CN114045259A - 一种抑制肿瘤干细胞的方法 - Google Patents
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Abstract
本发明属于细胞外泌体的抗肿瘤应用领域，涉及一种抑制肿瘤干细胞的方法，具体涉及通过视黄醛分子的T淋巴细胞外泌体囊泡特异性杀死肿瘤干细胞的方法。根据肿瘤干细胞的高乙醛脱氢酶的特性，再结合视黄醛与视黄酸的抗肿瘤活动的差别，通过乙醛脱氢酶的作用，实现两者的相互转化，最终实现抑制肿瘤干细胞的效果。T淋巴细胞的外泌体与视黄醛组成复合物，可以有效地将视黄醛递送到肿瘤细胞中，从而利用肿瘤干细胞的高ALDH活性将视黄醛有效的转化为对肿瘤干细胞有抑制和杀伤作用的视黄酸。外泌体囊泡具有非常弱的免疫原性，异体细胞对其无任何排斥作用，可以安全有效的对自体和异体使用。
Description
技术领域
本发明属于细胞外泌体的抗肿瘤应用领域，涉及一种抑制肿瘤干细胞的方法，具体涉及通过视黄醛分子的T淋巴细胞外泌体囊泡特异性杀死肿瘤干细胞的方法。
背景技术
外泌体是各种细胞分泌的具有重要细胞间通信和调控作用的小囊泡体。外泌体是近年来发现的细胞间信号通讯的重要途径，它是一种由胞内质膜性细胞器内质网和高尔基复合体主动合成并分泌到细胞外环境的直径在30-120nm左右的微型质膜囊体结构，是除细胞可溶性细胞因子外的重要旁分泌形式，exosome的特征主要有：（1）直径为30-100 nm；（2）具有来源细胞的胞质和脂质膜成分；（3）含有来源细胞特异性蛋白以及相关蛋白CD63，CD9，CD81等。（4）成分主要包括：microRNA、mRNA和蛋白等生物活性物质，具有减少细胞凋亡、减轻炎症反应、促进血管生成、抑制纤维化、提高组织修复潜力等重要生物学功能。研究表明干细胞外泌体可以作为细胞治疗的有利工具在组织损伤性疾病中发挥重要作用。类似于其他种类的干细胞，肌肉干细胞也可分泌大量具有修复功能的外泌囊泡体 (exosome)， 其中包含各种蛋白，多肽， 核酸和其他活性因子。间充质干细胞MSCs来源的exosome已被证实对肾脏损伤、心肌梗死、神经系统疾病等多个器官功能损伤具有明确保护作用，可以逆转细胞凋亡趋势，促进组织细胞损伤修复，与母体细胞无显著差异。同样，各种免疫细胞，包括CD8+的T淋巴细胞，也可分泌大量的外泌体。ALDH-high 的CD8+的T淋巴细胞，被发现具有比ALDH-low 的CD8+T淋巴细胞相对更强抗肿瘤的活性，而理论上ALDH-high 的T淋巴细胞的外泌体也会具有更强的抗肿瘤活性。
乙醛脱氢酶（ALDH）是醛脱氢酶的一种，在多种细胞谱系的干细胞和祖细胞中高效表达，包括造血细胞、乳腺细胞、内皮细胞、间充质细胞和神经细胞等。并且，大量研究表明ALDH在多种肿瘤干细胞中特异性的高表达，例如乳腺癌、前列腺癌、结肠癌、骨肉瘤等实体瘤以及白血病；ALDH已被广泛认定为肿瘤干细胞的标记性功能蛋白。ALDH在人体内催化有毒性的乙醛转化为无毒性的羧酸，从而来保持细胞内稳态，这对于干细胞和肿瘤干细胞的存活和自我更新尤为重要。ALDH在肿瘤干细胞中的高量表达也与其高效的抗氧化抗凋亡特性紧密相关。同时，ALDH活性高的免疫细胞，例如ALDH-high 的CD8+的T淋巴细胞，被发现具有比ALDH-low相对更强抗肿瘤的活性，而这种效果可能和ALDH-high的T淋巴细胞对肿瘤干细胞的特异性作用有关。
视黄醛也称维生素a醛，是视黄醇氧化后的衍生物。它是由β-胡萝卜素发生氧化断裂生成的。还原得到视黄醇，氧化得到视黄酸。视黄醛是视紫红质的辅基。包括视黄醇、视黄醛、视黄酸、视黄脂在内，它们都是具有维生素a活性的视黄醇类化合物。全反式视黄醛(all-trans retinal)是视黄酸的前体，可以通过各种脱氢酶（包括ALDH）氧化为视黄酸，而两者在肿瘤细胞中的作用有很大差异。视黄酸被发现具有抗肿瘤特性，包括诱导细胞的凋亡和抑制细胞的增殖，而视黄醛则没有明显的抗肿瘤活性。而理论上，由于癌症干细胞的ALDH活性较高，癌症干细胞可高效地将视黄醛转化为具有抗肿瘤活性的视黄酸。
