CN103880949B - 类载脂蛋白c-i及其在制备治疗肾母细胞瘤的药物中的应用 - Google Patents
类载脂蛋白c-i及其在制备治疗肾母细胞瘤的药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种新型的类载脂蛋白C‑I及其在制备预防或治疗肾母细胞瘤的药物中的应用,本发明提供的用于预防或治疗肾母细胞瘤的药物,其有效成分是由55个氨基酸组成的单链多肽蛋白质,即类载脂蛋白C‑I(SEQ ID No.1),可通过化学方法合成。实验表明,本发明的多肽蛋白质药物,通过抑制肾母细胞瘤的增殖与生长,达到治疗肾母细胞瘤的作用。本发明的多肽蛋白质药物可在治疗肾母细胞瘤中得到广泛应用。
Description
技术领域
本发明涉及生物医药领域,具体地说,涉及一种类载脂蛋白C-I及其在制备预防或治疗肾母细胞瘤的药物中的应用。
背景技术
肾母细胞瘤(Nephroblastoma),又名肾胚胎细胞瘤、wilms瘤(Wilms’Tumor,WT),是最常见的儿童肾脏肿瘤,其发病率在小儿腹部肿瘤中占首位。在发达国家患儿的五年生存率大约为80%,死亡率小于5%[1]。在不发达国家中,大约有近一千万患儿死于肾母细胞瘤,其中包括四百万新生儿[2]。肾母细胞瘤的治疗手段以手术切除为主,辅以化疗,但这种治疗手段仍难以完全解决转移和复发的问题,且化疗毒副反应严重,患儿难以承受。统计数据[3]表明:Ⅰ期肾母细胞瘤8年存活率达90.5%-98.5%,Ⅱ-Ⅲ期的肾母细胞瘤患儿8年存活率为73.4%-88.7%,而Ⅳ期肾母细胞瘤患儿的8年存活率则仅为45.0%-57.1%。由以上数据可以看出,II期及以上肾母细胞瘤患儿的治疗需要建立一种更高效、更安全的方法。因此,研究者一直在探索更为有效的、可最大限度上杀伤肿瘤细胞,同时不伤及正常细胞的生物治疗方法。
目前,肿瘤学研究的热点包括鉴定新的肿瘤标志物以提高早期诊断水平,筛选更有效的靶向药物以及患儿的个体化治疗等,蛋白质组学为上述研究提供了新的平台。蛋白质组技术可以从整体上,全面的、动态的、定量的分析比较正常及病变标本中蛋白质种类和数量的改变,有助于阐明蛋白质间的调控网络,从而有希望发现控制肿瘤进程的关键分子,为肿瘤的诊断、分型、药物研究带来新的思路和途径[4]。
同时,多肽和蛋白质类药物是目前医药研发领域中最活跃,进展最快的部分,是二十一世纪最有前途的产业之一。肿瘤的发生是多种原因作用的结果,但最终都要涉及癌基因或癌蛋白的表达调控。不同肿瘤产生时所需要的酶等调控因子不同,选择特异性肽作为肿瘤发生时所需的调控因子等,封闭其活性位点,可防止肿瘤发生。现在已发现很多肿瘤相关基因对肿瘤产生调控因子,筛选与这些靶点特异结合的多肽,已成为寻找抗癌药物的新热点。日本科学家发现一种多肽聚合物[5],由能够与HER2蛋白特异性结合的靶向肽与lytic肽通过肽键连接,该多肽聚合物具有高度细胞毒性作用,能够在对正常细胞无副作用的情况下杀死乳腺癌细胞,这种方法为治疗这一死亡率很高的恶性肿瘤开辟了新途径。
发明内容
本发明的目的是提供一种在肾母细胞瘤患儿血清中低表达的新型类载脂蛋白C-I。
本发明的另一目的是提供类载脂蛋白C-I在制备预防或治疗肾母细胞瘤的药物中的应用。
为了实现本发明目的,本发明的一种类载脂蛋白C-I,其氨基酸序列为:1)SEQ ID No.1;或2)SEQ ID No.1所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由1)衍生的蛋白。
