CN110205384B - Spz1的用途及其抑制剂的用途和药物筛选方法 - Google Patents

Spz1的用途及其抑制剂的用途和药物筛选方法 Download PDF

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CN110205384B
CN110205384B CN201910426342.2A CN201910426342A CN110205384B CN 110205384 B CN110205384 B CN 110205384B CN 201910426342 A CN201910426342 A CN 201910426342A CN 110205384 B CN110205384 B CN 110205384B
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张鹏
刘晓羽
许键
邱书奇
赵海亮
李红文
曾宪海
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Shenzhen City Longgang District Otolaryngology Hospital
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Abstract

本发明提供一种SPZ1作为靶点在筛选或制备用于预防和/或治疗胃癌的药物中的用途及其抑制剂的用途和对应的筛药方法。转录因子SPZ1与胃癌细胞凋亡密切相关，能间接下调介导胃癌细胞凋亡的Cyclin D1蛋白在胃癌细胞中的表达水平。实验结果表明，SPZ1抑制剂对体内外的胃癌细胞的凋亡有明显促进作用，能够抑制肿瘤生长。因此，本发明为抗胃癌新药的设计提供了新靶点，为抗肿瘤恶性增殖药物的开发和制备提供了新的思路。

Description

SPZ1的用途及其抑制剂的用途和药物筛选方法
技术领域
本发明涉及抗癌技术领域，特别涉及一种SPZ1的用途及其抑制剂的用途和药物筛选方法。
背景技术
胃癌是最常见的癌症类型之一。最新调查数据显示，胃癌是我国最常见的消化系统恶性肿瘤，其发病率和死亡率分别为于第二位和第三位。癌细胞最重要的生物学特征之一是其在体内快速且无限的增殖能力。正常细胞的增殖受自身或微环境的信号所调控，细胞接受足够的信号后才启动增殖程序，但通常认为癌细胞始终处于亢奋的增殖状态。胃癌细胞增殖与凋亡的紊乱是胃癌发生发展的主要原因。因此，研究胃癌细胞增殖的相关机制，寻求有效的抗肿瘤增殖措施进而抑制肿瘤生长进程已成为国内外的研究热点。研究人员发现细胞分裂增殖能力依赖于细胞周期蛋白的调控，而现有的研究结果提示细胞周期蛋白D1(Cyclin D1)的表达增加可能与肿瘤恶性程度密切相关。根据胃癌恶性增殖的产生机制，选择特殊靶点设计研发抗胃癌恶性增殖的药物进行靶向治疗，将对胃癌的治疗非常有益。
SPZ1(spermatogenic leucine zipper1)，即生精亮氨酸拉链蛋白1，属于碱性螺旋-环-螺旋-亮氨酸拉链(basic helix-loop-helix-leucine zipper，bHLH-Zip)结构的一类转录因子，最初发现于小鼠的精子文库之中，功能上与精子生成密切相关，并主要在胚胎发育阶段表达。在转基因鼠和裸鼠模型中，发现活化的SPZ1作为转录因子促进下游基因表达，其中就包括增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)。然而SPZ1与胃癌细胞增殖的相关性还有待进一步的研究。
发明内容
本发明所要解决的一个技术问题在于如何提供一种SPZ1作为靶点在筛选或制备用于预防和/或治疗胃癌的药物中的用途及其抑制剂的用途和对应的药物筛选方法。
本发明所采取的技术方案是：
SPZ1作为靶点在筛选或制备用于预防和/或治疗胃癌的药物中的用途。
根据本发明的实施例，治疗胃癌包括抑制胃癌细胞增殖、促进胃癌细胞凋亡中的至少一种。
SPZ1的抑制剂在制备用于预防和/或治疗胃癌的药物中的用途。
