CN109022462B - 一种PTEN基因的上游开放阅读框31aa-uORF核苷酸及其编码的多肽的应用 - Google Patents
一种PTEN基因的上游开放阅读框31aa-uORF核苷酸及其编码的多肽的应用 Download PDFInfo
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Abstract
本发明属于生物医药领域，具体涉及PTEN(phosphatase and tensin homolog)基因的上游开放读码框(uORF,up–stream open reading frame)及其编码多肽的应用。发明人发现了PTEN的5’UTR存在一个潜在的96个碱基的开放读码框(31aa‑uORF)，可以编码31个氨基酸的短肽(命名为PTEN‑31aa)，其在肿瘤的发生、发展过程中具有重要作用。本发明为与PTEN表达调控相关的疾病，尤其是神经胶质瘤提供了新的诊断、治疗方法和药物筛选平台。本发明还提供了一种多肽，用于PTEN表达调控相关的疾病治疗。
Description
技术领域
本发明属于生物医药领域，具体涉及PTEN(phosphatase and tensin homolog)基因的上游开放读码框(uORF,up–stream open reading frame)及其编码蛋白的应用。
背景技术
神经胶质瘤(脑胶质瘤)(Glioma)是一类起源于大脑的肿瘤，通常由胶质细胞病变后产生，是人类中枢神经系统中最常见的一类恶性肿瘤。WHO根据肿瘤的临床病理评价又把胶质瘤分为I-IV期，其中I期纤维型星型母细胞瘤病症最轻，而IV期多形性胶质母细胞瘤病症恶性程度最高，这是最常见应用范围最广的分期(Louis et al.,2007)。CBTRUS统计表明恶性胶质瘤在原发性恶性脑肿瘤中占有高达70％的比例，发病率大约为十万分之五。恶性胶质瘤的临床治愈率非常低，5年存活率仅仅高于胰腺癌和肺癌。在美国每年大约有一万个恶性胶质瘤患者接受治疗，其中大约有一半的病人能活过一年，而这些人中大约只有四分之一的能活过两年。近年来随着新的治疗手段的出现患者的存活期有明显延长(Nakazatoet al.,2008)。因此加强对恶性胶质瘤发病机制的研究，全面了解恶性胶质瘤的发病机理，才能最终寻找出疗效更好的新的治疗方案。
恶性胶质瘤的发病机制很复杂，目前尚未明确。但是有研究表明遗传性基因异常如NF(neurofibromatosis，1型和II型)以及TSC(tuberous sclerosis complex)基因突变会促进恶性神经胶质瘤的发展(Reuss et al.,2009)。当然很多不同的癌基因的异常表达也跟恶性胶质瘤的发展有关(Rader et al.,1993)。DNA修复基因：ERCC1，ERCC2以及XRCC1的遗传性的多态性会增加患胶质瘤的风险。也就是说DNA修复机制的改变或者缺失会促进胶质瘤的生成(Adel et al.,2014)。在胶质瘤中最常见的突变发生在异柠檬酸脱氢酶I和II(isocitrate dehydrogenase，IDH1，IDH2)基因上，有高达80％的低危型胶质瘤和中度恶性胶质瘤都在IDH1基因上携带同一个突变(Cohen et al.,2013)。一项对51例脑胶质瘤病人长期取样结果研究表明IDH1基因突变是最早期可以检测到的突变，它发生在P53基因突变和染色体lp/19q联合性缺失之前，所以说IDH1基因突变是突变的最早期助力(Watanabeet al.,2009)。