发明内容
本发明根据肿瘤干细胞的高乙醛脱氢酶的特性，再结合视黄醛与视黄酸的抗肿瘤活动的差别，通过乙醛脱氢酶的作用，实现两者的相互转化，最终实现抑制肿瘤干细胞的效果。
本发明的方案如下：
一种抑制肿瘤干细胞的方法，包括以下步骤：
（1）生产和收集高活性T淋巴细胞外泌体；
（2）整合外泌体和视黄醛；
（3）作用于肿瘤细胞。
优选地，所述的步骤（1）的过程为：
将T淋巴细胞悬浮培养至1x105细胞/mL，以乙醛脱氢酶ALDH活性为标准，筛选高ALDH1活性的T淋巴细胞，将T淋巴细胞导入3D培养系统进行培养扩增，收集培养液中的外泌体。
更进一步地，所述的步骤（1）的过程为：将T淋巴细胞初步培养扩增到约5x104细胞数量后，根据其ALDH的活性进行分离筛选，分离出含有20%以上的ALDH活性最高的细胞；经培养扩增至第4至6代时，转入3D的胶原蛋白制成的组织工程细胞骨架进行立体细胞培养，基础培养液是RPMI细胞培养基，其它添加剂包括胎牛血清，IL-2和CD137单克隆抗体；培养，外泌体在细胞培养液中的最终浓度可达到3 mg/ml~20 mg/ml；外泌体经过低温超高速离心提取获得。
优选地，所述的步骤（2）的过程为：将外泌体重新悬浮溶解于PBS中，加入视黄醛将混合物温育即得。
优选地，所述的视黄醛为全反式视黄醛。
优选地，所述的步骤（3）的过程为：将整合了视黄醛的外泌体溶解于生理盐水中，放入肿瘤细胞中；外泌体连续使用，进行考察肿瘤细胞的情况。
优选地，整合了视黄醛的外泌体在生理盐水中的最终浓度为40mg/ml~60mg/ml；每1~3天使用一次，连续使用14~28天。
本发明的有益效果
1、T淋巴细胞的外泌体与视黄醛组成复合物，可以有效地将视黄醛递送到肿瘤细胞中，从而利用肿瘤干细胞的高ALDH活性将视黄醛有效的转化为对肿瘤干细胞有抑制和杀伤作用的视黄酸。
2、外泌体囊泡具有非常弱的免疫原性，异体细胞对其无任何排斥作用，可以安全有效的对自体和异体使用。
3、抑制肿瘤干细胞效果好：本申请的外泌体是来自ALDH高活性的激活态的T淋巴细胞，本身具有生物活性和一定地对肿瘤细胞地趋向性，富含对肿瘤细胞的生长、凋亡、免疫调节等起调控作用的的微小RNA（miRNA）、生长因子和热休克蛋白等细胞因子。外泌体囊泡可以迅速的与肿瘤细胞膜融合，通过胞吞或胞饮方式将微小RNA、生长因子、热休克蛋白以及所携带的化合物等送入靶细胞，起效迅速。
4、外泌体囊泡区别于化学合成药物，可以被细胞自行吸收降解，对细胞无任何损伤。
附图说明
图1. 外泌体和视黄醛的复合物抑制骨肉瘤细胞（K7M2）的生长。（A）外泌体处理３天后，K7M2细胞的生长速度被明显降低。（B）外泌体处理2天后，正在凋亡的Propidiumiodide/PI+的细胞数目增加。
图2. 外泌体和视黄醛的复合物抑制小鼠骨肉瘤中肿瘤干细胞的生长和肿瘤的发展。（A）免疫荧光染色结果显示，外泌体复合物注射20天后，骨肉瘤中具有干细胞特性（Notch1+）的细胞减少，而成熟的血管（VWF+）的形成也被减弱；（B）骨肉瘤中表达骨肉瘤干细胞标记蛋白CD133的细胞数量被减少。
图３. 外泌体和视黄醛的复合物诱导小鼠骨肉瘤中细胞凋亡数目的增加。（A）H.E.染色显示，外泌体复合物注射20天后，骨肉瘤中发生更多的细胞凋亡；（Ｂ）RT-PCR结果显示，肿瘤中ALDH1、BCL-xL和BCL-2的表达降低，而Caspase３的表达升高。
具体实施方式