其中，抑制剂是指能够抑制SPZ1发挥功能的物质，其非限制性实例可以是抑制SPZ1相关基因的复制、转录(或转录后修饰)、翻译(或翻译后修饰)的核酸分子、蛋白质或化合物。例如，可以是siRNA、shRNA、dsRNA、miRNA、反义核酸等。
根据本发明的实施例，治疗胃癌包括抑制胃癌细胞增殖、促进胃癌细胞凋亡中的至少一种。
优选的，抑制剂为siRNA、shRNA中的任一种。
优选的，抑制剂为包含shRNA的重组载体或包括该重组载体的重组宿主细胞。
一种预防和/或治疗胃癌的药物或者是抑制胃癌细胞增殖的药物的筛选方法，使用包括SPZ1作为检测靶点进行筛选，其非限制性实例可以是，在筛选过程中进行SPZ1的表达测试以定量SPZ1的表达量。
根据本发明的实施例，治疗胃癌包括抑制胃癌细胞增殖、促进胃癌细胞凋亡中的至少一种。
本发明的有益效果是：
转录因子SPZ1与胃癌细胞凋亡密切相关，能间接下调介导胃癌细胞凋亡的CyclinD1蛋白在胃癌细胞中的表达水平。实验结果表明，SPZ1抑制剂对体内外的胃癌细胞的凋亡有明显促进作用，能够抑制肿瘤生长。因此，本发明为抗胃癌新药的设计提供了新靶点，为抗肿瘤恶性增殖药物的开发和制备提供了新的思路。
附图说明
图1是本发明的一个实施例的Western blot检测结果图。
图2是本发明的一个实施例的细胞增殖实验结果。A是细胞活力检测结果，B是细胞增殖检测结果。
图3是本发明的一个实施例的肿瘤大小结果图。左图是转染Lenti-control(shCTL)和Lenti-SPZ1 shRNA(shSPZ1)慢病毒质粒后的每组各5个重复在第15天取下时的肿瘤照片；右图是不同时间内肿瘤的生长曲线统计图，其中，上侧曲线是shCTL控制组，下侧曲线是shSPZ1实验组。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。
1.实验材料
细胞系：人胃癌细胞AGS，购自美国模式培养物寄存库(ATCC)。
SPZ1抑制剂：SPZ1的siRNA SPZ1#1(其靶向序列为SEQ ID No：1，5′-CCATTGCCTTATTCGAAAT-3′)和siRNA SPZ1#2(其靶向序列为SEQ ID No：2，5′-CCATCAAGTTACAGAACAA-3′)购自广州市锐博生物科技有限公司，SPZ1的抑制性质粒Lenti-SPZ1 shRNA(其靶向序列为SEQ ID No：3，5′-GCTGTCTGAGATGCCACCTTC-3′)购自汉恒生物科技(上海)有限公司。
试剂及试剂盒：CellTiter-Blue Cell Viability Aassay购自美国Promega公司，DharmaFECT 1 Transfection Reagent购自美国GE公司，BrdU Cell Proliferation ELISAkit购自美国Abcam公司，Anti-SPZ1购自美国GeneTex公司，Anti-Cyclin D1购自美国CST公司，Anti-ACTB购自美国Santa Cruz公司。
实验动物：荷瘤小鼠，其制备方法如下：取体外常规培养的AGS细胞，分为两组，一组转染Lenti-SPZ1 shRNA，另一组转染Lenti-control。按照每只小鼠2×106个细胞的接种量接种雄性裸鼠背部。
实验仪器：酶标仪购自美国Molecular Devices公司，5％CO2恒温细胞培养箱购自美国Thermo公司，蛋白电泳仪系统购自美国Bio-Rad公司，蛋白转印仪系统购自美国Bio-Rad公司。
2.实验方法
2.1转染实验
对AGS细胞分别转染siRNA contrl(siCTL)、siRNA SPZ1#1和siRNA SPZ1#2，转染48小时后收集细胞，通过Western blot检测SPZ1与Cyclin的表达量的关系。
2.2体外胃癌细胞抗增殖实验
对AGS细胞分别转染siRNA contrl(siCTL)、siRNA SPZ1#1和siRNA SPZ1#2，转染48小时后收集细胞。应用CellTiter-Blue Cell Viability Aassay检测细胞活力，应用BrdU Cell Proliferation ELISA kit检测细胞增殖。