除IDH1和IDH2外，外恶性胶质瘤的发生发展过程中还有很多其它基因突变，例如：1)在其他肿瘤中少见的EGFR(epidermal growth factor receptor)扩增或者重排在脑胶质瘤中却占有很高的比例(Zadeh et al.,2013)；2)在大多数肿瘤中比较常见的P53基因突变在各种类型的胶质瘤中出现的比例都非常高(例如：在低恶性程度的星形胶质瘤肿瘤中出现的比例高达60％)(Zheng et al.,2008)；3)PTEN(phosphatase and tensinhomolog)突变在原发性的胶质母细胞瘤中也达到了约30％(Zheng et al.,2008)；4)1p/19q染色体的杂合缺失(loss of heterozygosity)在不同类型的胶质瘤中出现的概率不同，在少突胶质母细胞瘤中出现的概率高达90％，但是在弥漫性星形细胞瘤中出现的概率却仅为百分之十五(Ducray et al.,2009)。尽管有不少研究都涉及到恶性胶质瘤的病因，但是具体详细的分子机制仍是未知，值得我们深入探究，从而为临床治疗提供更好的指导。
同源性磷酸酶-张力蛋白(Phosphatase and Tensin homolog，PTEN)的编码基因PTEN位于染色体10q23.3，是人类多种肿瘤(如脑癌、乳腺癌、前列腺癌)中最常见的因为缺失或突变而下调的基因之一(Li et al.,1997)。作为一个抑癌基因，PTEN在调控细胞生长、侵袭、凋亡、DNA损伤修复以及肿瘤细胞放化疗抵抗力等方面发挥着重要的作用(Dean etal.,2005；Koul,2008；Mellinghoff et al.,2005；Ming and He,2012；Ortega-Molina andSerrano,2013)。PTEN表达的缺失被认为是胶质瘤发展中的早期事件，而PTEN突变发生在60％的GBM中，是其最常见的基因改变(Bianco et al.,2003；Srividya et al.,2011)。PTEN的主要功能是PI3K/Akt通路的负调控因子。与PI3K作用相反，PTEN通过去磷酸化将PIP3转变为PI-4,5-P2，通过降低Akt的活化而抑制下游所有由Akt调控的信号通路(Trotman et al.,2006)。过往研究显示，在胶质瘤中，PI3K/Akt通路的活化直接影响胶质瘤的恶性程度，并在GBM的发展恶化过程中起关键作用(Rodriguez et al.,2011；Sonodaet al.,2001)。尽管过往研究显示抑癌基因PTEN在胶质瘤中具备重要的调控作用，但具体机制依然不明确，因此发现PTEN在胶质瘤中的所有活性变异体并探索其抑癌机制对于揭示胶质瘤的发生发展过程具有重要的意义。
目前胶质瘤治疗的标准方案是规范化诊疗，以手术治疗为主，手术治疗的原则是在尽可能保护肿瘤周围正常脑组织的原则下尽量最大限度的切除肿瘤，以达到降低患者术后复发和转移的概率、大大提高患者的生存率和生存质量的目的。当然除手术治疗外，结合多学科治疗，尤其是化疗、免疫、中药的等辅助手段对脑胶质瘤的治疗越来越受到关注。目前化疗治疗胶质瘤临床一线药物是烷基类化合物替莫唑胺(Temozolomid，TMZ)，具体原理为在生理PH条件下TMZ经快速非酶催化转变成活性化合物MTIC，MTIC可以将鸟嘌呤的06和N2位点烷基化从而发挥细胞毒性作用。但是患者对TMZ的敏感度取决于MGMT甲基转移酶的表达量，它们之间呈负相关，所以限制TMZ在临床的广泛应用。胶质瘤的免疫治疗方面可以从这几个方面下手：脑胶质瘤免疫抑制(如针对CDLA-4蛋白的抗体可以提高脑胶质瘤模型鼠的存活率，还有很多其他的靶向免疫调节蛋白的抗体正在临床试验中)、脑胶质瘤疫苗(临床试验中，例如ATL-DC疫苗治疗脑胶质瘤患者，32位受试者中有28位表现出免疫应答，大大提高临床治疗效果)。中药治疗胶质瘤目前还停留于整体治疗的阶段，通过调理集体的阴阳、气血、脏腑功能的平衡，从而在整体水平控制胶质瘤的发展与转移、减少复发、提高患者的生存期和生活质量。还有一些中药化合物单体在临床前的细胞实验上也显示了很好的抗胶质瘤活性，中药的质量标准化、安全性和毒性问题不解决，将大大限制其应用于临床治疗。
