CN116286634A - 类干细胞化诱导培养基及其制备方法和应用 - Google Patents
类干细胞化诱导培养基及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于细胞生物学技术领域,具体涉及一种类干细胞化诱导培养基及其制备方法和应用。类干细胞化诱导培养基包括类干细胞化诱导组分、细胞生长营养组分、维生素与细胞因子组分和其他组分。本发明可以实现CD8+ T细胞向类干细胞化转变,转变后的细胞具备干细胞的特性,回输动物体或者人体后,细胞具备更强的肿瘤杀灭能力,同时消减CD8+ T细胞衰竭和T细胞免疫抑制问题。
Description
技术领域
本发明属于细胞生物学技术领域,具体涉及一种类干细胞化诱导培养基及其制备方法和应用。
背景技术
根据国际癌症研究所发布的全球癌症统计最新报告,2020年全球新增了1930万名癌症患者,其中因癌症而死亡的人数接近1000万。近几年,随着化疗、放疗、分子靶向疗法的不断发展,免疫治疗逐渐崭露头角。与其它的治疗方法不同,它尝试通过病人的免疫系统来控制和消除肿瘤。
肿瘤免疫治疗已经成为继手术、化疗、放疗之后一种有效的疗法,如治疗复发或难治性大B细胞淋巴瘤的CAR-T细胞疗法KTE-19,已获美国FDA批准。与其他疗法不同的是,肿瘤免疫治疗是通过重新启动并维持抗肿瘤免疫循环,恢复机体正常的抗肿瘤免疫反应,从而控制、清除肿瘤的一种治疗方法。大部分已确立的免疫治疗是利用免疫细胞治疗肿瘤。随着对肿瘤发展与免疫逃逸之间的紧密联系的认识不断加深,免疫效应细胞在肿瘤生物学及治疗中的重要作用已引起人们的关注。细胞免疫治疗在癌症治疗中起着重要作用,可以有效地控制一些化疗不成功的肿瘤,包括那些巨块“肿瘤”。目前,在临床研究中所使用的免疫治疗的效应细胞主要包括自然杀伤细胞(NK)、肿瘤浸润淋巴细胞(TIL)、DC-T细胞和树突状细胞(DC)等。这种治疗模式中提供的细胞可经多种方法处理,纯化细胞、基因工程修饰或生长因子活化,亦或是经病毒预处理制备成疫苗等。美国食品药品监督管理局(FDA)在2010年批准了首个用于肿瘤治疗的细胞免疫治疗产品Provenge(或称Sipuleucel-T),用来治疗没有症状或者症状轻微的、转移性、去势治疗抵抗(即内分泌治疗失败)的前列腺癌。
当前CD8+ T细胞或者γδT细胞等在采用单独细胞过继免疫治疗或与PD-1抗体药物联合应用方面展现出良好的治疗效果。但是越来越多的研究发现浸润性T细胞存在细胞衰竭现象,使得免疫治疗效果受到影响,这迫切需要找寻一种解决T细胞衰竭和提升T细胞杀灭肿瘤能力,用于免疫治疗的T细胞存活更长时间并进行复制和生长的方法。
最新的研究(Vodnala SK, Eil R, Kishton RJ, Sukumar M, Yamamoto TN, HaNH, et al. T cell stemness and dysfunction in tumors are triggered by acommon mechanism. Science. 2019; 363.)发现在高钾条件下生长的T细胞能保持T细胞的“干性”:稳定持久增殖、自我更新,机体回输成活率高、肿瘤清除能力强及其他多能性。类干细胞化的T细胞在肿瘤细胞清除方面具有更高的颗粒酶和INF-γ的分泌能力。同样高乳酸(乳酸根离子≥40mM)处理后的T细胞与高钾处理后的T细胞结果一致(Feng Q, Liu Z,Yu X, Huang T, Chen J, Wang J, et al. Lactate increases stemness of CD8 + Tcells to augment anti-tumor immunity. Nat Commun. 2022; 13: 4981.)。但是目前在CD8+ T细胞类干细胞转化的过程中存在如下不足:(1)尚无针对诱导CD8+ T细胞干性化的试剂或培养基,也无成熟的细胞诱导时间和诱导节点(即细胞何时添加钾离子和乳酸等处理效果更佳、处理时间多长最佳等数据和方案);(2)高K+或高乳酸单独诱导时长需要8-10天,很容易造成CD8+ T细胞老化或衰竭,不利于后期在CART细胞、基因编辑等方面的使用;(3)单独使用高K+或乳酸不能形成促进CD8+ T细胞类干细胞化的协同效应。因此,CD8+ T细胞在干性化的研究和应用过程中受到一定程度阻碍。
