CN106928325A - 增强肝癌细胞对cik细胞杀伤敏感性的人工多肽及其生物制品 - Google Patents
增强肝癌细胞对cik细胞杀伤敏感性的人工多肽及其生物制品 Download PDFInfo
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Abstract
本发明公开了增强肝癌细胞对CIK细胞杀伤敏感性的人工多肽及其生物制品，该人工多肽为包含式(Ⅲ)氨基酸序列的多肽：ETLGQPDAK(Xa)PCFQEDPMA(Xb)GTDCMELGIWN(Ⅲ)；其中，Xa和Xb选自M,Y,L,V,W或E。本发明提供的人工多肽可以有效增强肝癌细胞对CIK细胞杀伤敏感性，可以制备成药学上可以接受的生物制品，以实现使用少量的CIK细胞就发挥较优的免疫治疗效果。
Description
技术领域
本发明属于多肽领域，涉及多肽在细胞免疫治疗的应用，具体涉及增强肝癌细胞对CIK细胞杀伤敏感性的人工多肽及其生物制品。
背景技术
原发性肝癌(以下简称肝癌)是世界范围内最常见的恶性肿瘤之一，每年大约有63万的新发病例，而且近年来发病率有上升趋势。肝癌患者的病程短，临床上较难早期发现，确诊时往往病情已进入中、晚期，病死率高。目前肝癌的治疗主要是以手术切除、肝移植、局部消融、化学治疗栓塞及其他局部区域治疗、分子靶向治疗等，但这些方法的治疗效果并不理想，因为肝癌患者体内天然免疫、特异性免疫功能低下，根治性手术切除率低，手术后复发率高，放射、化学治疗疗效差，易发生肝内播散及肝外转移，因此急需寻找新的有效方法来治疗肝癌。生物治疗已经成为肿瘤综合治疗中的第四种模式，越来越受到重视。
目前用于肿瘤生物治疗的免疫活性细胞主要有肿瘤浸润性淋巴细胞(TIL)、树突状细胞(DC)以及细胞因子诱导的杀伤细胞(CIK)等。CIK细胞是一种新型的免疫活性细胞，其主要效应细胞的表面标志为CD3+CD56+，与其他过继性免疫治疗细胞相比，具有增殖速度快、杀瘤活性高、杀瘤谱广等优点。研究表明，过继细胞免疫治疗对抑制肝癌的复发和转移、提高患者治愈率、改善患者生活质量、延长生存期都具有重要作用。
如何用较少的CIK细胞就发挥较优的免疫治疗效果是科研工作者的一个重要研究方向。
发明内容
本发明的目的在于提供一种增强肝癌细胞对CIK细胞杀伤敏感性的人工多肽及其生物制品，以使用较少的CIK细胞就发挥较优的免疫治疗效果。
实现本发明上述目的技术方案如下：
一种包含式(Ⅲ)氨基酸序列的多肽：
ETLGQPDAK(Xa)PCFQEDPMA(Xb)GTDCMELGIWN； (Ⅲ)
其中，Xa和Xb选自M,Y,L,V,W或E。
优选地，Xa和Xb选自M或W。
优选地，所述多肽的氨基酸序列如下：
ETLGQPDAKMPCFQEDPMAMGTDCMELGIWN(SEQ ID NO.1)；
ETLGQPDAKWPCFQEDPMAWGTDCMELGIWN(SEQ ID NO.2)；
ETLGQPDAKMPCFQEDPMAWGTDCMELGIWN(SEQ ID NO.3)；
ETLGQPDAKWPCFQEDPMAMGTDCMELGIWN(SEQ ID NO.4)。
优选地，多肽N和/或C末端被化学修饰。
优选地，所述多肽的N末端被乙酰化修饰，C末端被酰胺化修饰。
优选地，所述多肽为游离形式或药学上可以接受的成盐形式，包括盐酸盐、硫酸盐、磷酸盐、磺酸盐、乙酸盐、柠檬酸盐和酒石酸盐。
上述多肽在增强肝癌细胞对CIK细胞杀伤敏感性方面的应用。
一种生物制品，含有上述多肽和药学上可以接受的辅料。
上述生物制品在增强肝癌细胞对CIK细胞杀伤敏感性方面的应用。
本发明的突出优点：
本发明提供的人工多肽可以有效增强肝癌细胞对CIK细胞杀伤敏感性，可以制备成药学上可以接受的生物制品，以实现使用少量的CIK细胞就发挥较优的免疫治疗效果。
附图说明
图1为各组CIK细胞对HepG2的杀伤率比较。
具体实施方式
下面就结合实施例具体介绍本发明的实质性内容，由于篇幅原因，实验过程的描述无法做到非常详细，凡是实验中未详细描述的部分均为本领域技术人员熟知的常规操作。