下面将结合具体实施例来详细说明本发明，在此本发明的示意性实施例以及说明用来解释本发明，但并不作为对本发明的限定。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：
一种抑制肿瘤干细胞的方法，具体步骤如下：
(1)高活性T淋巴细胞外泌体的生产和收集：将T细胞悬浮培养至1x105细胞/mL乙醛脱氢酶，以ALDH1的活性为标准，运用流式细胞仪筛选高ALDH1活性的T细胞（约2x104），将T淋巴细胞导入3D培养系统进行培养扩增，连续培养72h后低温超高速离心获得外泌体囊泡。基础培养液是RPMI 1640 细胞培养基，其它添加剂质包括胎牛血清最终体积分数为15％、40 IU/mL IL-2和0.1 ng/ml的CD137单克隆抗体；细胞培养48h 后，加入终浓度为0.1ng/ml的γ-干扰素（IFN-γ），继续培养24h后，外泌体在细胞培养液中的最终浓度达到3mg/mL；收集细胞培养液再经过低温超高速离心提取获得干细胞外泌体囊泡；获得的外泌体测定蛋白含量后冻存于-80℃冰箱；
(2)外泌体和视黄醛的整合: 将外泌体重新悬浮溶解于PBS中，加入2mg/ml的视黄醛(全反式视黄醛，all-trans retinal)， 将混合物在 37℃ 摇动温育 2小时。
实施例2
一种抑制肿瘤干细胞的方法，具体步骤如下：
(1)高活性T淋巴细胞外泌体的生产和收集：将T细胞悬浮培养至1x105细胞/mL乙醛脱氢酶，以ALDH1的活性为标准，运用流式细胞仪筛选高ALDH1活性的T细胞（约2x104），将T淋巴细胞导入3D培养系统进行培养扩增，连续培养72h后低温超高速离心获得外泌体囊泡。基础培养液是RPMI 1640 细胞培养基，其它添加剂质包括胎牛血清最终体积分数为10％、30 IU/mL IL-2和0.2 ng/ml的CD137单克隆抗体；细胞培养48h 后，加入终浓度为0.1ng/ml的γ-干扰素（IFN-γ），继续培养24h后，外泌体在细胞培养液中的最终浓度达到10mg/mL；收集细胞培养液再经过低温超高速离心提取获得干细胞外泌体囊泡；获得的外泌体测定蛋白含量后冻存于-80℃冰箱；
(2)外泌体和视黄醛的整合: 将外泌体重新悬浮溶解于PBS中，加入1mg/ml的视黄醛(全反式视黄醛，all-trans retinal)， 将混合物在 37℃ 摇动温育 2小时。
实施例3
一种抑制肿瘤干细胞的方法，具体步骤如下：
(1)高活性T淋巴细胞外泌体的生产和收集：将T细胞悬浮培养至1x105细胞/mL乙醛脱氢酶，以ALDH1的活性为标准，运用流式细胞仪筛选高ALDH1活性的T细胞（约2x104），将T淋巴细胞导入3D培养系统进行培养扩增，连续培养72h后低温超高速离心获得外泌体囊泡。基础培养液是RPMI 1640 细胞培养基，其它添加剂质包括胎牛血清最终体积分数为20％、50 IU/mL IL-2和0.05 ng/ml的CD137单克隆抗体；细胞培养基的总体积约是10mL培养液/CM3细胞骨架）；细胞培养48h 后，加入终浓度为0.1ng/ml的γ-干扰素（IFN-γ），继续培养24h后，外泌体在细胞培养液中的最终浓度达到20mg/mL；收集细胞培养液再经过低温超高速离心提取获得干细胞外泌体囊泡；获得的外泌体测定蛋白含量后冻存于-80℃冰箱；
(2)外泌体和视黄醛的整合: 将外泌体重新悬浮溶解于PBS中，加入3mg/ml的视黄醛(全反式视黄醛，all-trans retinal)， 将混合物在 37℃ 摇动温育 2小时。
实施效果例
一、外泌体对体外培养肿瘤细胞的处理：将整合了视黄醛的外泌体以一定的浓度重悬溶解于细胞培养液中（约50mg/mL），用来培养骨肉瘤细胞K7M2。 基础培养液是DMEM细胞培养基，其它添加剂质包括胎牛血清最终体积分数为15％、40 IU/mL IL-2和0.1 ng/ml的CD137单克隆抗体；每天换一次新的培养液，培养３天后，将细胞固定进行细胞生长等特性的观察分析；