本发明还提供编码所述类载脂蛋白C-I的基因。
本发明还提供含有编码所述类载脂蛋白C-I基因的载体、转基因细胞系和工程菌。
本发明还提供所述类载脂蛋白C-I在制备预防或治疗肾母细胞瘤的药物中的应用。
本发明进一步提供一种用于预防或治疗肾母细胞瘤的药物,其有效成分为类载脂蛋白C-I。
发明人在前期研究中应用蛋白质组学技术完成了肾母细胞瘤血清和组织蛋白质标记物筛查及特异性标记物鉴定,鉴定出M/Z值为6455.5的特异性多肽蛋白质标记物,经检测发现该特异性多肽蛋白质标记物与载脂蛋白C-I的覆盖比达55.56%,将其命名为类载脂蛋白C-I肽[6],这种标记物在肾母细胞瘤患儿血清中低表达,在正常儿童血清中高表达。发明人用蛋白质组学技术筛选肾母细胞瘤特异性多肽蛋白质标记物类载脂蛋白C-I肽的同时,构建了肾母细胞瘤血清蛋白质指纹图谱诊断模型[7],肾母细胞瘤血清蛋白质指纹图谱临床分期模型[8],肾母细胞瘤血清蛋白质指纹图谱预后监测模型[9],发现类载脂蛋白C-I肽会随着病情的发展而不同,根治性手术切除肿瘤后患儿血清中该标记物表达量接近正常儿童水平,姑息性手术切除肿瘤后患儿血清中该标记物表达量无变化。因此,类载脂蛋白C-I肽可以作为肾母细胞瘤血清特异性标记物,用于前期诊断肾母细胞瘤并作为肾母细胞瘤治疗预后监测指标。
根据以上研究推测,类载脂蛋白C-I肽与肾母细胞瘤的发生发展可能存在某种生物学关系。而多肽和蛋白质类药物是人体内源性物质或针对生物体内调控因子研发而得,通过参与、介入、促进或抑制人体内或细菌病毒中生理生化过程而发挥作用。相比化疗药物,多肽和蛋白质类药物具有副作用低、药效高、针对性强,不会蓄积于体内而引起中毒。本发明以开发一种能够治疗肾母细胞瘤的多肽蛋白质药物为目标,对类载脂蛋白C-I肽对肾母细胞瘤生物学特性的影响进行了进一步研究。
鉴于类载脂蛋白C-I肽在肾母细胞瘤患儿血清中的特异表达:肾母细胞瘤患儿血清中低表达,在正常儿童血清中高表达;根治性手术切除肿瘤后患儿血清中该标记物表达量接近正常儿童水平,姑息性手术切除肿瘤后患儿血清中该标记物表达量无变化。本发明首先通过化学合成方法,合成出类载脂蛋白C-I肽,由长度55个氨基酸的多肽序列组成,通过高效液相色谱HPLC对其进行纯化,使其纯度达到99%以上;临床手术中,完整切除III/IV期肾母细胞瘤志愿者的瘤体后,剖开瘤体,选择生长良好的肿瘤标本,进行原代培养,培养出稳定的细胞系以后进行鉴定为肾母细胞瘤细胞。再使用荧光标记物FITC标记类载脂蛋白C-I肽,将标记有FITC的类载脂蛋白C-I肽与肾母细胞瘤细胞共培养48h,在荧光显微镜下观察类载脂蛋白C-I肽在细胞上的表达情况,结果表明该多肽蛋白质能够与肾母细胞瘤细胞相结合(图1)。
本发明进行类载脂蛋白C-I肽干预肾母细胞瘤细胞生长的体内试验。经类载脂蛋白C-I肽干预的肾母细胞瘤细胞列为观察组,未经类载脂蛋白C-I肽干预的肾母细胞瘤细胞列为对照组:计算出细胞倍增时间,绘制细胞生长曲线;经CCK8染色试剂处理后,酶标仪下获得相关数据计算出细胞生长活性曲线和类载脂蛋白C-I肽IC50\IC80抑制浓度;流式细胞仪技术检测观察组与对照组的细胞生长周期和凋亡率。结果显示,类载脂蛋白C-I肽能够抑制肾母细胞瘤细胞的增殖。