发明内容
本发明的目的是提供一种类干细胞化诱导培养基,可以实现CD8+ T细胞向类干细胞化转变,转变后的细胞具备干细胞的特性,回输动物体或者人体后,细胞具备更强的肿瘤杀灭能力,同时消减CD8+ T细胞衰竭和T细胞免疫抑制问题;另外,转变后的细胞也可以用于CART细胞生产和基因组编辑细胞生产,解决CD8+ T细胞在肿瘤治疗过程中持久性问题;本发明同时提供了类干细胞化诱导培养基的制备方法和应用。
本发明所述的类干细胞化诱导培养基包括类干细胞化诱导组分、细胞生长营养组分、维生素与细胞因子组分和其他组分;其中,
类干细胞化诱导组分为乳酸钾、乳酸、乙醇胺、磷酸乙醇胺、CDP-乙醇胺和磷脂酰乙醇胺;
细胞生长营养组分为葡萄糖、氨基酸与氨基酸盐混合物、胆固醇、亚麻酸、亚油酸、花生四烯酸和棕榈酸;
维生素与细胞因子组分为D-泛酸钙、盐酸吡哆醛、盐酸硫胺素、肌醇、生物素、叶酸、核黄素、维生素C磷酸镁、生育酚、维生素A醋酸酯、甲萘醌、人血白蛋白、转铁蛋白和胰岛素;
其他组分为氯化钠、乙磺酸、盐酸腐胺、超氧化物歧化酶、碳酸氢钠、酚红和水。
所述的氨基酸与氨基酸盐混合物为α-甘氨酸、L-α-丙氨酸、L-α-精氨酸盐酸盐、L-α-天冬酰胺、L-α-天冬氨酸、L-α-胱氨酸二盐酸盐、L-α-谷氨酸、L-α-谷氨酰胺、L-α-盐酸组氨酸、L-α-异亮氨酸、L-α-亮氨酸、L-α-赖氨酸盐酸盐、L-α-甲硫氨酸、L-α-苯丙氨酸、L-α-脯氨酸、L-α-丝氨酸、L-α-苏氨酸、L-α-色氨酸、L-α-酪氨酸二钠和L-α-缬氨酸的混合物。
所述的类干细胞化诱导组分的组成如下,以类干细胞化诱导培养基的体积计:
乳酸钾40-45mM、乳酸5-10mM、乙醇胺0.5-2mM、磷酸乙醇胺1-2mM、CDP-乙醇胺1-2mM和磷脂酰乙醇胺1-3mM。
所述的细胞生长营养组分的组成如下,以类干细胞化诱导培养基的体积计:
葡萄糖25-30mM、氨基酸与氨基酸盐混合物、胆固醇0.1-0.12mM、亚麻酸0.2-0.25mM、亚油酸0.2-0.25mM、花生四烯酸0.3-0.5mM和棕榈酸0.1-0.13mM;其中,氨基酸与氨基酸盐混合物的组成为α-甘氨酸0.4-0.5mM、L-α-丙氨酸0.4-0.5mM、L-α-精氨酸盐酸盐0.4-0.5mM、L-α-天冬酰胺0.4-0.5mM、L-α-天冬氨酸0.4-0.5mM、L-α-胱氨酸二盐酸盐0.6-0.7mM、L-α-谷氨酸0.4-0.5mM、L-α-谷氨酰胺2-3mM、L-α-盐酸组氨酸0.6-0.7mM、L-α-异亮氨酸0.6-0.7mM、L-α-亮氨酸0.6-0.7mM、L-α-赖氨酸盐酸盐1-2mM、L-α-甲硫氨酸0.4-0.5mM、L-α-苯丙氨酸0.6-0.7mM、L-α-脯氨酸0.4-0.5mM、L-α-丝氨酸0.4-0.5mM、L-α-苏氨酸0.6-0.7mM、L-α-色氨酸0.6-0.7mM、L-α-酪氨酸二钠0.6-0.7mM和L-α-缬氨酸0.6-0.7mM。
所述的维生素与细胞因子组分的组成如下,以类干细胞化诱导培养基的体积计:
D-泛酸钙0.02-0.03mM、盐酸吡哆醛0.02-0.03mM、盐酸硫胺素0.02-0.03mM、肌醇0.001-0.002mM、生物素0.001-0.002mM、叶酸0.001-0.002mM、核黄素0.002-0.003mM、维生素C磷酸镁3-4.5mM、生育酚0.01-0.02mM、维生素A醋酸酯0.001-0.002mM、甲萘醌0.01-0.02mM、人血白蛋白10-12mM、转铁蛋白0.01-0.02mM和胰岛素0.02-0.03mM。