上游开放读码框(uORF,up-stream open reading frame)是一类位于mRNA 5`非翻译区的开放读码框。uORF可以调控真核生物基因表达(Lovett et al.,1996)。通常来说上游开放读码框的翻译都会抑制其相应母本基因的表达(Vilela et al.,2003)。在细菌中，上游开放读码框也叫作先导肽，是在研究调控参与氨基酸合成或者转移的基因时首次发现的(Lovett et al.,1996)。随着测序精度加深以及5`UTR序列数据库的完善(Pesoleet al.,1999)，通过一些算法我们可以预测到数以万计的uORF，理论上uORF可以出现在任意一个mRNA的上游(McCarthy et al.,1998；Vilela et al.,1998)。当然它们的确认以及具体功能还需大量生物实验验证。通常来说，在各种生物中uORF都主要参与调控一些关键的细胞生物学过程(Vilela et al.,2003)目前发现或者证明的uORF一般出现在一些调控关键细胞学过程基因的上游，参与调控下游的母体mRNA对应基因的表达。例如：酿酒酵母中调控细胞周期的G1细胞周期蛋白CLN3(Polymenis et al.,1997)、人中调控凋亡的BCL2(Harigai et al.,1996)、人中的重要癌基因MDM-2(Brown et al.,1999)、小鼠中莫洛尼氏肉瘤癌基因c-mos(Steel et al.,1997)。这些研究结果表明有功能的uORF通常都参与调控细胞生长凋亡等至关重要的生理过程，不同的uORF在不同的物种或者生理过程会起不同的作用(McCarthy et al.,1998；Vilela et al.,1998；Vilela et al.,1999)。虽然目前在酿酒酵母以及真菌等研究背景较简单的生物中发现有不少的uORF，但是还有更多未知的uORF等待我们去研究发现，随着实验手段和分析方法的进步，我们将会发现越来越多的uORF并确定它们的生理功能，也许它们除了这种顺式调控作用还会有更复杂的调控模式等待我们发现。
最近有两个课题组分别在Cell metabolism和Science上发表文章，都表明在PTEN基因的5`UTR上存在新的转录起始位点，编码PTEN的一个新剪切体，分别命名为PTEN-A和PTEN-long。其中发表在Cell metabolism上的PTEN-A被证明可以参与调控线粒体呼吸作用从而调控细胞代谢；而发表在Science上的PTEN-long可以分泌到细胞外被另外的细胞摄取调控细胞存活促进肿瘤细胞死亡(Liang et al.,2014；Hopkins et al.,2013)。这两个课题组关于PTEN新ORF的的研究为我们提供了新的研究灵感。因为PTEN的5`UTR比较长，有大约1000bp的核苷酸，那么我们就思考PTEN的上游是否还存在别的转录起始位点，更或者存在一个新的小开放读码框呢？查阅文献我们发现Han等人在03年研究发现如果在肿瘤细胞中转入小鼠PTEN的5`UTR，会使细胞周期停滞从而抑制肿瘤细胞生长。Han团队认为这是由于5`UTR区域不同长短而引起启动子活性不一样造成(Han et al.,2013)
发明内容
本发明在人PTEN基因(NCBI号：NM_000314)的5`UTR区域发现一段开放96个碱基的开放读码框，命名为31aa-uORF，可以编码成31个氨基酸的短肽，我们命名为：PTEN-31aa(图1)。该微肽的分子量大约为4-5KD。所述的31aa-uORF具有SEQ ID NO：1所述的核苷酸序列；所述的PTEN-31aa具有SEQ ID NO：2所述的氨基酸序列。
一方面，本发明提供了一种核酸片段31aa-uORF或其编码的多肽PTEN-31aa。
另一方面，本发明提供了PTEN基因的上游开放阅读框31aa-uORF核苷酸序列、或与其编码同等氨基酸序列的核苷酸序列、或其编码的多肽PTEN-31aa在制备治疗或预防肿瘤的药物中的应用。
