CN113049798A - Pstk在制备用于诊断急性器官损伤的产品中的应用及试剂盒 - Google Patents

Pstk在制备用于诊断急性器官损伤的产品中的应用及试剂盒 Download PDF

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CN113049798A
CN113049798A CN202110254636.9A CN202110254636A CN113049798A CN 113049798 A CN113049798 A CN 113049798A CN 202110254636 A CN202110254636 A CN 202110254636A CN 113049798 A CN113049798 A CN 113049798A
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郑栋
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Zheng Dong
Suzhou University
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Abstract

本发明涉及一种PSTK在制备用于诊断急性器官损伤的产品中的应用及试剂盒,其公开了PSTK的新用途,顺铂能够引起肾脏组织中PSTK蛋白表达含量下降,而在顺铂引起的急性器官损伤中,PSTK蛋白是唯一一个与硒蛋白合成相关的表达下调的蛋白。因此,PSTK对顺铂引发的器官损伤具有诊断作用,从而为临床急性器官损伤的诊断和预后提供了新的指标和途径。

Description

PSTK在制备用于诊断急性器官损伤的产品中的应用及试剂盒
技术领域
本发明涉及一种PSTK在制备用于诊断急性器官损伤的产品中的应用及试剂盒,属于生物医药技术领域。
背景技术
顺铂,是一种含铂的抗癌药物,即顺式-二氯二氨合铂(II),为橙黄色或黄色结晶性粉末,微溶于水、易溶于二甲基甲酰胺,在水溶液中可逐渐转化成反式和水解。临床上对卵巢癌、前列腺癌、睾丸癌、肺癌、鼻咽癌、食道癌、恶性淋巴瘤、头颈部鳞癌、甲状腺癌及成骨肉瘤等多种实体肿瘤均能显示疗效。
但是,单次中、大剂量用药后,偶会出现轻微、可逆的肾功能障碍,可出现微量血尿。多次高剂量和短期内重复用药,会出现不可逆的肾功能障碍,严重时肾小管坏死,导致无尿和尿毒症。然而,目前对顺铂引起的急性氧化性肾损伤没有便捷、精确的诊断方法。
基于上述情况,特提出本申请。
发明内容
本发明的目的在于提供一种PSTK在制备用于诊断急性器官损伤的产品中的应用及试剂盒,能对顺铂引起的急性氧化性肾损伤进行快速诊断。
为达到上述目的,本发明提供如下技术方案:一种PSTK在制备用于诊断顺铂引起的急性器官损伤的试剂盒和药物中的应用。
进一步地,所述急性器官损伤为急性氧化性肾损伤。
进一步地,所述急性器官损伤由顺铂引起。
进一步地,通过检测所述PSTK的表达水平对顺铂引起的急性器官损伤进行诊断。
进一步地,所述产品包括试剂盒和药物。
本发明还提供一种用于诊断急性器官损伤的试剂盒,所述试剂盒包括用于检测PSTK mRNA的探针。
进一步地,所述探针包括Pstk-T和Pstk-B,所述Pstk-T和Pstk-B的序列分别如SEQID NO:1和SEQ ID NO:2所示。
进一步地,所述试剂盒还包含dNTP混合溶液、Mg2+溶液、DNA聚合酶、PCR反应缓冲液、PCR去离子水。
进一步地,所述试剂盒用于诊断由顺铂引起的急性器官损伤。
本发明还提供一种根据所述的试剂盒在急性器官损伤预后情况判断中的应用。
与现有技术相比,本发明的有益效果在于:本申请的PSTK在制备用于诊断急性器官损伤的产品中的应用及试剂盒对血清中PSTK的表达水平进行研究,在顺铂引起的急性氧化性肾损伤中发现顺铂能够引起肾脏组织PSTK蛋白表达含量下降,进而导致体内硒蛋白的含量下降,无法起到减轻氧化应激引起的细胞损伤的作用。故,本申请的PSTK在制备用于诊断急性器官损伤的产品中的应用及试剂盒对顺铂引发的器官损伤具有诊断作用,从而为临床急性器官损伤的诊断和预后提供了新的指标和途径。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1A为实施例一中A、B、C三组小鼠的肾组织的情况示意图;
图1B和图1C为在实施例一中CD3、CD45、CD68在A、B、C三组小鼠的染色情况;
图2A为实施例一中A、B、C三组小鼠的RT-PCR结果示意图;
图2B和图2C为实施例一中A、B、C三组小鼠的免疫印迹图;
图3A为实施例二中顺铂致急性肾损伤人体血清中PSTK的表达水平;
图3B和3C为实施例二中顺铂治疗后的人体足细胞增殖实验结果示意图。
具体实施方式
下面结合附图和实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
需要说明的是:本发明的“上”、“下”、“左”、“右”、“内”、“外”等用语只是参考附图对本发明进行说明,不作为限定用语。
PSTK是一种磷酸丝氨酰转运核糖核酸激酶,发明人通过对单细胞真核生物功能研究发现PSTK在三磷酸腺苷和镁离子的作用下特异性地对丝氨酰磷酸化,使丝氨酰转运核糖核酸磷酸化[seryl-tRNA(Sec)]变成O-磷酸丝氨酸转运核糖核酸,形成硒代半胱氨酸中间转运核糖核酸[selenocysteinyl-tRNA(Sec)]从而合成硒代半胱氨酸蛋白(Sec)。PSTK是唯一依赖于tRNA用大分子为底物的P-环激酶家族的成员。由于PSTK是硒代半胱氨酸合成的必须成分,与硒蛋白或硒酶合成有关,而对PSTK基因敲除线虫进行功能验证,结果发现:PSTK基因敲除线虫生存时间显著缩短。结果表明,通过调控PSTK细胞内硒蛋白水平能够减少细胞内ROS的生成,从而减轻氧化应激引起的细胞损伤。
实施例一
本实施例从上海实验动物中心(中国上海)购买了4-6周龄的雄性C57BL/6小鼠进行微阵列分析。在顺铂暴露试验中,将小鼠分为3组(n=10),A组:正常对照组;B组:采用单次腹腔注射顺铂20mg/kg的方法,每小时一次,连续给药6h,C组:采用单次腹腔注射顺铂20mg/kg的方法,每小时一次,连续给药24h;小鼠平均体重21g。按照中华人民共和国国家科学委员会制定的指导方针饲养和护理所有啮齿动物,在22℃和55%相对湿度的12小时昼夜循环环境下培育至少1周,并在该过程中充分得到营养补充。
在样本量较小的情况下,本实施例利用基于RVM的F检测法筛选对照组和实验组中差异表达的基因。通过p值阈值和FDR分析确定差异表达基因,显著基因的阈值取p<0.05和FDR<0.05。
与对照组相比,顺铂给药后6h和24h组小鼠均出现肾部炎症。接受顺铂的啮齿动物表现为肾结构塌陷,间质水肿。这些发现与组织病理学评分相对应(分别p<0.01和p<0.05)(图1A)。同时,通过CD3、CD45和CD68染色,单核细胞的显著浸润进一步证实了肾损伤(图1B和1C)。
本实施例采用实时RT-PCR检测PSTK在肺组织中的表达变化。与对照组相比,在6小时和24小时,PSTK的表达(mRNA)在统计学上显著降低(p<0.05)。24小时和6小时组之间存在差异(p<0.05)(图2A)。免疫印迹法测定肾组织PSTK蛋白水平。6小时组和24小时组之间存在差异(p<0.05),与正常组相比,6小时组和24小时组的PSTK蛋白再次显著降低(p<0.01)(图2B和图2C)。
本实施例在检测PSTK表达水平时采用本申请的试剂盒,主要包括Pstk-T和Pstk-B探针,其序列分别如SEQ ID NO:1和SEQ ID NO:2所示。
实施例二
本实施例研究了顺铂致急性肾损伤人体血清中PSTK的表达水平和顺铂治疗后人体足细胞中的细胞增殖活性情况。如图A所示,在顺铂导致的急性肾损伤人体血清中,PSTK表达水平有显著降低。如图3B和C所示,足细胞的细胞增殖活性在PSTK过度表达后显著增加。相反,PSTK基因敲除后,细胞增殖显著减少。因此,PSTK对顺铂诱导的足细胞损伤具有明显的预防作用。
综上所述:本申请的PSTK在制备用于诊断急性器官损伤的产品中的应用及试剂盒对血清中PSTK的表达水平进行研究,在顺铂引起的急性氧化性肾损伤中发现顺铂能够引起肾脏组织PSTK蛋白表达含量下降,进而导致体内硒蛋白的含量下降,无法起到减轻氧化应激引起的细胞损伤的作用。故,本申请的PSTK在制备用于诊断急性器官损伤的产品中的应用及试剂盒对顺铂引发的器官损伤具有诊断作用,从而为临床急性器官损伤的诊断和预后提供了新的指标和途径。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Figure BDA0002967554580000061
序列表
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<212> DNA
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aattcaaaaa agctcatgga gagaaagata gatctcttga atctatcttt ctctccatga 60
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Claims (10)