所述的其他组分的组成如下,以类干细胞化诱导培养基的体积计:
氯化钠80-100mM、乙磺酸50-60mM、盐酸腐胺3-5mM、超氧化物歧化酶1-2mM、碳酸氢钠80-90mM、酚红0.5-0.8mM和水余量。
本发明所述的类干细胞化诱导培养基的制备方法是将类干细胞化诱导组分、细胞生长营养组分、维生素与细胞因子组分和除水以外的其他组分溶于水后,过滤,得到类干细胞化诱导培养基。
所述的水为超纯水。
所述的过滤是采用0.22μm过滤膜进行过滤。
本发明所述的类干细胞化诱导培养基的制备方法具体是首先将类干细胞化诱导组分、细胞生长营养组分、维生素与细胞因子组分和除水以外的其他组分溶于部分超纯水中,然后定容至1L,采用0.22μm过滤膜过滤除菌,过滤后保存于4℃环境备用,得到类干细胞化诱导培养基。
本发明所述的类干细胞化诱导培养基的应用是T淋巴细胞筛选富集后得到CD8+ T细胞,将CD8+ T细胞加入到类干细胞化诱导培养基中培养,得到类干细胞化的CD8+ T细胞。
所述的T淋巴细胞为对数生长期的T淋巴细胞。
所述的培养时间为48-72h,优选为72h。
本发明所述的类干细胞化诱导培养基的应用具体是培养T淋巴细胞至细胞进入对数生长期,采用磁珠筛选富集CD8+ T细胞,离心收集细胞并进行计数,用类干细胞化诱导培养基重悬1×106-1×107个细胞,转移至5% CO2浓度、37℃的二氧化碳培养箱中培养48-72h,即获得类干细胞化的CD8+ T细胞。
本发明所述的类干细胞化诱导培养基的诱导机制如图1所示,40-45mM K+降低细胞的线粒体中柠檬酸含量,致使乙酰辅酶A在细胞核中含量下降,导致Ifng基因(γ-干扰素)启动子区组蛋白乙酰化水平下降,促进细胞自噬发生,降低线粒体中三羧酸循环反应;同时,乙醇胺、磷酸乙醇胺、CDP-乙醇胺和磷脂酰乙醇胺加强甘乃迪信号通路(Kennedypathway),该通路促进吞噬包形成吞噬体,协同加强细胞自噬,最终实现CD8+ T细胞类干细胞化。本发明为了加快细胞类干细胞化进程,在高K+和甘乃迪信号通路协同加强的基础上,采用高乳酸(乳酸钾与乳酸总含量≥40mM)抑制组蛋白去乙酰化酶活性,提升T细胞干性化标志基因Tcf7表达水平,进一步加强CD8+ T细胞类干细胞化。
本发明的类干细胞化诱导培养基中高K+和高乳酸能够通过不同途径促进CD8+ T细胞向类干细胞化转变,乙醇胺、磷酸乙醇胺、CDP-乙醇胺和磷脂酰乙醇胺等通过增强细胞自噬,协同提升CD8+ T细胞向类干细胞化转变。
本发明的类干细胞化诱导培养基中细胞生长营养组分、维生素与细胞因子组分和其他组分主要用于维持细胞的正常生长。
本发明的有益效果如下:
T淋巴细胞在细胞药物制备、临床疾病治疗时面临细胞衰竭、繁殖能力差、杀伤力弱、机体发挥时效短等问题,为了提升T细胞稳定持久增殖、自我更新、机体回输成活率、肿瘤清除能力及其他多能性等干细胞特性,本发明构建了体系完整的用于CD8+ T细胞类干细胞化转变的诱导培养基。
(1)本发明提供了一种针对诱导CD8+ T细胞干性化的培养基,确定了最佳的细胞诱导时间和诱导节点;
(2)本发明将现有的高K+或高乳酸单独诱导时长从8-10天缩短至48-72h,避免长时间的培养造成CD8+ T细胞老化或衰竭,有利于后期在CART细胞、基因编辑等方面的使用;
(3)本发明联合使用高K+、高乳酸(乳酸钾与乳酸总含量≥40mM)以及乙醇胺、磷酸乙醇胺、CDP-乙醇胺和磷脂酰乙醇胺,通过三种调控途径实现促进CD8+ T细胞类干细胞化的协同效应。
本发明的培养基解决了CD8+ T细胞在使用中的诸多问题,可以用于肿瘤疾病及其他疾病的防治,并能为类似新型细胞培养基的开发提供参考。
附图说明
图1是本发明的高K+、高乳酸和甘乃迪信号通路(Kennedy pathway)协同作用示意图。
图2是培养的野生型T细胞图。
图3是野生型CD8+ T细胞和类干细胞化CD8+ T细胞的代谢物检测结果图。