进而本发明设计动物试验,对BALB/c裸鼠皮下种植肾母细胞瘤细胞,成功构建荷瘤鼠后,未经干预的荷瘤裸鼠列为对照组,使用类载脂蛋白C-I肽治疗的荷瘤裸鼠列为观察组。实验结果:观察组裸鼠的肿瘤较对照组裸鼠的肿瘤体积明显缩小;观察组裸鼠的体重和健康状况较对照组裸鼠的体重、健康状况和精神状态明显改善;观察组裸鼠的肿瘤切片病理检查显示出现不同程度的凋亡坏死情况,对照组裸鼠的肿瘤切片病理检查显示仍以活性较好的增生期肾母细胞瘤细胞为主。以上结果表明类载脂蛋白C-I肽能够有效地治疗肾母细胞瘤,且安全性高、副作用小、药效高。
由此,本发明提出一种治疗肾母细胞瘤的多肽蛋白质药物,其氨基酸序列及相关信息如表1所示。
表1 类载脂蛋白C-I肽氨基酸序列及相关信息
本发明的药物形式为类载脂蛋白C-I肽氨基酸序列单链化合物,可溶解于纯水、生理盐水或PBS缓冲液中,常温下可配制成浓度0.1-1.0μmol/ml的试剂,体外使用剂量为0.2μmol/ml,直接加至细胞培养体系中;体内使用剂量初步为4mg/kg,对于小鼠模型可采用腹腔注射,尾静脉注射等方法。
本发明提供的一种用于预防或治疗肾母细胞瘤的药物,其有效成分是由55个氨基酸组成的单链多肽蛋白质,即类载脂蛋白C-I(SEQID No.1),可通过化学方法合成。实验表明,本发明的多肽蛋白质药物,通过抑制肾母细胞瘤的增殖与生长,达到治疗肾母细胞瘤的作用。本发明的多肽蛋白质药物可在治疗肾母细胞瘤中得到广泛应用。
附图说明
图1为本发明标记有FITC的类载脂蛋白C-I肽与肾母细胞瘤共同培养48h后在两种显微镜下观察同一视野的结果;A.普通相差显微镜下观察,20×10倍镜;B.荧光显微镜下观察,20×10倍镜。
图2为本发明实施例2中不同浓度梯度的类载脂蛋白C-I肽干预肾母细胞瘤细胞生长的曲线图。
图3为本发明实施例2中不同浓度梯度的类载脂蛋白C-I肽干预肾母细胞瘤细胞后,细胞倍增时间对比。
图4为本发明实施例3中不同浓度梯度的类载脂蛋白C-I肽对肾母细胞瘤细胞生长的抑制。
图5为本发明实施例4中肾母细胞瘤细胞正常培养以及类载脂蛋白C-I肽在IC50、IC80浓度下干预肾母细胞瘤细胞48h后,流式细胞仪检测细胞凋亡率的结果。
图6为本发明实施例6中类载脂蛋白C-I肽腹腔注射至荷瘤小鼠模型体内,治疗6周后解剖观察组裸鼠,测得的肿瘤直径。
图7为本发明实施例6中将生理盐水注射至荷瘤小鼠模型体内,6周后解剖对照组裸鼠,测得的肿瘤直径。
图8为本发明实施例6中对照组与观察组裸鼠肿瘤直径对比。p<0.05,观察组与对照组之间具有统计学意义。
图9为本发明实施例6中对照组与观察组裸鼠肿瘤体积对比。p<0.05,观察组与对照组之间具有统计学意义。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1 FOMC-保护氨基酸固相合成法合成类载脂蛋白C-I肽
本发明采用Fomc-Ser(tbu)-Wang-Resin树脂作为载体,具体合成工艺步骤如下:1)用DCM将树脂充分溶胀后,然后用DMF清洗树脂几遍;2)使用适当浓度的DBLK,将Fomc-保护基团脱出,使用DMF清洗树脂数遍,洗去DBLK残余液;3)称取适合的缩合剂和活化剂以及C端第二个Fomc-保护氨基酸(Fomc-Asp(otbu)-OH)进行偶联,偶联完毕后使用茚三酮检测法进行检测确保连接比较完全,再使用DMF清洗几遍,洗去残留的各种残基和活化剂缩合剂;4)依类载脂蛋白C-I肽氨基酸序列将各氨基酸逐个进行偶联,重复上述步骤2)、3);5)将所有的氨基酸连接结束后,重复上述步骤2),脱去最后的FOMC-保护基团;6)用切割液裂解树脂、氨基酸混合物,去除树脂和氨基酸保护基团,得到粗品;7)质谱质谱仪检测类载脂蛋白C-I肽粗品,确认产品正确;8)高效液相色谱分离提纯粗品类载脂蛋白C-I肽,至纯度>99%,其氨基酸序列如SEQ ID No.1所示。