另一方面，本发明还提供31aa-uORF或其编码的多肽PETN-31aa的检测试剂在制备用于肿瘤诊断和/或预后判断的试剂中的应用；优选地，所述的肿瘤诊断和/或预后判断的试剂包括：用于检测31aa-uORF的探针，或扩增31aa-uORF的引物，或者抗PTTN-31aa蛋白的抗体。
另一方面，本发明还提供了一种用于治疗或预防肿瘤的药物组合物，该药物组合物含有：31aa-uORF核苷酸序列、或与其编码同等氨基酸序列的核苷酸序列、或其编码的多肽PETN-31aa；任选地，所述的药物组合物还可以包括一种或多种药用赋形剂或药用载体，所述的载体如慢病毒等。
另一方面，本发明还提供了一种用于肿瘤诊断和/或预后判断的试剂盒，所述试剂盒含有：用于检测31aa-uORF的探针，或扩增31aa-uORF的引物，或者抗PTTN-31aa蛋白的抗体。
另一方面，本发明还提供了编码PTEN-31aa的表达载体，以及表达该载体的宿主细胞，以及他们用于肿瘤抑制/预防/治疗的用途。
另一方面，本发明还提供了一种通过化学合成，或者重组表达产生多肽，其具有SEQ ID NO：2所示的氨基酸序列。经实验证实，无论是化学合成的多肽或者是重组表达产生的多肽，均具有抑制肿瘤的功能。所述的肿瘤为PTEN调控相关的肿瘤；所述的PTEN调控具体为：PTEN参与PI3K/Akt通路的调控；优选地，所述的肿瘤为胶质瘤；更优选地，为神经胶质瘤。
本发明中，其中所述的肿瘤为PTEN调控相关的肿瘤。
或者，所述的肿瘤为脑癌、乳腺癌、前列腺癌；更优选的，所述的肿瘤为神经胶质瘤。
PTEN基因是人类多种肿瘤(如脑癌、乳腺癌、前列腺癌等)中最常见的因为缺失或突变而下调的基因之一(Li et al.,1997)。作为一个抑癌基因，PTEN在调控细胞生长、侵袭、凋亡、DNA损伤修复以及肿瘤细胞放化疗抵抗力等方面发挥着重要的作用。而上游开放读码框(uORF,up-stream open reading frame)可以调控其下游基因表达。因此，可以推知，本发明中PTEN的uOFR(31aa-uORF)，及其编码的微肽(PTEN-31aa)，在与PTEN调控的相关疾病中，可能发生调控作用，从而发挥抑瘤作用。
其中，所述的PTEN调控具体为：PTEN参与PI3K/Akt通路的负调控。PTEN通过去磷酸化将PIP3转变为PI-4,5-P2，通过降低Akt的活化而抑制下游所有由Akt调控的信号通路(Trotman et al.,2006)。而在胶质瘤中，PI3K/Akt通路的活化直接影响胶质瘤的恶性程度，并在GBM的发展恶化过程中起关键作用(Rodriguez et al.,2011；Sonoda et al.,2001)。因此PTEN被认为是一种重要的通过消极地调节磷脂酰肌醇3-激酶(PI3K)信号通路的肿瘤抑制剂。
一方面，本发明通过制备PTEN-31aa特异性抗体，我们在293和U251细胞内检测识别了该uORF编码的微肽。同时还发现该微肽在正常脑胶质细胞中的表达量远高于脑胶质瘤细胞(图2)。
一方面，本发明根据31aa-uORF碱基序列设计慢病毒过表达载体，包装过表达慢病毒载体并感染U251神经胶质瘤细胞，筛选得到稳定过表达31aa-uORF编码微肽的胶质瘤细胞系，并验证其细胞生物学功能。在U251神经胶质瘤细胞中过表达31aa-uORF编码微肽可以明显抑制胶质瘤细胞U251的生长、增殖以及克隆形成等(图3)。
一方面，人工合成31aa-uORF编码微肽可以进入U251神经胶质瘤细胞并且能够明显抑制U251胶质瘤细胞的生长增殖(图4)。
一方面，本发明通过动物学实验体内验证31aa-uORF编码微肽的抗肿瘤能力。小鼠皮下接种200万U251神经胶质瘤细胞，三十天后用化学合成的31aa-uORF编码微肽(50ug/瘤子)处理移植瘤，隔天给药，发现与对照多肽(将31aa-uORF编码多肽序列随机打乱后合成对照多肽)相比化学合成31aa-uORF编码微肽可以明显抑制U251神经胶质瘤移植瘤的生长和形成，说明31aa-uORF编码微肽在动物水平也显示很好地抗肿瘤活性(图5)。