图4 是野生型CD8+ T细胞和类干细胞化CD8+ T细胞的标记蛋白Tcf7的表达分析图。
图5是本发明培养基各组分协同作用分析图,以Tcf7 mRNA水平为观察点。
图6是治疗过程中小鼠体温变化图。
图7是治疗过程中小鼠体重变化图。
图8是治疗过程中小鼠瘤块体积变化图。
图9是肝脏和肾脏病理变化图。
图10是肿瘤小鼠存活率实验结果图。
实施方式
以下结合实施例对本发明做进一步描述。
实施例1
(1)T淋巴细胞分离与激活
① CD3+抗体包被平板
于6孔板中加入2ml/孔的PBS缓冲液,然后加入CD3+抗体至终浓度10μg/ml,置于37℃CO2培养箱孵育2小时,而后吸去PBS,用新鲜的预热PBS清洗平板2次,再用无血清RPMI-1640培养基清洗1次。
②成熟 T淋巴细胞分离与培养
1、抽取C57BL/6小鼠新鲜血液1mL,马上加入含有10μL肝素抗凝剂的1.5mL离心管中。为保持淋巴细胞的活性,采血后马上进行分离。
2、将1mL新鲜血液转移至15mL无菌离心管中,加入3mL的PBS或0.9%NaCl溶液稀释血液。稀释血液可降低红细胞的凝聚,提高淋巴细胞收获量。
3、取4mL淋巴细胞分离液加入另一个新的15mL无菌离心管中,并升温到室温。
4、用巴斯德玻璃吸管吸取步骤2中的4ml稀释后的血液样品,沿管壁缓慢铺到淋巴细胞分离液上面,不要打乱液层界面。
5、水平转子2000r/min或700×g离心20min,室温20℃。存放2h以上的血液应离心30min。
6、离心后管底是红细胞,中间层是分离液,最上层是血浆。血浆层与分离液之间是一薄层较致密的白膜,即为淋巴细胞层。用吸管直接插入到白膜层(淋巴细胞层)并吸取该层,放入另一试管中。
7、加10ml Hank’s液稀释步骤6中分离的淋巴细胞,250×g离心10min,弃上清,得到淋巴细胞沉淀。重复上述步骤2次,除去血小板和抗凝物质,最终得到淋巴细胞沉淀。
③ T淋巴细胞激活与繁殖
用2mL RPMI-1640全培养基重悬②中步骤7得到的淋巴细胞沉淀,转移至①中的CD3+抗体包被的6孔板中,置于CO2培养箱培养24小时,而后加入白介素-2(终浓度1000U/ml)刺激细胞生长,所得结果见图2,繁殖起来的细胞即为野生型T淋巴细胞,一般3天后进入对数生长期。
(2)类干细胞化诱导培养基配制
选取类干细胞化诱导培养基各组分,根据组分溶解特点分批次溶于800mL超纯水中,各组分充分溶解后,加超纯水定容至1L,然后用0.22μm过滤膜过滤除菌,培养基各组分最终浓度乳酸钾40mM、乳酸6mM、乙醇胺1mM、磷酸乙醇胺1mM、CDP-乙醇胺1mM、磷脂酰乙醇胺2mM;葡萄糖25mM、氨基酸与氨基酸盐混合物(α-甘氨酸0.4mM、L-α-丙氨酸0.4mM、L-α-精氨酸盐酸盐0.5mM、L-α-天冬酰胺0.4mM、L-α-天冬氨酸0.4mM、L-α-胱氨酸二盐酸盐0.7mM、L-α-谷氨酸0.4mM、L-α-谷氨酰胺2mM、L-α-盐酸组氨酸0.6mM、L-α-异亮氨酸0.6mM、L-α-亮氨酸0.7mM、L-α-赖氨酸盐酸盐1mM、L-α-甲硫氨酸0.5mM、L-α-苯丙氨酸0.6mM、L-α-脯氨酸0.4mM、L-α-丝氨酸0.4mM、L-α-苏氨酸0.6mM、L-α-色氨酸0.7mM、L-α-酪氨酸二钠0.6mM、L-α-缬氨酸0.6mM)、胆固醇0.1mM、亚麻酸0.2mM、亚油酸0.2mM、花生四烯酸0.3mM、棕榈酸0.1mM;D-泛酸钙0.02mM、盐酸吡哆醛0.02mM、盐酸硫胺素0.02mM、肌醇0.001mM、生物素0.001mM、叶酸0.001mM、核黄素0.002mM、维生素C磷酸镁3mM、生育酚0.01mM、维生素A醋酸酯0.001mM、甲萘醌0.01mM、人血白蛋白10mM、转铁蛋白0.01mM、胰岛素0.02mM;氯化钠100mM、乙磺酸50mM、盐酸腐胺3mM、超氧化物歧化酶1mM、碳酸氢钠80mM、酚红0.8mM。
