CN106519007B - 一种单链多肽及其在制备用于预防和治疗胃癌的药物中的应用 - Google Patents
一种单链多肽及其在制备用于预防和治疗胃癌的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种单链多肽，其氨基酸序列为SEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQAR，分子量为4344g/mol。本发明的单链多肽在制备用于预防和治疗胃癌的药物中的应用，其药物中的有效成分为WSGC肽。本发明的用于预防和治疗胃癌的药物形式为单链化合物，可溶解于纯水、生理盐水或PBS缓冲液中。实验证明，本发明制备的多肽药物通过抑制胃癌细胞的增殖与生长，达到治疗胃癌的作用，并可在预防和治疗胃癌中得到广泛应用。
Description
技术领域
本发明涉及生物医药领域，尤其是涉及一种单链多肽，本发明还涉及该多肽在制备用于预防和治疗胃癌的药物中的应用。
背景技术
胃癌在全球范围内发病率高，死亡率高，明确诊断时大多已到晚期，5年生存率低于25%，且目前缺乏有效的治疗手段。传统小分子药物的非特异性毒性限制了其大范围的临床应用及市场推广，而多肽类药物由于具有不蓄积中毒、相对成本低、生物活性强、副作用小、作用靶点专一等优点，日渐引起制药企业及学术界的关注，已广泛涉足抗肿瘤药物、心脑血管药物、抗病毒多肽、抗菌性活性肽以及疫苗、诊断试剂盒等领域。生物技术的进步使药物化学研究发生了很大的转变：过去占主导地位的是合成化学和天然产物的筛选，目前生物分子学已成为药物化学发展的推动力，而肽类药物的产品开发和生物分子学基础研究又有着天然的联系。通过体内、体外实验，结果发现：联合应用HER2及VEGF模拟肽对乳腺癌有着明显的抗肿瘤及抑制肿瘤血管生成的作用。日本科学家发现了HER2裂解混合肽，该肽对宫颈癌及乳腺癌细胞系均有较强的细胞毒性作用，甚至对曲妥珠单抗和拉帕替尼耐药的肿瘤细胞系亦有作用，而对正常的细胞系却无明显细胞毒效应；HER2裂解混合肽可以裂解过表达HER2的肿瘤细胞膜，对正常细胞的细胞膜却无裂解作用；通过静脉途经注射用药，混合肽可明显抑制无胸腺小鼠乳腺癌模型的肿瘤生长。上述研究成果也为治疗胃癌这一死亡率较高的恶性肿瘤开辟了新的途径。
发明内容
本发明的目的在于提供一种单链多肽，本发明还提供该多肽在制备用于预防和治疗胃癌的药物中的应用。
为实现上述目的，本发明可采取下述技术方案：
本发明所述的单链多肽由40个氨基酸组成；其氨基酸序列为SEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQAR，简称WSGC肽，其分子量为4344g/mol。
本发明的单链多肽在制备用于预防和治疗胃癌的药物中的应用，其药物中的有效成分为WSGC肽。
本发明所述的特异性WSGC肽应用蛋白质组学技术可在胃癌患者血清中鉴定出，该肽在胃癌患者术前、术后及复发患者血清中呈动态性表达。鉴于该肽在在胃癌患者血清中的特异性表达，本发明首先通过FOMC-保护氨基酸固相合成法合成由40个氨基酸组成的WSGC肽，通过高效液相色谱(HPLC)对其进行纯化，使其纯度达到95%以上。
本发明的用于治疗胃癌的药物形式为单链化合物，可溶解于纯水、生理盐水或PBS缓冲液中。实验证明，本发明制备的多肽药物通过抑制胃癌细胞的增殖与生长，达到治疗胃癌的作用，并可在预防和治疗胃癌中得到广泛应用。
附图说明
图1为标记有FITC的WSGC肽与胃癌细胞共同培养48h后在两种显微镜下观察同一视野的结果。
图2为不同浓度梯度的WSGC肽干预胃癌细胞的生长曲线图。
图3为不同浓度梯度的WSGC肽对胃癌细胞生长的抑制率。
图4为未干预的胃癌细胞及WSGC肽在IC50、IC80浓度下干预胃癌细胞48h后流式细胞仪检测细胞凋亡图。
图5为未干预的胃癌细胞及WSGC肽在IC50、IC80浓度下干预胃癌细胞48h后流式细胞仪检测细胞周期图。
图6为典型的对照组和实验组持续腹腔注射生理盐水和WSGC肽6周后效果图。
图7为对照组和实验组持续腹腔注射生理盐水和WSGC肽6周后荷瘤鼠肿瘤体积对比。
具体实施方式
下面对本发明做更加详细的说明。其中所用的技术手段为本领域的常规手段，所用原料均为市售商品。
一、本发明单链多肽（WSGC肽）的氨基酸序列及物理特性见下表1。
表1
。
该单链多肽的制备方法如下：
1）采用Wang树脂作为载体，用二氯甲烷（DCM）将树脂充分溶胀后，用N-二甲基甲酰（DMF）清洗树脂几遍；
2）使用适当浓度的20%哌啶将9-芴甲氧羰基（Fomc）基团脱出，使用DMF清洗树脂数遍，洗去残余液；
3）称取适量的O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸（TBTU）及C端第二个Fomc-保护氨基酸进行偶联，偶联完毕后使用茚三酮检测法确保连接完全，再使用DMF清洗2遍，洗去残留的各种残基和缩合剂；
