CN107523541A - 一种以白术内酯ⅱ为促增殖剂的cik细胞培养基 - Google Patents
一种以白术内酯ⅱ为促增殖剂的cik细胞培养基 Download PDFInfo
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Abstract
本发明公开了一种以白术内酯Ⅱ为促增殖剂的CIK细胞培养基，包括无血清培养基，该无血清培养基中含有有效浓度的白术内酯Ⅱ。所述无血清培养基为RPMI 1640，所述白术内酯Ⅱ在无血清培养基中的浓度为10‑100μM。本发明发现白术内酯Ⅱ可以提高CIK细胞的增殖率，提高CIK细胞对肿瘤细胞的杀伤活性，可以制备成CIK细胞培养基用于CIK细胞的培养增殖，以保证患者在放射治疗和化学治疗后能够回输足量的CIK细胞。
Description
技术领域
本发明属于细胞培养领域，涉及CIK细胞的诱导培养，具体涉及一种以白术内酯Ⅱ为促增殖剂的CIK细胞培养基。
背景技术
过继性细胞免疫治疗是指将免疫细胞分离，在体外诱导扩增产生大量免疫细胞后，将细胞回输到患者体内进行治疗的一种手段。细胞因子诱导的杀伤细胞(CIK)是一群具有细胞毒性的T淋巴细胞，为膜蛋白分子表型特点为CD3+CD56+的异质群体细胞，具有T淋巴细胞强大的抗瘤活性、自然杀伤(NK)细胞的非主要组织相容性复合体限制性及对正常细胞低毒性的优点，可清除手术、放化疗后残余的癌细胞及微小病灶，降低放、化疗毒副作用，提高生存质量，延长生存期。目前，国内在肿瘤免疫治疗方面以CIK细胞治疗最为广泛。
对于CIK细胞免疫治疗，临床上采用体外培养手段使CIK细胞大量增殖。为保证患者在放射治疗和化学治疗后能够回输足量的CIK细胞，有必要开发一种既能诱导CIK细胞成熟促进其增殖效率又不干扰其杀瘤活性的细胞培养基。
白术内酯Ⅱ为白术中的一种化合物，与白术内酯Ⅰ和白术内酯Ⅲ为白术中的主要内酯成分。其化学结构式如下，生源上白术内酯Ⅲ为白术内酯Ⅰ的氧化产物，白术内酯Ⅱ为白术内酯Ⅲ的脱水产物。通常认为，白术内酯Ⅰ和白术内酯Ⅲ为白术的主要活性成分，是其抗炎和抗癌有效成分(参考文献：白术内酯类成分及其药理作用研究进展，中国药房2012年第23卷第39期)。
申请人发现白术内酯Ⅱ有诸多特别的、不同于白术内酯Ⅰ和白术内酯Ⅲ的活性。
发明内容
本发明的目的在于提供一种以白术内酯Ⅱ为促增殖剂的CIK细胞培养基。
实现本发明上述目的技术方案如下：
一种以白术内酯Ⅱ为促增殖剂的CIK细胞培养基，包括无血清培养基，该无血清培养基中含有有效浓度的白术内酯Ⅱ。
优选地，所述无血清培养基为RPMI 1640。
优选地，所述白术内酯Ⅱ在无血清培养基中的浓度为10-100μM。
白术内酯Ⅱ在制备用于促进CIK细胞增殖、提高其杀瘤活性的试剂方面的用途。
本发明的突出优点：
本发明发现白术内酯Ⅱ可以提高CIK细胞的增殖率，增强CIK细胞对肿瘤细胞的杀伤活性，可以制备成CIK细胞培养基用于CIK细胞的培养增殖，以保证患者在放射治疗和化学治疗后能够回输足量的CIK细胞。
附图说明
图1为各组CD3+CD56+表型细胞(即CIK细胞)的占比比较；
图2为各组细胞对乳腺癌细胞的杀伤率(效靶比20:1)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
白术内酯Ⅱ、白术内酯Ⅰ和白术内酯Ⅲ自制，纯度大于98％。
二、实验方法
1、外周血单个核细胞(PBMC)的分离
采集健康志愿者外周抗凝全血，加入Ficoll-Plaque淋巴细胞分离液并进行密度梯度离心(2500r/min，25min)，提取白膜层，PBS洗涤3遍。
2、CIK细胞的培养及扩增
将PBMC悬浮于RPMI 1640完全培养液(含10％FBS)中，调整至1×106/ml，接种至培养皿中。按如下组别进行分组，并在培养液中添加不同种类和剂量的成分进行培养：
常规培养组(细胞因子诱导)：第0天加入IFN-γ1000IU/ml，置37℃、5％CO2培养箱培养。24小时后，加入IL-21000IU/ml，抗人CD3单克隆抗体(CD3McAb)50ng/ml。此后每隔3天根据情况添加CD3单抗、IL-2及新鲜培养液，置于37℃、5％CO2培养箱中培养连续培养14天。
白术内酯Ⅱ组(细胞因子半量+白术内酯Ⅱ)：与常规培养组相比，添加细胞因子的种类和时间相同，区别仅在于所有细胞因子减半，并在第0天时加入50μM白术内酯Ⅱ，以后添加的新鲜培养液均含有50μM白术内酯Ⅱ；
白术内酯Ⅰ组(细胞因子半量+白术内酯Ⅱ)：与白术内酯Ⅱ组相比，仅将白术内酯Ⅱ换成白术内酯Ⅰ；
白术内酯Ⅲ组(细胞因子半量+白术内酯Ⅲ)：与白术内酯Ⅱ组相比，仅将白术内酯Ⅱ换成白术内酯Ⅲ；
3、细胞表型分析及细胞增殖测定
各组于培养14天后，用台盼蓝拒染法计数细胞，用流式细胞仪检测CIK细胞的比例及细胞表型，并计数CIK细胞总数，每组每次各查4个复孔，计算平均值。
4、肿瘤杀伤活性测定