1.一种PSTK在制备用于诊断急性器官损伤的产品中的应用。
2.如权利要求1所述的应用,其特征在于,所述急性器官损伤为急性氧化性肾损伤。
3.如权利要求2所述的应用,其特征在于,所述急性器官损伤由顺铂引起。
4.如权利要求3所述的应用,其特征在于,通过检测所述PSTK的表达水平对顺铂引起的急性器官损伤进行诊断。
5.如权利要求1所述的应用,其特征在于,所述产品包括试剂盒和药物。
6.一种用于诊断急性器官损伤的试剂盒,其特征在于,所述试剂盒包括用于检测PSTKmRNA的探针。
7.如权利要求6所述的试剂盒,其特征在于,所述探针包括Pstk-T和Pstk-B,所述Pstk-T和Pstk-B的序列分别如SEQ ID NO:1和SEQ ID NO:2所示。
8.如权利要求6所述的试剂盒,其特征在于,所述试剂盒还包含dNTP混合溶液、Mg2+溶液、DNA聚合酶、PCR反应缓冲液、PCR去离子水。
9.如权利要求6所述的试剂盒,其特征在于,所述试剂盒用于诊断由顺铂引起的急性器官损伤。
10.根据权利要求6至8中任一项所述的试剂盒在急性器官损伤预后情况判断中的应用。
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CN109453380A (zh) * 2018-09-29 2019-03-12 浙江大学 optineurin蛋白在制备诊断和治疗急性肝损伤产品中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6641807B1 (en) * 1998-08-13 2003-11-04 Merck & Co., Inc. Adenoviral vectors encoding erythropoietin and their use in gene therapy
WO2014205555A1 (en) * 2013-06-28 2014-12-31 British Columbia Cancer Agency Branch Methods and uses for diagnosis and treatment of prostate cancer
WO2016073179A2 (en) * 2014-10-23 2016-05-12 The Trustees Of The University Of Pennsylvania Novel chronotherapy based on circadian rhythms
CN109453380A (zh) * 2018-09-29 2019-03-12 浙江大学 optineurin蛋白在制备诊断和治疗急性肝损伤产品中的应用

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