(3)CD8+ T细胞收集
待步骤(1)中T细胞(3×108-5×108个细胞)繁殖进入对数生长期,采用IPHASE CD8+ T Cells, Negative Selection试剂盒(货号:082A04.21,北京汇智和源生物技术有限公司)筛选富集CD8+ T细胞(1×106-1×107个),转接入类干细胞化诱导培养基培养72h,然后取少许细胞(1×104-2×104个细胞),检测糖酵解代谢产物具体为葡萄糖-6-磷酸(G-6-P)、1,6-二磷酸果糖(F-1,6-BP)和乳酸(Lactate),检测氨基酸具体为丝氨酸(Serine)、赖氨酸(Lysine)和蛋氨酸(Methionine)。通过所检测的氨基酸和糖酵解代谢产物评价CD8+ T细胞的类干细胞化转变情况(Vodnala SK, Eil R, Kishton RJ, Sukumar M, Yamamoto TN,Ha NH, et al. T cell stemness and dysfunction in tumors are triggered by acommon mechanism. Science. 2019; 363.)。
检测结果见图3。图3中糖酵解代谢物浓度单位为mM,氨基酸浓度单位为μM。代谢物在类干细胞化T细胞中含量下降。从代谢物水平变化说明本发明的培养基在培养CD8+ T细胞72h后类干细胞化形成。
(4)类干细胞化Tcf7标记蛋白表达分析
①制冰、配制细胞裂解液(裂解液:蛋白酶抑制剂为1ml:10μl)并预冷。
②收集细胞加入细胞裂解液(细胞:细胞裂解液为0.2g:1.5ml)。
③蛋白含量定量并SDS-PAGE凝胶蛋白电泳。
④转膜:将浓缩胶与分离胶分离,保留分离胶。剪下大约粗细为2-3cm的PVDF膜,长度与分离胶接近,放入甲醇中活化3-5min,根据目标蛋白分子量大小在Marker条带上找到对应大概位置,将PVDF膜放在相应位置。转模采用三明治方法,夹子由白到黑为海绵、滤纸、PVDF膜、分离胶、滤纸、海绵,将转膜的夹子夹紧放入转膜槽,放入冰盒,倒入4℃1×转模液,由负极向正极跑,打开电源,恒流200mA,转模2.5h。
⑤封闭:将转模后的PVDF膜放入含5%脱脂奶粉的TBST缓冲液中,常温摇床1.5h。
⑥一抗孵育:配制含1%脱脂奶粉的TBST缓冲液,按一抗(Anti-TCF7L2, ab272235;GAPDH,ab9485)说明书的稀释浓度加入一抗,4℃摇床孵育13h。
⑦洗膜:一抗孵育结束,用TBST洗膜,每次5-10min,5次。
⑧二抗孵育:洗膜结束后,配制含1%脱脂奶粉的TBST缓冲液,按二抗(ab205718)说明书的稀释浓度加入二抗,常温摇床孵育1h。
⑨洗膜:二抗孵育结束,用TBST洗膜,每次5-10min,5次。
⑩封膜和曝光:打开暗夹,平铺保鲜膜并固定,按试剂A和试剂B各500µL,以1:1的比例配制好ECL发光液(北京全式金生物技术股份有限公司,EasySee® Western BlotKit)(注意避光),将PVDF膜平铺在保鲜膜上,滴发光液,等其反应5min左右,盖上保鲜膜并去除气泡。进入暗室,如果荧光明显,取出X光片4张,平铺到暗夹中,合上盖子3-5min,取出X光片放入显影液中显影5-10min,取出用清水清洗后再放入定影液中5-10min,取出再次清洗,晾干保存。如果荧光不明显,取出X光片4张,平铺到暗夹中,合上盖子4h,后面操作同上。
结果如图4所示,与野生型T细胞相比,类干细胞化CD8+ T细胞的标记蛋白Tcf7的表达显著上调,说明CD8+ T细胞向类干细胞化转变成功。
(5)各组分协同效应分析