采用实时无标记细胞分析系统(real time cellular analysis，RTCA)评估CIK细胞的杀伤活性。其原理为加入靶细胞后，靶细胞通过细胞和微电极之间的相互作用产生电信号。加入CIK细胞后，CIK细胞作用于靶细胞导致细胞形态、黏附能力等状态发生综合变化。RTCA系统监测实时变化，以电信号的形式反映出来。如果CIK细胞最终导致靶细胞死亡，信号降低至零；反之，对信号没有影响。具体操作过程：加入CIK细胞前1d，靶细胞(乳腺癌细胞)按照1×105/ml的密度接种于6孔培养板；按效靶比20:1，将CIK细胞加入培养孔。基线扫描、设定细胞检测程序、设定实验孔靶细胞和效应细胞名称及数目，共培养1d后，收集实验数据，计算细胞杀伤活率。CIK细胞杀伤活率＝[靶细胞对照组吸光度值－(靶细胞实验组吸光度值－效应细胞对照组吸光度值)]/靶细胞对照组吸光度值×100％。
5、统计学处理
用SPSS 19.0统计软件进行统计学分析，数据采用均数±标准差表示，组间比较采用单因素方差分析，P＜0.05表示差异有统计学意义。
三、实验结果
1、CIK细胞体外增殖及细胞表型分析
培养14天后，各组细胞总数无显著差异，但是各组CD3+CD56+表型细胞(即CIK细胞)比例明显不同，CIK细胞增殖倍数如表1和图1所示。
该结果表明，白术内酯Ⅱ可以显著促进CIK细胞的体外增殖，在细胞因子减半(可以节省昂贵的细胞因子，节约培养成本)的情况下，体外增殖倍数显著优于常规培养方法。
表1各组CD3+CD56+表型细胞(即CIK细胞)的占比比较
2、肿瘤杀伤活性
测定结果如表2和图2所示。与常规培养组相比，白术内酯Ⅰ组和白术内酯Ⅲ组细胞对乳腺癌细胞的杀伤率显著降低(可能是因为CD3+CD56+表型细胞比例不及常规培养组)，白术内酯Ⅱ组细胞对乳腺癌细胞的杀伤率显著提高。
表2各组细胞对乳腺癌细胞的杀伤率(效靶比20:1)
| 常规培养组 | 白术内酯Ⅱ组 | 白术内酯Ⅰ组 | 白术内酯Ⅲ组 | |
| 杀伤率(％) | 64.3±6.5 | 88.7±7.4 | 50.4±6.2 | 54.8±6.5 |
上述实施例证明，白术内酯Ⅱ可以提高CIK细胞的增殖率，增强CIK细胞对肿瘤细胞的杀伤活性，可以制备成CIK细胞培养基用于CIK细胞的培养增殖，以保证患者在放射治疗和化学治疗后能够回输足量的CIK细胞。
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。
Claims (4)
1.一种以白术内酯Ⅱ为促增殖剂的CIK细胞培养基，包括无血清培养基，其特征在于：该无血清培养基中含有有效浓度的白术内酯Ⅱ。
2.根据权利要求1所述的CIK细胞培养基，其特征在于：无血清培养基为RPMI 1640。
3.根据权利要求1所述的CIK细胞培养基，其特征在于：所述白术内酯Ⅱ在无血清培养基中的浓度为10-100μM。
4.白术内酯Ⅱ在制备用于促进CIK细胞增殖、提高其杀瘤活性的试剂方面的用途。
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| CN109055314A (zh) * | 2018-08-29 | 2018-12-21 | 青海七彩花生物科技有限公司 | 一种促进cik细胞增殖并增强其杀瘤活性的化合物 |
| CN109097330A (zh) * | 2018-08-29 | 2018-12-28 | 青海七彩花生物科技有限公司 | 一种化合物用于促进cik细胞增殖并增强其杀瘤活性的用途 |
| CN112094811A (zh) * | 2020-09-30 | 2020-12-18 | 广东康盾生物工程技术有限公司 | 一种car-t细胞的复苏方法 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109055314A (zh) * | 2018-08-29 | 2018-12-21 | 青海七彩花生物科技有限公司 | 一种促进cik细胞增殖并增强其杀瘤活性的化合物 |
| CN109097330A (zh) * | 2018-08-29 | 2018-12-28 | 青海七彩花生物科技有限公司 | 一种化合物用于促进cik细胞增殖并增强其杀瘤活性的用途 |
| CN109055314B (zh) * | 2018-08-29 | 2021-11-02 | 山东兴瑞生物科技有限公司 | 一种促进cik细胞增殖并增强其杀瘤活性的化合物 |
| CN109097330B (zh) * | 2018-08-29 | 2021-12-17 | 上海智荃生物科技有限公司 | 一种化合物用于促进cik细胞增殖并增强其杀瘤活性的用途 |
| CN112094811A (zh) * | 2020-09-30 | 2020-12-18 | 广东康盾生物工程技术有限公司 | 一种car-t细胞的复苏方法 |
| CN112094811B (zh) * | 2020-09-30 | 2023-04-07 | 广东康盾创新产业集团股份公司 | 一种car-t细胞的复苏方法 |
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