一、实验材料
本发明涉及的多肽通过本领域公知的标准多肽固相合成技术合成，采用芴甲氧羰基(Fmoc)N端保护策略。按照树脂固相合成的方法依次连接相应氨基酸，期间依次脱去Fmoc-保护基团，然后切肽，获得粗品，粗品经C18柱分离纯化，即可制得式(Ⅲ)所示的多肽。
HPLC和MS检测确证了本发明多肽的结构，且纯度均≥98％。
多肽Ⅲ-1至多肽Ⅲ-4的氨基酸序列如下(N末端→C末端)，末端未经化学修饰：
ETLGQPDAKMPCFQEDPMAMGTDCMELGIWN(多肽Ⅲ-1，SEQ ID NO.1)；
ETLGQPDAKWPCFQEDPMAWGTDCMELGIWN(多肽Ⅲ-2，SEQ ID NO.2)；
ETLGQPDAKMPCFQEDPMAWGTDCMELGIWN(多肽Ⅲ-3，SEQ ID NO.3)；
ETLGQPDAKWPCFQEDPMAMGTDCMELGIWN(多肽Ⅲ-4，SEQ ID NO.4)。
人肝癌细胞系HepG2为本公司长期保存。重组人干扰素rhIFN-γ、重组人白细胞介素rhIL-2、CD3鼠抗人单克隆抗体、CIK细胞分离液、PBS、无血清培养基MD-CM-STMN、RPMI1640培养基、胎牛血清FBS、MTT、DMSO、胰酶均购自南京建成生物。
二、实验方法
1、外周血单个核细胞制备
无菌抽取健康人外周血约50mL，肝素抗凝(肝素15IU/mL)，用等体积PBS稀释外周血。按1：1的体积比缓慢加入到含有细胞分离液的50mL离心管中，2000r/min离心20min。取中间环状乳白色单个核细胞层，移入50mL离心管中，加入适量PBS，1800r/min离心8min，弃上清，洗涤2次，细胞计数。
2、CIK细胞诱导培养
用含有5％FBS的MD-CM-STMN细胞培养基调整单个核细胞密度为1×106个/mL，接种20mL于培养瓶中，细胞总数为2×107，当天(第0天)加入rhIL-21000U/mL、rhIFN-γ1000U/mL、CD3鼠抗人单克隆抗体5mg/L，放置37℃，5％CO2培养箱中开始培养。第3天开始补加MD-CM-STMN细胞培养基，同时补充CD3鼠抗人单克隆抗体、rhIFN-γ、rhIL-2，用量同前，此后每3d添加新鲜培养基，补加rhIFN-γ，用量相同，rhIL-2用量减半为500U/mL。
3、CIK细胞增殖测定
台盼蓝染色方法，用细胞计数板分别于培养第0、5、8、13、20天对细胞进行计数，重复3次取平均值。
4、肿瘤细胞系的培养
将HepG2细胞接种于培养瓶中，加入含有10％FBS的RPMI 1640，贴壁细胞用0.25％的胰酶消化。取对数生长期的细胞，接种于96孔板，进行体外杀伤实验。
5、MTT法测定多肽Ⅲ-1～Ⅲ-4对HepG2细胞的细胞毒作用
取对数生长期HepG2细胞，调整细胞悬液密度为5×103个/mL，接种于96孔板，每孔200μL。待细胞贴壁后换液，给药组分别加入5μM多肽Ⅲ-1～Ⅲ-4，对照组加入培养液，每组均设3个平行孔，放置在37℃、5％CO2培养箱中作用48h后，各给药组及对照组每孔均分别加入20μLMTT(5g/L)，继续放置在37℃、5％CO2培养箱中培养4h，弃上清，每孔加入200μLDMSO，37℃下摇床振荡10min，酶标仪测定490nm处吸光度(A)值。
6、MTT法测定CIK对HepG2细胞杀伤作用
将培养至第13天的CIK细胞作为效应细胞，进行体外杀伤实验。调整HepG2细胞浓度5×103个/mL，每孔100μL，分为Ⅲ-1增敏组、Ⅲ-2增敏组、Ⅲ-3增敏组、Ⅲ-4增敏组和常规组，Ⅲ-1～Ⅲ-4增敏组分别添加5μM多肽Ⅲ-1～Ⅲ-4，常规组正常培养，放置在37℃、5％CO2培养箱中培养至细胞贴壁后换液，按效靶比10：1将效应细胞加入培养孔中，同时另设单独靶细胞(靶细胞+培养液)和单独效应细胞(效应细胞+培养液)为阴性对照，放置在37℃、5％CO2培养箱中作用48h后，加入MTT(5g/L)，20μL/孔，继续放置在37℃、5％CO2培养箱中培养4h，2000r/min离心5min，翻板弃上清，加入DMSO 150μL/孔，震荡，待沉淀溶解，放于酶标仪中，490nm测光密度(OD)值，按如下公式计算杀伤活性：杀伤率(％)＝{1-[(实验组OD值-单独效应细胞OD值)/单独靶细胞OD值]}×100％。