用实施例1所得外泌体和视黄醛复合物产品对骨肉瘤细胞进行处理后，比较对照组，外泌体和视黄醛的复合物可有效抑制肿瘤细胞的生长。结果如下图1。
二、外泌体对小鼠肿瘤的处理:将整合了视黄醛的外泌体以一定的浓度重悬溶解于生理盐水中（约50mg/mL），注射入小鼠的骨肉瘤发生区。外泌体连续注射20天 ，收取少量组织进行病理观察。以冷冻切片和免疫组化等方法检测比较外泌体注射的效果。组织病理方面的改变可通过H.E. 染色和免疫荧光染色（Notch1-干细胞标记蛋白, vWF-血管细胞标记蛋白, CD133-肿瘤干细胞标记蛋白）等方法鉴定，在光镜下观察肿瘤组织的病理变化；PCR检测肿瘤中相关细胞因子表达变化，结果见图2、3。
综上所述，本发明提供一种利用T细胞外泌体结合视黄醛对肿瘤干细胞进行抑制的方法和步骤，可以有效降低肿瘤干细胞的活性和生长能力。本发明制备的产品与传统干细胞不同，无任何活体细胞成分，便于运输和储存，可以在冰箱中长期保存，用于帮助抑制肿瘤细胞生长，具有极佳的临床实验应用价值。
Claims (7)
1.一种抑制肿瘤干细胞的方法，其特征在于，包括以下步骤：
（1）生产和收集高活性T淋巴细胞外泌体；
（2）整合外泌体和视黄醛；
（3）作用于肿瘤细胞。
2.根据权利要求1所述的抑制肿瘤干细胞的方法，其特征在于，所述的步骤（1）的过程为：
将T淋巴细胞悬浮培养至1x105细胞/mL，以乙醛脱氢酶ALDH活性为标准，筛选高ALDH1活性的T淋巴细胞，将T淋巴细胞导入3D培养系统进行培养扩增，收集培养液中的外泌体。
3.根据权利要求1或2所述的抑制肿瘤干细胞的方法，其特征在于，所述的步骤（1）的过程为：将T淋巴细胞初步培养扩增到约5x104细胞数量后，根据其ALDH的活性进行分离筛选，分离出含有20%以上的ALDH活性最高的细胞；经培养扩增至第4至6代时，转入3D的胶原蛋白制成的组织工程细胞骨架进行立体细胞培养，基础培养液是RPMI细胞培养基，其它添加剂包括胎牛血清，IL-2和CD137单克隆抗体；培养，外泌体在细胞培养液中的最终浓度可达到3mg/ml~20 mg/ml；外泌体经过低温超高速离心提取获得。
4.根据权利要求1所述的抑制肿瘤干细胞的方法，其特征在于，所述的步骤（2）的过程为：将外泌体重新悬浮溶解于PBS中，加入视黄醛将混合物温育即得。
5.根据权利要求4所述的抑制肿瘤干细胞的方法，其特征在于，所述的视黄醛为全反式视黄醛。
6.根据权利要求1所述的抑制肿瘤干细胞的方法，其特征在于，所述的步骤（3）的过程为：将整合了视黄醛的外泌体溶解于生理盐水中，放入肿瘤细胞中；外泌体连续使用，进行考察肿瘤细胞的情况。
7.根据权利要求6所述的抑制肿瘤干细胞的方法，其特征在于，整合了视黄醛的外泌体在生理盐水中的最终浓度为40mg/ml~60mg/ml；每1~3天使用一次，连续使用14~28天。
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102070481A (zh) * | 2009-06-11 | 2011-05-25 | 辽宁利锋科技开发有限公司 | 具有金刚烷结构药物曲金刚胺及其衍生物和类似物抗肿瘤新适应症的应用 |
| CN102526011A (zh) * | 2012-01-19 | 2012-07-04 | 苏州大学 | 视黄酸在制备治疗乳腺癌药物中的应用 |
| KR20120095320A (ko) * | 2011-02-18 | 2012-08-28 | 가톨릭대학교 산학협력단 | 레티날 또는 레티노산을 유효성분으로 포함하는 자연살해세포의 독성 억제를 위한 세포보호용 조성물 |