在本发明中，所述的“多肽”、“微肽”、“短肽”应当理解为具有同样的含义，用来表述氨基酸片段。
除了具有上述SEQ ID NO：2所述的氨基酸序列的多肽，其余现有技术中常规修饰本发明序列的多肽，也应当理解为具有本发明所述的肿瘤抑制作用。如多肽治疗剂可能具有短的循环半衰期和蛋白水解降解以及低溶解度。为了改善本发明的生物药物的药代动力学和药效动力学特性，可以实施诸如操纵氨基酸序列等方法以降低或提高免疫原性和降低蛋白水解裂解；可以将肽融合或偶联到免疫球蛋白和血清蛋白，诸如白蛋白；还可以并入到用于生物药物(诸如本发明的肽)和抗体的药物递送载体中以保护并减慢释放；并且还设想了偶联到天然或合成聚合物。具体地，对于合成聚合物偶联，还设想了聚乙二醇化或酰化，诸如N-酰化、S-酰化、酰胺化等。
如本文中所使用的，“药用赋形剂或药用载体”包括任何和所有的溶剂、分散介质、包衣、抗细菌和抗真菌剂、等渗剂和吸收延迟剂等。将此种介质和试剂用于药物活性物质的用途在本领域中是众所周知的。除非任何常规介质或药剂与活性成分不相容，否则可以预期其在治疗组合物中的使用。补充活性成分也可以并入到该组合物中。将此种介质和试剂用于药物活性物质的用途在本领域中是众所周知的。除非任何常规介质或药剂与活性成分不相容，否则可以预期其在治疗组合物中的使用。补充活性成分也可以并入到本发明所述的药物组合物中。
药物化合物可以以方便的方式给予，例如通过口服、静脉内(在水溶性的情况)、肌内、皮下、腹腔内、经鼻、皮内或栓剂途径或植入(例如，使用缓慢释放分子通过腹膜内途径或通过使用细胞或者慢病毒并继转移到接受者。根据给药途径，可能需要将活性组分包覆在材料中以保护其不受酶、酸和可能失活所述成分的其它天然条件的作用。
本发明的有益效果：
1.本发明首次发现了在PTEN的5’端存在上游小开放读码框(31aa-uORF)，可以编码31个氨基酸的短肽(PTEN-31aa)。制备PTEN-31aa的抗体发现31aa-uORF确实可以在胶质瘤细胞和正常脑胶质细胞中内源表达。同时检测多种胶质瘤样本和正常样本中31aa-uORF的表达量发现，与正常脑组织相比，胶质瘤组织样本中31aa-uORF的表达量明显减少。
2.更另人兴奋的是，无论是在体外实验，还是在活体动物实验身上；无论是重组表达的微肽(PTEN-31aa)，还是人工合成的微肽(PTEN-31aa)，均可以明显抑制胶质瘤细胞的生长和增殖。
3.本发明还提供了一种人工合成或者重组构建载体表达的多肽，该微肽的分子量特别小，可以很容易进入胶质瘤细胞和渗透到肿瘤组织，也能够很好的透过血脑屏障，治疗神经胶质瘤。
4.本发明基于新发现的上游开放读码框(31aa-uORF)，及其编码的微肽(PTTN-31aa)，可以为肿瘤治疗，尤其是神经胶质瘤的临床治疗或预防/提供一种新的多肽药物、新的思路和策略。
附图说明
图1显示了31aa-uORF在PTEN mRNA上位置示意图。
图2显示了31aa-uORF编码微肽的预测及内源表达鉴定；
A：31aa-uORF在PTEN mRNA上定位的示意图；
B：31aa-uORF编码微肽在不同物种中同源性分析；
C：31aa-uORF编码微肽特异性抗体western检测31aa-uORF在细胞内的内源性表达；
D：31aa-uORF编码微肽在正常胶质细胞和不同脑胶质瘤细胞中的表达量。
图3显示了31aa-uORF在胶质瘤细胞U251中作为抑癌多肽起作用；
A：过表达31aa-uORFU251细胞分别流式检测细胞周期；
B：对A的统计柱状图；其中，柱状图从上到下依次表示G2(DNA合成后期)，S(DNA合成期)，G1(DNA合成前期)；
C：检测CCK-8的OD值测生长曲线的变化；
D：平板克隆(CFA)和soft agar(SA)检测细胞的克隆形成能力；
E：对CFA和soft agar的定量。