7、统计学方法
采用SPSS 19.0统计软件分析，数据以均数±标准差表示，P＜0.05具有统计学意义。
三、实验结果
1、CIK细胞增殖
CIK细胞在培养第5天细胞增殖速度加快，第5-13天为快速增殖时间段，在第20天细胞增殖达到原种植量的约115倍，在第20天细胞增殖达到原种植量的约180倍。
2、多肽Ⅲ-1～Ⅲ-4对HepG2细胞的细胞毒作用
多肽Ⅲ-1～Ⅲ-4给药组与对照组的吸光度(A)值无显著差异，证明5μM多肽Ⅲ-1～Ⅲ-4对HepG2细胞无明显细胞毒作用。结果见表1。
表1多肽Ⅲ-1～Ⅲ-4给药组与对照组的吸光度值的比值
| Ⅲ-1 | Ⅲ-2 | Ⅲ-3 | Ⅲ-4 | |
| A给药/A对照 | 0.98 | 1.03 | 0.99 | 0.97 |
3、多肽Ⅲ-1～Ⅲ-4增强肝癌细胞对CIK细胞杀伤敏感性
从表2和图1可以看出，各增敏组CIK细胞对HepG2细胞的杀伤率均显著高于常规组。已知5μM多肽Ⅲ-1～Ⅲ-4对HepG2细胞无明显细胞毒作用，各增敏组CIK细胞对HepG2细胞杀伤率的提高只能归结于多肽Ⅲ-1～Ⅲ-4提高了HepG2细胞对CIK细胞的杀伤敏感性，使用多肽Ⅲ-1～Ⅲ-4对靶细胞HepG2细胞增敏后，效靶比不变的情况下，CIK细胞对HepG2细胞的杀伤力显著提高。
表2各组CIK细胞对HepG2细胞的杀伤率(效靶比10:1)
本发明提供的人工多肽可以有效增强肝癌细胞对CIK细胞杀伤敏感性，可以制备成药学上可以接受的生物制品，以实现使用少量的CIK细胞就发挥较优的免疫治疗效果。
上述实施例是对本发明实质性内容的体现，用于更好地解释本发明，但本领域技术人员应当知晓，不应将本发明的保护范围局限于上述具体的实施例。
SEQUENCE LISTING
<110> 南京盖斯夫医药科技有限公司
<120> 增强肝癌细胞对CIK细胞杀伤敏感性的人工多肽及其生物制品
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<170> PatentIn version 3.3
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Claims (9)
1.一种包含式(Ⅲ)氨基酸序列的多肽：
ETLGQPDAK(Xa)PCFQEDPMA(Xb)GTDCMELGIWN； (Ⅲ)
其中，Xa和Xb选自M,Y,L,V,W或E。
2.根据权利要求1所述的多肽，其特征在于：Xa和Xb选自M或W。
3.根据权利要求2所述的多肽，其特征在于，氨基酸序列如下：
ETLGQPDAKMPCFQEDPMAMGTDCMELGIWN (SEQ ID NO.1)；
ETLGQPDAKWPCFQEDPMAWGTDCMELGIWN (SEQ ID NO.2)；
ETLGQPDAKMPCFQEDPMAWGTDCMELGIWN (SEQ ID NO.3)；
ETLGQPDAKWPCFQEDPMAMGTDCMELGIWN (SEQ ID NO.4)。
4.根据权利要求3所述的多肽，其特征在于：多肽N和/或C末端被化学修饰。
5.根据权利要求4所述的多肽，其特征在于：所述多肽的N末端被乙酰化修饰，C末端被酰胺化修饰。
6.根据权利要求3所述的多肽，其特征在于：所述多肽为游离形式或药学上可以接受的成盐形式，包括盐酸盐、硫酸盐、磷酸盐、磺酸盐、乙酸盐、柠檬酸盐和酒石酸盐。
7.权利要求1-6任一所述的多肽在增强肝癌细胞对CIK细胞杀伤敏感性方面的应用。
8.一种生物制品，含有权利要求1-6任一所述的多肽和药学上可以接受的辅料。
9.权利要求8所述的生物制品在增强肝癌细胞对CIK细胞杀伤敏感性方面的应用。
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