将野生型CD8+ T细胞分为未处理野生型细胞组、高K+组、高乳酸组、甘乃迪信号途径组分组和本发明培养基组共计5组,分别接种在T75细胞培养瓶中,每组接种细胞数目1×107-2×107个细胞,每组3个重复。未处理野生型细胞组采用的培养基组分为不含类干细胞化诱导组分,其它组分同实施例1培养基组分;高K+组采用的培养基组分为将类干细胞化诱导组分替换为40mM 乳酸钾,其它组分同实施例1培养基组分;高乳酸组采用的培养基组分为将类干细胞化诱导组分替换为40mM 乳酸,其它组分同实施例1培养基组分;甘乃迪信号途径组分组采用的培养基组分为将类干细胞化诱导组分替换为2mM磷脂酰乙醇胺、1mM乙醇胺、1mM磷酸乙醇胺和1mM CDP-乙醇胺,其它组分同实施例1培养基组分;本发明培养基组采用的培养基组分为实施例1培养基组分。
细胞每隔24h,取1×105-2×105个细胞提取全RNA,采用实时定量PCR检测Tcf7基因的表达变化,具体程序如下:使用RNAprep pure Cell/Bacteria Kit 试剂盒提取各组细胞总RNA,使用PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time)takara逆转录试剂盒合成cDNA。设计目的基因Tcf7和内参基因GAPDH的引物。引物序列见表1,引物由TAKARA宝生物工程(大连)有限公司合成。
利用SYBR GREEN染料检测各组细胞Tcf7表达情况,并以GAPDH基因作为内参照。利用SYBR Premix Ex TaqII染料法荧光定量试剂盒,反应体系为20μL,包括:
1) 按表2组分配制Real time PCR反应液(在冰上进行),每个反应做3个平行;
反应在Stepone Plus Real Time PCR System Cycler (Applied Biosystem)上进行,反应程序为:95℃15min预变性,95℃ 15s变性,53℃ 30s退火,68℃ 35s延伸,溶解曲线在60-90℃由机器自行完成,各组Tcf7相对于对照(未处理野生型细胞组)的表达通过GAPDH RNA进行校正。
2)数据使用Real Time PCR System Cycler 自带软件进行分析,Ct值及相对表达率(表达倍数,图5纵坐标)依照Pfaffl计算方法进行。
图5结果显示,高K+和高乳酸均能显著促进CD8+ T细胞向类干细胞化转变,高K+效果强于高乳酸,但是高K+需要10天以上,而高乳酸需要7天;另外,甘乃迪信号途径组分单独使用诱导细胞发生类干细胞化效果较低,可能是仅仅促进了细胞自噬,对于启动类干细胞化关键基因能力不足;当类干细胞化诱导成分联合使用后(本发明培养基),CD8+ T类干细胞化标注基因Tcf7表达从48h开始显著提升,72h后表达进入稳定期,结果说明,本发明三种诱导组分联合使用,协同效应显著,极大地缩短诱导时间。
(6)类干细胞化CD8+ T细胞回输荷瘤小鼠杀伤肿瘤细胞能力研究
1)类干细胞化CD8+ T细胞药效学评价
动物:C57BL/6,female,6周
模型:构建MC38小鼠结直肠癌细胞荷瘤小鼠模型
模型构建过程为:培养一定数量生长状况良好的小鼠结直肠癌MC38细胞,收集细胞进行下一步实验。将从北京维通利华实验动物技术有限公司购买的6周龄雌鼠随机分为两组:肿瘤模型组(60只)和对照组(10只),60只肿瘤模型组于每只小鼠右大腿根部皮下接种2×106个细胞,10只对照组注射生理盐水以便于观察注射部位瘤块不是因为注射过程发生感染而引起以及观察注射部位瘤块的变化。待接种肿瘤细胞7天后,一般即可出现肉眼可见肿瘤,每隔三日测量肿瘤体积,包括瘤体的长径与短径,肿瘤体积:V=(a×b2)/2(a为肿瘤的最长径,b为肿瘤的最短径)。
给药方式:待小鼠皮下肿瘤体积为50mm3左右时,从60只肿瘤模型组中选40只瘤块大小一致且小鼠生长状态良好的肿瘤模型小鼠,分为5组,每组8只,开始分组给药,药物为野生型CD8+ T细胞和本发明的类干细胞化CD8+ T细胞,每两周取血一次。具体给药方式见表3。
2)药效学结果
①体温检测