图4显示了化学合成的31aa-uORF编码多肽能够进入U251细胞并明显抑制胶质瘤细胞生长；
A：31aa-peptide浓度：10μM；加入U251细胞，4小时后PBS洗掉多余多肽，免疫荧光检测多肽进细胞能力；
B：细胞继续培养72小时CCK-8检测细胞的活力。
图5显示了化学合成的31aa-uORF编码多肽可以明显抑制裸鼠U251皮下移植瘤的生长。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
实施例1 31aa-uORF编码微肽的内源表达与鉴定
通过ORF finder(http://www.bioinformatics.org/sms/orf_find.html)在PTENmRNA(NM_000314)第一个内含子区域的5`UTR发现一个96nt的开放读码框(Open ReadingFrame，ORF)，可以编码一个31个氨基酸的短肽，我们命名为：31aa-uORF，其编码的短肽命名为(PTTN-31aa)。通过蛋白质分子量预测软件http://www.bio-soft.net/sms/prot_ mw.html预测蛋白的分子量约为4-5KD。根据31aa-uORF的氨基酸序列设计特异识别该微肽并能用western blot方法检测的多克隆抗体。具体方法如下：通过化学合成多肽的方法，合成31aa-uORF其中一段TRLRSVLSSRKLQP氨基酸序列作为免疫原，注射并免疫新西兰大白兔，然后纯化目的抗体用于检测细胞内的31aa-uORF编码微肽。CRISPR/Cas9在基因组上特异敲除31aa-uORF中间的一段，从基因组水平上沉默31aa-uORF微肽的表达，得到31aa-uORF敲除的293细胞系。用31aa-uORF特异性抗体通过western检测31aa微肽的内源表达及变化。
SEQ ID NO：1
homo-31aa-uORF(96nt)核苷酸序列
ATGTGGCGGGACTCTTTATGCGCTGCGGCAGGATACGCGCTCGGCGCTGGGACGCGACTGCGCTCAGTTCTCTCCTCTCGGAAGCTGCAGCCATGA
SEQ ID NO：2
homo-31aa-uORF编码的氨基酸序列(PTTN-31aa)
MWRDSLCAAAGYALGAGTRLRSVLSSRKLQP
详细方法：
1)CRISPR/Cas9敲除细胞系的建立
根据31aa-uORF的序列设计若干gRNA的序列分别靶向31aa-uORF的不同区域，特异性删除31aa-uORF的一段，获得基因组敲除31aa-uORF的细胞系。
具体的gRNA序列信息如下：
SEQ ID NO：3
31aa-SgRNA1:5’-CAACUCUCAAACUUCCAUCA-3’
SEQ ID NO：4
31aa-SgRNA2:5’-UCAUGGCUGCAGCUUCCGAG-3’
SEQ ID NO：5
31aa-SgRNA3:5’-CUCGGAAGCUGCAGCCAUGA3’
(克隆时加了g在5’-末端，以利于hU6启动子高效启动)
将pX330-Puro-PTEN-SgRNA瞬时转染入HEK293T细胞，测序检测其效率，结果表明31aa-SgRNA2为高效率SgRNA，选择其进行后续实验。用puro霉素筛选转染31aa-SgRNA2的HEK293T细胞，然后挑选阳性克隆，其中的一个克隆细胞，编号为E11号克隆删除了九个碱基(具体序列为：GAGAGGAG)，发生移码突变。符合我们要求，获得基因组敲除31aa-uORF的细胞系。
2)细胞培养
HEK-293T细胞购于ATCC(
CRL-11268TM)，培养于DMEM(Gibico，8113281)加10％的胎牛血清(FBS，Gibco,10099)、10U/mL的青霉素-链霉素(Gibco,15140-122)的培养基，并放置在一个5％CO2、37℃恒温湿润的细胞培养培养箱中培养。细胞每三天传代一次，传代比例1：4。