| US20170079942A1 (en) * | 2014-05-21 | 2017-03-23 | National Institute Of Advanced Industrial Science And Technology | Cancer stem cell proliferation inhibitor |
| CN108103026A (zh) * | 2017-12-05 | 2018-06-01 | 四川省肿瘤医院 | 用于肿瘤免疫治疗的γδ-T细胞外泌体及其制备方法 |
| CN110840812A (zh) * | 2019-11-27 | 2020-02-28 | 沣潮医药科技（上海）有限公司 | 胎体来源的外泌体在皮肤调节产品中的用途 |
| US20220152151A1 (en) * | 2019-02-19 | 2022-05-19 | Direct Biologics, Llc | Acellular intravenous infusion including mesenchymal stem cell growth factors and exosomes |
-
2021
- 2021-11-08 CN CN202111312546.7A patent/CN114045259B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102070481A (zh) * | 2009-06-11 | 2011-05-25 | 辽宁利锋科技开发有限公司 | 具有金刚烷结构药物曲金刚胺及其衍生物和类似物抗肿瘤新适应症的应用 |
| KR20120095320A (ko) * | 2011-02-18 | 2012-08-28 | 가톨릭대학교 산학협력단 | 레티날 또는 레티노산을 유효성분으로 포함하는 자연살해세포의 독성 억제를 위한 세포보호용 조성물 |
| CN102526011A (zh) * | 2012-01-19 | 2012-07-04 | 苏州大学 | 视黄酸在制备治疗乳腺癌药物中的应用 |
| US20170079942A1 (en) * | 2014-05-21 | 2017-03-23 | National Institute Of Advanced Industrial Science And Technology | Cancer stem cell proliferation inhibitor |
| CN108103026A (zh) * | 2017-12-05 | 2018-06-01 | 四川省肿瘤医院 | 用于肿瘤免疫治疗的γδ-T细胞外泌体及其制备方法 |
| US20220152151A1 (en) * | 2019-02-19 | 2022-05-19 | Direct Biologics, Llc | Acellular intravenous infusion including mesenchymal stem cell growth factors and exosomes |
| CN110840812A (zh) * | 2019-11-27 | 2020-02-28 | 沣潮医药科技（上海）有限公司 | 胎体来源的外泌体在皮肤调节产品中的用途 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115044543A (zh) * | 2022-08-17 | 2022-09-13 | 山东卓东生物科技有限公司 | 一种提高衰老人体来源肌肉干细胞活性的方法 |
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