实施例2 类载脂蛋白C-I肽干预肾母细胞瘤细胞后对生长曲线和细胞倍增时间的分析
选择对数生长期的肾母细胞瘤细胞重悬浮,分别加入数个24孔培养板,接种密度为2×104个/孔。接种1天后,取3孔细胞进行计数,作为初始细胞数,将类载脂蛋白C-I肽按由低到高的浓度梯度加入上述培养板每孔的细胞悬液中,被干预的细胞悬液作为观察组,未干预的细胞悬液作为对照组。每隔24h取每种不同浓度的观察组细胞悬液以及对照组细胞悬液各3孔,用台盼蓝染料染色,滴入细胞计数板内在光学显微镜下进行计数,连续7天,绘制出细胞生长曲线(图2)。根据所绘制的细胞生长曲线通过帕特森公式计算得到类载脂蛋白C-I肽干预48h后细胞倍增时间(图3)。实验结果显示:未经干预的肾母细胞瘤细胞数量在72小时达到5.741×105个,细胞倍增时间为16.14小时;经过药物浓度1×10-2μmol/ml的类载脂蛋白C-I肽干预后,肾母细胞瘤细胞数量在72小时仅达到0.65×105个,倍增时间延长至66.98小时。类载脂蛋白C-I肽对肾母细胞瘤细胞的生长增殖起到抑制作用。
实施例3 类载脂蛋白C-I肽干预肾母细胞瘤细胞后对细胞活性和半数抑制率的分析
选择对数生长期的肾母细胞瘤细胞重悬浮,分别加入数个96孔培养板,每孔板100μL介质,接种密度为5×104个/孔。接种1天后,将多肽蛋白质药物按0、5、10、15、20、25、30×10-2μmol/ml的浓度分别加入上述培养板每孔的细胞悬液中,被干预的细胞悬液作为观察组,未干预的细胞悬液作为对照组。24h、48h和72h后,分别使用CCK8试剂染色,2h后在酶标仪下,用450nm/630nm双波长光波检测,根据存活细胞数,绘制出校正曲线(图4)。根据校正曲线,计算出类载脂蛋白C-I肽在肾母细胞瘤细胞系中的24h、48h和72h半数抑制浓度(IC50)和80%抑制浓度(IC80),结果如表2所示。
表2 类载脂蛋白C-I肽在肾母细胞瘤细胞系中的24h、48h和72h半数抑制浓度(IC50)和80%抑制浓度(IC80)
实施例4 类载脂蛋白C-I肽干预肾母细胞瘤细胞后流式细胞仪分析细胞凋亡
选择对数生长期的肾母细胞瘤细胞重悬浮,分别加入数个6孔培养板,每孔板1200μL介质,接种密度为5×104个/孔。接种1天后,将类载脂蛋白C-I肽按48h后IC50和IC80的浓度分别加入上述培养板的各孔中作为观察组,无干预的孔内细胞作为对照组。48小时后,收集对照组细胞,多肽蛋白质药物处理后的观察组细胞低温PBS冲洗2次后,与100μL 1×结合缓冲液混合,室温下加入细胞凋亡Annexin-V/PI双染试剂(annexin-V/PI(BD Biosciences)double staining solution)继续培养15min。用流式细胞仪检测染色细胞并分析凋亡细胞百分比(图5)。结果显示未经干预的和经过浓度IC50、IC80的类载脂蛋白C-I肽干预后的肾母细胞瘤细胞在48小时早期凋亡率分别为8.4%、22.3%和69.6%,由此可见类载脂蛋白C-I肽高效诱导肾母细胞瘤细胞的早期凋亡。
实施例5 类载脂蛋白C-I肽干预肾母细胞瘤细胞后采用流式细胞仪分析细胞生长周期