3)Western blot
用RAPA提取细胞总蛋白，BCA蛋白定量法对提取的蛋白进行定量；配置5％SDS-PAGE浓缩胶、15％SDS-PAGE分离胶，上样总蛋白为100微克；采用80V 20分钟、150V 1h跑蛋白电泳；转膜采用100V转膜2h；5％的脱脂牛奶封闭1h；31aa-uORF兔抗抗体(1:500)、β-actin抗体(abcam货号ab197345)(1:3000)；孵育4度过夜；第二天采用兔二抗(1:10000)常温孵育1h，TBST洗涤5遍每次5min，然后发光，显影，定影。
实验结果：
通过制备31aa-uORF特异性抗体，我们在293和U251细胞内检测识别了该uORF编码的微肽(图2)。同时还发现该微肽在正常脑胶质细胞中的表达量远高于脑胶质瘤细胞，提示该多肽可能具有抑癌功能(图2)。
实施例2 31aa-uORF微肽细胞生物学功能的确定
1)过表达31aa-uORF稳定细胞系的构建
合成过表达31aa-uORF-GFP的载体(载体骨架pCDH-CMV-MCS-EF1-Puro)，与慢病毒骨架载体PSPAX2,PMD2G按照4：3：1比例转染到HEK-293T细胞中包装慢病毒。48小时和72小时候分别收取带慢病毒的培养基上清然后感染U251细胞(加8ug/ml polybrene增加感染效率)，puromycin(1mg/mL)筛选三天后撤掉嘌呤霉素，然后正常扩增细胞。细胞转染步骤：293T细胞50万个细胞接种于6孔板培养板中，24h细胞贴壁后可进行转染；转染前，将100微升无血清培养基DMEM和质粒制备成混合液；将100微升无血清培养基DMEM和5微升(2ug质粒/5uL lipo2000)liop2000脂质体均匀混合，做成脂质体混合液；将上述两种混合液等比例混合，室温放置20min；按照转染试剂操作说明书操作；最终6孔板中的孔终体积为1毫升，转染6小时候，换成正常培养基(10％胎牛血清加90％DMEM培养基加1％青霉素链霉素)1毫升，细胞培养条件37度，5％二氧化碳。
2)流式测定细胞周期
U251神经胶质瘤细胞和慢病毒过表达31aa-uORF细胞150万个细胞接种于T25培养瓶中，细胞贴壁后再培养24小时，培养于DMEM(Gibico，8113281)加10％的胎牛血清(FBS，Gibco,10099)、10U/mL的青霉素-链霉素(Gibco,15140-122)的培养基，并放置在一个5％CO2、37℃恒温湿润的细胞培养培养箱中培养。胰酶消化细胞离心后PBS洗一遍，再离心去上清，沉淀重悬于1mL的PBS中，然后逐滴加入3mL﹣20℃预冷的100％乙醇固定细胞30min，离心，PBS洗一遍后将沉淀重悬于25ug/mL的溴化丙啶(PI，sigma)/PBS溶液中，避光染色30min后上机检测细胞周期分布情况。
3)CCK-8检测细胞增殖
将U251-GFP对照细胞或者U251-31aa-uORF-GFP细胞以2000个每孔的数量接种于96孔板中，每个样品5个重复，贴壁后每24h加入每孔加入10μL CCK-8(Dojindo，CK04)试剂37℃培养1-4h后检测450λm的吸光度(OD)。吸光度值与细胞数目成正比。
4)平板克隆形成实验(CFA，clone formation assay)
将U251-GFP对照细胞或者U251-31aa-uORF-GFP细胞以2000个每孔的数量接种于六孔板，在一个5％CO2、37℃恒温湿润的细胞培养培养箱中连续培养两周后结晶紫染色后拍照，同时统计形成克隆的数目。至少三次重复实验。
5)软琼脂克隆形成实验(soft agar assay)
先在六孔板中加入2mL0.6％(在DMEM中配)的低熔点琼脂糖，凝固后再加入含有20000细胞的2mL 0.35％(在DMEM中配)的低熔点琼脂糖，轻轻混匀然后在一个5％CO2、37℃恒温湿润的细胞培养培养箱中连续培养两周后光学显微镜下拍照并统计克隆形成数目。至少三次重复实验。