2.3体内胃癌细胞生长抑制实验
对AGS细胞分别转染Lenti-SPZ1 shRNA和Lenti-control慢病毒质粒。按照每只小鼠2×106个细胞的接种量接种雄性裸鼠背部，15天后取下肿瘤，拍照并测量肿瘤的大小。
3.实验结果
3.1转染实验结果
图1是本发明的一个实施例的Western blot检测结果图。如图1所示，在AGS细胞中，与对照组siCTL相比，SPZ1的两条siRNA均可以显著敲低SPZ1的表达，抑制Cyclin D1的表达。该实验结果表明转录因子SPZ1间接促进细胞周期蛋白Cyclin D1的表达。
3.2体外胃癌细胞抗增殖实验结果
图2是本发明的一个实施例的细胞增殖实验结果。A是细胞活力检测结果，B是细胞增殖检测结果。如图2所示，与对照组相比，转染了SPZ1抑制剂(siRNA SPZ1#1和siRNASPZ1#2)的实验组的AGS细胞的相对存活率和BrdU渗入率都有了显著的下降。这说明，SPZ1抑制剂可以抑制胃癌细胞的细胞活力和增殖能力。该实验结果表明转录因子SPZ1会促进胃癌细胞增殖的发生。
3.3体内胃癌细胞生长抑制实验结果
图3是本发明的一个实施例的肿瘤大小结果图。左图是转染Lenti-control(shCTL)和Lenti-SPZ1 shRNA(shSPZ1)慢病毒质粒后的每组各5个重复在第15天取下时的肿瘤照片；右图是不同时间内肿瘤的生长曲线统计图，其中，上侧曲线是shCTL控制组，下侧曲线是shSPZ1实验组。如图3所示，shSPZ1实验组的肿瘤明显更小，而且生长速度更慢，与对照组相比具有显著性差异，这说明SPZ1抑制剂Lenti-SPZ1 shRNA能明显抑制肿瘤生长。该实验结果表明，SPZ1抑制剂对体内胃癌具有明显的抑制作用，且对实验动物无毒副作用，进一步提示SPZ1抑制剂可为促胃癌凋亡药物的开发和制备提供新的思路。
综合上述实验结果可知，SPZ1的抑制剂对体内和体外的胃癌细胞的凋亡都有明显的促进作用，能够抑制肿瘤生长。因此，本发明为抗胃癌新药的设计提供了新靶点，为抗肿瘤恶性增殖药物的开发和制备提供了新的思路。
显然，以上所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求书的保护范围为准。
SEQUENCE LISTING
<110> 张鹏 深圳市龙岗区耳鼻咽喉医院
<120> SPZ1的用途及其抑制剂的用途和药物筛选方法
<160> 3
<170> PatentIn version 3.5
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<212> DNA
<213> 人工序列
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Claims (1)

1. SPZ1的抑制剂在制备用于抑制人胃癌细胞AGS的增殖或生长的药物中的用途，所述抑制剂为siRNA、shRNA中的任一种。
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EP1862180A4 (en) * 2005-03-25 2010-09-15 Takeda Pharmaceutical PROPHYLACTIC AND THERAPEUTIC AGENT AGAINST CANCER
CA2525255A1 (en) * 2005-11-03 2007-05-03 Jumi Shin Minimalist bzip proteins and uses thereof
KR100856108B1 (ko) * 2007-02-28 2008-09-02 한국생명공학연구원 Ｚｆｐ91의 활성을 조절하는 분자를 함유하는 항암제,항염증제, 면역질환치료제 또는 허혈성 질환 치료제
CN103937872A (zh) * 2013-01-23 2014-07-23 上海市东方医院 Crm1在胃癌诊断与治疗中的应用
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