于第一次用药前先用电子体温计酒精消毒后插入小鼠肛门测量,然后于各组用药后连续七日的同一时间测量各组小鼠的肛温,结果见图6,图6中的温度差是指组内用药前和用药后的温度差。图6显示,与第1组(未处理对照组)相比,第4组和第5组尾静脉注射类干细胞化CD8+ T细胞后体温逐日升高,第四日体温最高,然后又回落,而第3组小鼠体温上升不明显。以上结果说明第4组和第5组尾静脉注射类干细胞化CD8+ T细胞后,类干细胞化CD8+ T细胞杀灭小鼠体内MC38小鼠结直肠癌细胞,从而导致体温上升,而第3组因尾静脉注射细胞数量较少、以及第2组野生型CD8+ T细胞等都对小鼠体内MC38小鼠结直肠癌细胞杀灭效果较低,所以体温变化不明显。
②体重检测
从各组小鼠平均体重变化来看(图7),除第五组外,其它各组小鼠体重随着天数增加总体上出现体重下降,然后死亡(图中后续天数未再显示,代表已经死亡),而第五组在最后阶段体重略有上升,说明该剂量的类干细胞化CD8+ T细胞显著控制住了肿瘤细胞的生长。
③肿瘤大小检测
用游标卡尺测量小鼠肿瘤的长短径,根据公式计算肿瘤体积,绘制各组小鼠平均肿瘤体积变化曲线,肿瘤体积:V=(a×b2)/2(a为肿瘤的最长径,b为肿瘤的最短径)。类干细胞化CD8+ T细胞治疗组小鼠肿瘤瘤块体积明显缩小,其中高剂量组(第5组)最明显,见图8。
3)类干细胞化CD8+ T细胞治疗安全性初评
取第1组和第5组的小鼠的肝脏、肾脏固定于固定液,经冲洗、脱水机脱水、石蜡包埋、HE染色等步骤,光学显微镜下观察病理组织切片。如图9所示,第1组(未处理对照组)肝细胞形态结构正常,无病理现象;肾小球及肾小管结构正常,无病理变化。从类干细胞化CD8+ T细胞(表3第5组)回输后模型小鼠与未处理对照组小鼠肝、肾组织切片对比图可以看出,类干细胞化CD8+ T细胞对机体没有毒性。
(7)类干细胞化CD8+ T细胞持久稳定性评价
动物:C57BL/6,female,6周
模型:构建MC38小鼠结直肠癌细胞荷瘤小鼠模型
模型构建过程为:培养一定数量生长状况良好的小鼠结直肠癌MC38细胞,收集细胞进行下一步实验。将从北京维通利华实验动物技术有限公司购买的6周龄雌鼠随机分为两组:肿瘤模型组(30只)和对照组(5只),30只肿瘤模型组于每只小鼠右大腿根部皮下接种2×106个细胞,5只对照组注射生理盐水。待接种肿瘤细胞7天后,一般即可出现肉眼可见肿瘤,每隔三日测量肿瘤体积,包括瘤体的长径与短径,肿瘤体积:V=(a×b2)/2(a为肿瘤的最长径,b为肿瘤的最短径)。
给药方式:待小鼠皮下肿瘤体积为50mm3左右时,从30只肿瘤模型组中选24只瘤块大小一致且小鼠生长状态良好的肿瘤模型小鼠,分为3组,每组8只,开始分组给药,药物为PBS缓冲液、野生型CD8+ T细胞和本发明的类干细胞化CD8+ T细胞,在第1天和第7天分两次给药。具体给药方式见表4。
结果如图10所示,PBS对照组(第1组)肿瘤模型小鼠,在第16天全部死亡,野生型CD8+ T细胞治疗组(第2组)在第23天小鼠也全部死亡,而类干细胞化CD8+ T细胞组(第3组)至第60天死亡率为37.5%(3/8),可见经本发明培养基处理后的CD8+ T 细胞具有持久增殖、自我更新、机体回输成活率高等特点。
本发明的培养基在48-72h内可以将CD8+ T细胞转变成类干细胞化的T细胞,使其具备干细胞的特性,回输肿瘤小鼠后具备更强的杀灭肿瘤能力,可以解决肿瘤(实体瘤)免疫治疗过程中免疫抑制的问题、解决T细胞衰竭和繁殖能力差等问题。
Claims (10)
1.一种类干细胞化诱导培养基,其特征在于包括类干细胞化诱导组分、细胞生长营养组分、维生素与细胞因子组分和其他组分;其中,
类干细胞化诱导组分为乳酸钾、乳酸、乙醇胺、磷酸乙醇胺、CDP-乙醇胺和磷脂酰乙醇胺;
细胞生长营养组分为葡萄糖、氨基酸与氨基酸盐混合物、胆固醇、亚麻酸、亚油酸、花生四烯酸和棕榈酸;
维生素与细胞因子组分为D-泛酸钙、盐酸吡哆醛、盐酸硫胺素、肌醇、生物素、叶酸、核黄素、维生素C磷酸镁、生育酚、维生素A醋酸酯、甲萘醌、人血白蛋白、转铁蛋白和胰岛素;