实验结果：
在U251神经胶质瘤细胞中，过表达31aa-uORF编码微肽可以明显抑制胶质瘤细胞U251的生长、增殖以及克隆形成等(图3)。
实施例3验证微肽进细胞的能力
将U251细胞以50％的密度接种于载玻片上，加不同浓度的连接有FITC荧光基团的PTEN-31aa微肽处理细胞(微肽以DMSO溶解，浓度为20μg/μL,5mM)4h，然后福尔马林固定并与荧光显微镜下观察拍照。发现对于PTEN-31aa微肽而言10μM浓度就能在4h内顺利进入U251细胞，并且视野内所有细胞都有荧光(图4)。
实施例4动物水平验证31aa-uORF微肽的抑癌功能
将U251细胞以200万每个点接种于裸鼠的左右侧。三十天后观察瘤子的形成，然后将瘤子按照大小平均分成对照组和实验组(每组五只裸鼠，十个瘤子)，对照组隔天打对照多肽，实验组隔天打31aa-uORF多肽。每个瘤子打25ug人工合成多肽。给药前测量瘤子的长径和短径，给药7次后处死裸鼠并取瘤子拍照和称重。
瘤子体积＝1/2*长径*短径的平方
实验结果如图5所示，对照多肽(将31aa-uORF编码多肽序列随机打乱后合成对照多肽)相比化学合成31aa-uORF编码微肽可以明显抑制U251神经胶质瘤移植瘤的生长和形成，说明31aa-uORF编码微肽在动物水平也显示很好地抗肿瘤活性。
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Claims (11)
1.一种多肽，其特征在于，所述的多肽的氨基酸序列如SEQ ID NO ：2所示。
2.如权利要求1所述的多肽，其特征在于，所述的多肽通过化学合成；或者，所述的多肽通过重组表达产生。
3.一种核酸片段，其编码如权利要求1所述的多肽。
4.一种核酸片段，其特征在于，所述核酸片段的核苷酸序列如SEQ ID NO ：1所示。
5.如权利要求1或2所述的多肽或如权利要求3或4所述的核酸片段在制备治疗肿瘤的药物中的应用；所述的肿瘤为神经胶质瘤。
6.如权利要求1或2所述的多肽或如权利要求3或4所述的核酸片段的检测试剂在制备用于肿瘤诊断和/或预后判断的试剂中的应用；所述的肿瘤为神经胶质瘤。
7.如权利要求6所述的应用，其特征在于，所述的肿瘤诊断和/或预后判断的试剂包括：用于检测所述核酸片段的探针，或扩增所述核酸片段的引物，或者抗所述多肽的抗体。
8.一种用于治疗肿瘤的药物组合物，该药物组合物含有如权利要求1或2所述的多肽或如权利要求3或4所述的核酸片段；所述的肿瘤为神经胶质瘤。
9.如权利要求8所述的药物组合物，其特征在于，所述的药物组合物还含有一种或多种药用赋形剂或药用载体。
10.一种用于肿瘤诊断和/ 或预后判断的试剂盒，所述试剂盒含有：用于检测如权利要求3或4所述核酸片段的探针，或扩增如权利要求3或4所述核酸片段的引物，或者抗如权利要求1或2所述多肽的抗体；所述的肿瘤为神经胶质瘤。
11.编码权利要求1或2所述的多肽的表达载体、或含有该表达载体的宿主细胞，在制备治疗肿瘤的药物中的应用；所述的肿瘤为神经胶质瘤。
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| CN101988091A (zh) * | 2009-08-07 | 2011-03-23 | 芮屈生物技术（上海）有限公司 | 一种pten基因的原位杂交检测试剂盒及其检测方法和应用 |
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| CN101988091A (zh) * | 2009-08-07 | 2011-03-23 | 芮屈生物技术（上海）有限公司 | 一种pten基因的原位杂交检测试剂盒及其检测方法和应用 |
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