选择对数生长期的肾母细胞瘤细胞重悬浮,分别加入数个6孔培养板,每孔板1200μL介质,接种密度为5×104个/孔。接种1天后,将类载脂蛋白C-I肽按48h后IC50和IC80的浓度加入上述培养板作为观察组,无干预的细胞悬液作为对照组。48小时后,收集对照组细胞,类载脂蛋白C-I肽段处理后的观察组细胞(IC50和IC80),用低温PBS冲洗2次后,加入300μL 1×结合缓冲液混匀,在室温下加入RNaseA和PI继续培养15min。用流式细胞仪检测出生长周期及各阶段细胞百分比(表3)。结果显示:经过浓度IC50、IC80的类载脂蛋白C-I肽干预后的肾母细胞瘤细胞在G0/G1期上比未经干预的肾母细胞瘤细胞延长,证明类载脂蛋白C-I肽能够诱导G0/G1期阻滞。
表3 经过浓度IC50、IC80的类载脂蛋白C-I肽干预后的肾母细胞瘤细胞在生长周期及各阶段细胞百分比
*表示p<0.05,IC50、IC80与对照组之间有统计学意义。
实施例6 动物体内模型试验验证类载脂蛋白C-I肽治疗肾母细胞瘤疗效
采用4周龄雄性BALB/c Nude无胸腺小鼠,在标准化SPF级动物实验室内饲养。建立稳定的12小时昼夜更替环境。所有操作均在无菌环境下进行。所有程序均经动物伦理委员会批准。肾母细胞瘤细胞重悬浮于1:1血清培养基和基底膜基质中,调整细胞浓度为1×107个/0.2ml。消毒裸鼠皮肤后,向左侧前肢腋窝皮下接种0.2ml细胞混合液,每3天观测一次,持续35天。最后将裸鼠分为2组,每天向腹腔内给药,持续6周,观察组注射剂量为4mg/Kg的类载脂蛋白C-I肽;对照组注入相应体积生理盐水。类载脂蛋白C-I肽干预4周后,处死裸鼠,取出瘤体测量体积、直径,根据相关数据作统计学对比(图6-9)。结果显示,经类载脂蛋白C-I肽治疗后的肾母细胞瘤荷瘤小鼠模型肿瘤体积和直径明显减小,由此可见,类载脂蛋白C-I肽对肾母细胞瘤能够起到有效的治疗作用。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
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Claims (1)
1.类载脂蛋白C-I在制备预防或治疗肾母细胞瘤的药物中的应用,其中,所述类载脂蛋白C-I的氨基酸序列如SEQ ID No.1所示。
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Identification of Apolipoprotein C-I as a Potential Wilms’Tumor Marker after Excluding Inflammatory Factors;Junjie Zhang et al;《Int.J.Mol.Sci.》;20140912;第15卷;16186-16195 * |
Identification of novel serum biomarkers in child nephroblastoma using proteomics technology;Qian Zhang et al;《Mol Biol Rep》;20100406;第38卷;631-638 * |
蛋白质组学技术在小儿肾母细胞瘤临床中的应用研究;张谦;《中国博士学位论文全文数据库医药卫生科技辑》;20110615(第06期);全文 * |
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