4）依WSGC肽氨基酸序列将各氨基酸逐个进行偶联，重复上述步骤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WSGC肽粗品，确认产品正确；
8）采用高效液相色谱(HPLC)分离提纯粗品WSGC肽，至纯度>95%。
二、WSGC肽与胃癌细胞的融合
完整切除III/IV期胃癌志愿者的瘤体，在剖开的瘤体中选择生长良好的肿瘤标本，进行原代培养并鉴定为胃癌细胞系。使用荧光标记物FITC标记WSGC肽，将标记有FITC的WSGC肽与胃癌细胞共培养48h，在荧光显微镜下观察WSGC肽与细胞的融合情况，结果显示该多肽能够与胃癌细胞相结合，如图1所示，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;C.A+B合并图。
三、WSGC肽干预胃癌细胞生长的体外试验
将经WSGC肽干预的胃癌细胞列为实验组，未经WSGC肽干预的胃癌细胞列为对照组：绘制细胞生长曲线，如图2所示。结果显示：WSGC肽对胃癌细胞的生长增殖起到抑制作用。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（如图3所示）和WSGC肽24h、48h和72h半数抑制浓度(IC50)和80%抑制浓度(IC80)，结果如下表2所示。
表2 WSGC肽对胃癌细胞系生长抑制的24h、48h和72h半数抑制浓度（IC50）和80%抑制浓度(IC80)
流式细胞仪检测实验组与对照组的细胞凋亡率(如图4所示，A：WSGC肽浓度为0；B：WSGC肽浓度为IC50；C：WSGC肽浓度为IC80)和生长周期(如图5所示，A：WSGC肽浓度为0；B：WSGC肽浓度为IC50；C：WSGC肽浓度为IC80)，结果显示：未经干预的和经过浓度IC50、IC80的WSGC肽干预后的胃癌细胞在48小时的凋亡率分别为2.84%、42.05%和70.44%，WSGC肽可高效诱导胃癌细胞的凋亡；经过浓度IC50、IC80的WSGC肽干预后的胃癌细胞在S期的细胞比例高于未经干预的胃癌细胞，WSGC肽能够诱导S期阻滞，结果如下表3所示。
表3 IC50及IC80浓度的WSGC肽干预后的胃癌细胞不同细胞周期细胞比例(%,±s)
注：*** p<0.001，IC80、IC50与对照组之间比较均有统计学意义。
四、对本发明的WSGC肽设计动物实验，相关操作符合实验动物伦理规范。
对BALB/c无胸腺小鼠皮下种植胃癌细胞，成功构建荷瘤鼠。将腹腔注射生理盐水(4ml/kg)的荷瘤鼠列为对照组，腹腔注射WSGC肽(4mg/Kg)治疗的荷瘤鼠列为实验组。持续6周后结果显示：实验组小鼠的肿瘤体积较对照组明显缩小（图6中A为对照组，B为实验组；图7中p<0.0001，差异具有统计学意义）；实验组小鼠的体重和健康状况较对照组明显改善。以上结果表明WSGC肽能够有效地治疗胃癌，且副作用小。
Application Project
-------------------
<110> 王家祥 宋东建
<120> 一种单链多肽及其在制备用于预防和治疗胃癌的药物中的应用
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 40
<212> PRT
<213> Homo sapiens
<400> 1
Ser Glu Ala Glu Asp Ala Ser Leu Leu Ser Phe Met Gln Gly
Tyr Met Lys His Ala Thr Lys Thr Ala Lys Asp Ala Leu Ser
Ser Val Gln Glu Ser Gln Val Ala Gln Gln Ala Arg
Claims (1)
1.一种单链多肽在制备用于治疗胃癌的药物中的应用，其特征在于：该单链多肽由40个氨基酸组成，其氨基酸序列为SEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQAR。
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| US20120177610A1 (en) * | 2007-09-19 | 2012-07-12 | Kieu Hoang | Manufacturing and Purification Processes of Complex Protein found in Fraction IV to make a separated Apo, Transferrin , and Alpha 1 Anti strepsin (A1AT) or A combined Transferrin / Apo/Human Albumin/A1AT and all new found proteins |
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