其他组分为氯化钠、乙磺酸、盐酸腐胺、超氧化物歧化酶、碳酸氢钠、酚红和水。
2.根据权利要求1所述的类干细胞化诱导培养基,其特征在于所述的氨基酸与氨基酸盐混合物为α-甘氨酸、L-α-丙氨酸、L-α-精氨酸盐酸盐、L-α-天冬酰胺、L-α-天冬氨酸、L-α-胱氨酸二盐酸盐、L-α-谷氨酸、L-α-谷氨酰胺、L-α-盐酸组氨酸、L-α-异亮氨酸、L-α-亮氨酸、L-α-赖氨酸盐酸盐、L-α-甲硫氨酸、L-α-苯丙氨酸、L-α-脯氨酸、L-α-丝氨酸、L-α-苏氨酸、L-α-色氨酸、L-α-酪氨酸二钠和L-α-缬氨酸的混合物。
3.根据权利要求1所述的类干细胞化诱导培养基,其特征在于所述的类干细胞化诱导组分的组成如下,以类干细胞化诱导培养基的体积计:
乳酸钾40-45mM、乳酸5-10mM、乙醇胺0.5-2mM、磷酸乙醇胺1-2mM、CDP-乙醇胺1-2mM和磷脂酰乙醇胺1-3mM。
4.根据权利要求1所述的类干细胞化诱导培养基,其特征在于所述的细胞生长营养组分的组成如下,以类干细胞化诱导培养基的体积计:
葡萄糖25-30mM、氨基酸与氨基酸盐混合物、胆固醇0.1-0.12mM、亚麻酸0.2-0.25mM、亚油酸0.2-0.25mM、花生四烯酸0.3-0.5mM和棕榈酸0.1-0.13mM;其中,氨基酸与氨基酸盐混合物的组成为α-甘氨酸0.4-0.5mM、L-α-丙氨酸0.4-0.5mM、L-α-精氨酸盐酸盐0.4-0.5mM、L-α-天冬酰胺0.4-0.5mM、L-α-天冬氨酸0.4-0.5mM、L-α-胱氨酸二盐酸盐0.6-0.7mM、L-α-谷氨酸0.4-0.5mM、L-α-谷氨酰胺2-3mM、L-α-盐酸组氨酸0.6-0.7mM、L-α-异亮氨酸0.6-0.7mM、L-α-亮氨酸0.6-0.7mM、L-α-赖氨酸盐酸盐1-2mM、L-α-甲硫氨酸0.4-0.5mM、L-α-苯丙氨酸0.6-0.7mM、L-α-脯氨酸0.4-0.5mM、L-α-丝氨酸0.4-0.5mM、L-α-苏氨酸0.6-0.7mM、L-α-色氨酸0.6-0.7mM、L-α-酪氨酸二钠0.6-0.7mM和L-α-缬氨酸0.6-0.7mM。
5.根据权利要求1所述的类干细胞化诱导培养基,其特征在于所述的维生素与细胞因子组分的组成如下,以类干细胞化诱导培养基的体积计:
D-泛酸钙0.02-0.03mM、盐酸吡哆醛0.02-0.03mM、盐酸硫胺素0.02-0.03mM、肌醇0.001-0.002mM、生物素0.001-0.002mM、叶酸0.001-0.002mM、核黄素0.002-0.003mM、维生素C磷酸镁3-4.5mM、生育酚0.01-0.02mM、维生素A醋酸酯0.001-0.002mM、甲萘醌0.01-0.02mM、人血白蛋白10-12mM、转铁蛋白0.01-0.02mM和胰岛素0.02-0.03mM。
6.根据权利要求1所述的类干细胞化诱导培养基,其特征在于所述的其他组分的组成如下,以类干细胞化诱导培养基的体积计:
氯化钠80-100mM、乙磺酸50-60mM、盐酸腐胺3-5mM、超氧化物歧化酶1-2mM、碳酸氢钠80-90mM、酚红0.5-0.8mM和水余量。
7.一种权利要求1-6任一所述的类干细胞化诱导培养基的制备方法,其特征在于将类干细胞化诱导组分、细胞生长营养组分、维生素与细胞因子组分和除水以外的其他组分溶于水后,过滤,得到类干细胞化诱导培养基。
8.一种权利要求1-6任一所述的类干细胞化诱导培养基的应用,其特征在于T淋巴细胞筛选富集后得到CD8+ T细胞,将CD8+ T细胞加入到类干细胞化诱导培养基中培养,得到类干细胞化的CD8+ T细胞。
9.根据权利要求8所述的类干细胞化诱导培养基的应用,其特征在于所述的T淋巴细胞为对数生长期的T淋巴细胞。
10.根据权利要求8所述的类干细胞化诱导培养基的应用,其特征在于所述的培养时间为48-72h。
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