4B)。并进行了细胞功能的检测,使用Annexin-V-FITC凋亡检测试剂盒来测量细胞凋亡,利用划痕实验检测细胞迁移能力,以及用Matrigel和Trans-well实验检测细胞侵袭能力。结果表明,逆转录转座基因L1-ATP8B1的高表达使肺鳞癌细胞凋亡数量减少(图4C),划痕修复能力增强(图4D),侵袭潜能提高(图4E)。
本发明又在细胞水平上,以逆转录转座基因L1-ATP8B1为治疗靶点,应用逆转录转座抑制剂奈韦拉平进行处理,初步观察该方法的治疗效果。结果表明,加入药物后,明显抑制了逆转录转座基因L1-ATP8B1的表达水平(图4A),并且降低了肺鳞癌细胞的增殖能力(图4B),细胞凋亡数量增加(图4C),并降低了细胞的迁移能力(图4D)和侵袭能力(图4E)。
5.研究表明,肿瘤细胞中产生的活性氧通常高于正常细胞,高水平的活性氧可以激活许多促癌通路,引起肿瘤的发生和转移。本发明进一步检测了逆转录转座基因L1-ATP8B1对活性氧的影响。活性氧检测结果显示,高表达逆转录转座基因L1-ATP8B1的H520细胞中活性氧含量显著高于对照细胞,且高水平的活性氧被逆转录转座抑制剂奈韦拉平所抑制(图5A)。本发明又在细胞水平上,基于逆转录转座基因L1-ATP8B1产生高水平活性氧,应用抗氧化剂N-乙酰半胱氨酸进行处理,初步观察该方法的治疗效果。结果表明,加入N-乙酰半胱氨酸后,明显降低了肺鳞癌细胞的增殖能力(图5B),细胞凋亡数量增加(图5C),并降低了细胞的迁移能力(图5D)和侵袭能力(图5E)。
6.为了在体内验证逆转录转座基因L1-ATP8B1对于肺鳞癌发生发展的影响,本发明将处于对数生长期的对照肺鳞癌细胞系H520,以及构建的高表达L1-ATP8B1基因的H520细胞(H520OV-NC和H520OV-L1-ATP8B1),皮下接种NOD/SCID小鼠(6-8周龄,18-22g)腹股沟部位,构建不同动物模型。动物模型构建成功后,每3天观察肿瘤生长情况,连续观察4-6周,成瘤后每天称量小鼠体重,并测量肿瘤长短径,按照公式计算肿瘤大小,绘制小鼠肿瘤生长曲线和小鼠生存曲线。通过比较对照组和实验组小鼠的体重、成瘤率、成瘤时间和成瘤大小,发现接种H520OV-L1-ATP8B1细胞的小鼠与对照小鼠的体重没有明显差异,但接种H520OV-L1-ATP8B1细胞的小鼠成瘤时间较对照组短,成瘤体积也显著大于对照组(图6)。本申请进一步地检测奈韦拉平和N-乙酰半胱氨酸的治疗效果,发现单独注射奈韦拉平的治疗组小鼠成瘤体积减小,而奈韦拉平和N-乙酰半胱氨酸两药联合的治疗效果更加显著。通过将奈韦拉平与不同浓度的N-乙酰半胱氨酸(75mg/kg/天,150mg/kg/天和300mg/kg/天)联合治疗,发现奈韦拉平与150mg/kg/天、300mg/kg/天浓度的N-乙酰半胱氨酸联合的治疗效果优于与75mg/kg/天浓度的N-乙酰半胱氨酸联合,但奈韦拉平与300mg/kg/天N-乙酰半胱氨酸联合的毒性强,引起小鼠体重急剧下降。综合考虑治疗效果与药物毒性,本申请确定的最优配比为奈韦拉平(100mg/kg/天)与150mg/kg/天N-乙酰半胱氨酸联合用药(图6)。
本具体实施方式的实施例均为本发明的较佳实施例,并非依此限制本发明的保护范围,故:凡依本发明的结构、形状、原理所做的等效变化,均应涵盖于本发明的保护范围之内。
序列表
<110> 天津市肿瘤医院(天津医科大学肿瘤医院)
<120> 逆转录转座基因L1-ATP8B1及其作为肺鳞癌标志物的用途和治疗肺鳞癌的药物
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Claims (10)
1.一种逆转录转座基因L1-ATP8B1,其特征在于:所述逆转录转座基因L1-ATP8B1的核酸序列如SEQ ID NO.1所示。
2.一种权利要求1所述逆转录转座基因L1-ATP8B1表达水平的检测试剂在制备诊断肺鳞癌产品中应用。
3.根据权利要求2所述的应用,其特征在于:所述检测试剂为逆转录转座基因L1-ATP8B1的上游检测引物和下游检测引物,所述上游检测引物的核酸序列如SEQID NO.2所示,所述下游检测引物的核酸序列如SEQ ID NO.3所示。
4.一种权利要求1所述逆转录转座基因L1-ATP8B1表达水平的抑制剂在制备治疗肺鳞癌产品中应用。
5.根据权利要求4所述的应用,其特征在于:所述抑制剂选自逆转录转座抑制剂和抗氧化剂中的一种或两种。
6.根据权利要求5所述的应用,其特征在于:所述逆转录转座抑制剂为奈韦拉平;所述抗氧化剂为N-乙酰半胱氨酸。
7.一种权利要求1所述逆转录转座基因L1-ATP8B1的检测引物,其特征在于:所述检测引物包括上游检测引物和下游检测引物,所述上游检测引物的核酸序列如SEQ ID NO.2所示,所述下游检测引物的核酸序列如SEQ ID NO.3所示。
8.一种治疗肺鳞癌的药物,其特征在于:所述药物以逆转录转座基因L1-ATP8B1为治疗靶点,所述逆转录转座基因L1-ATP8B1的核酸序列如SEQ ID NO.1所示。
9.根据权利要求8所述的一种治疗肺鳞癌的药物,其特征在于:所述药物选自逆转录转座抑制剂和抗氧化剂中的一种或两种。
10.根据权利要求9所述的一种治疗肺鳞癌的药物,其特征在于:所述逆转录转座抑制剂为奈韦拉平;所述抗氧化剂为N-乙酰半胱氨酸。
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