CN108354946A - 一种人体免疫复合因子的制备方法及应用 - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
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- A—HUMAN NECESSITIES
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
本发明提出了一种人体免疫复合因子的制备方法,包括人外周血单个核细胞的分离、人外周血单个核细胞的培养与诱导、细胞的纯化除杂以及细胞的破碎和产物的提取。其作用机理是将外周血(包括脐血)中的单个核细胞(主要是淋巴细胞)培养扩增,在一定条件下诱导其产生有效的免疫复合因子,包括多肽及各种小分子物质,所以临床效果更明显,作用更全面。另外,由于排除了外源性细胞的输入,输入后不会引起发热、乏力、肌肉酸痛等副作用,所以安全性高,免疫功能更全面。本发明制备的单核细胞诱导冻融提取物,使用方便、易于保存和运输,可同时作为提高机体免疫力、抗衰老及治疗肿瘤等疾病的有效生物制剂。
Description
技术领域
本发明涉及生物免疫领域,特别是指一种人体免疫复合因子的制备方法和应用。
背景技术
PBMC(peripheral blood mononuclear cell),外周血单个核细胞,顾名思义,其主要细胞类型为血液里具有单个核的细胞,包括淋巴细胞(T/B)、单核细胞、吞噬细胞、树突状细胞和其他少量细胞类型,其中淋巴细胞占很大一部分。
现有生物免疫治疗领域的技术,主要是通过对干细胞、间充质干细胞、淋巴细胞等基因改造,再将这些细胞输入机体,包括自体免疫细胞或异体过继性免疫疗法的细胞输入治疗,包括细胞因子诱导后的杀伤细胞(即CIK)、各种基因转染改造后的免疫细胞、免疫检查点阻断剂(CTLA-4阻断剂、PD-1/PD-L1阻断剂等)、树突状细胞疫苗等都是已知的免疫领域较为成熟的技术。目前干细胞、PD-1、PD-L1及CAR-T等是生物免疫治疗领域的热点。上述技术,由于外源性基因改造及借助病毒转染后的细胞输入,可能会带来明显的副作用(包括发热、乏力、肌肉酸痛等),使得产品安全性有待提高;另外,治疗的效果以及应用范围往往难以确定。干细胞、PD-1及CAR-T等生物治疗,是诱导细胞功能单向分化,属于特定的靶向治疗,所以作用单一。特别是细胞输入机体后,细胞内的活性物质并不能够从细胞核与细胞质中完全释放出来,甚至可能被细胞内的各种酶分解,最终透过细胞膜释放的有效物质减少,从而达不到理想的治疗效果。
发明内容
本发明所要解决的技术问题,就是提出一种人体免疫复合因子的制备方法。此外,本发明还提出了上述人体免疫复合因子作为免疫制剂在临床治疗上的应用范围。
为解决上述技术问题,本发明采用以下技术方案予以实现:
一种人体免疫复合因子的制备方法,包括如下步骤:
1)人外周血单个核细胞的分离:通过仪器单采或人工抽取人外周血,肝素抗凝处理及Ficoll密度梯度离心法分离得到单个核细胞;
2)人外周血单个核细胞的培养与诱导:将所得的单个核细胞重悬并置于AIM-V无血清培养基培养,调整细胞浓度为1×106个/mL,同时加入植物血凝素使得植物血凝素在体系中所占含量为10-100ug/mL,继续置于37℃、5%的CO2孵箱中培养,以后隔日补加AIM-V无血清培养液,培养5-10天,使细胞浓度至少达到1×107个/mL,收获细胞;
3)细胞的纯化除杂:将收获的细胞用生理盐水洗涤三次,以除去培养液中所有成分;
4)细胞破碎和产物收集:在收集的细胞中加入生理盐水,将细胞稀释成2×106个/mL悬液,经超声粉碎并重复冻融裂解处理后,离心收集上清即为免疫复合因子,冷冻保藏即可。
作为优选地,所述外周血包括脐血。
作为优选地,所述步骤2)和步骤4)中离心操作的条件为4℃、2000rpm/min,离心时间为5分钟。
作为优选地,所述步骤2)中,加入的植物血凝素在体系中所占含量为50ug/mL。
作为优选地,所述步骤3)中,用生理盐水洗涤后的细胞需检测以排除细菌、真菌、内毒素污染。
作为优选地,所述步骤4)中,超声粉碎的条件为15-25kHz,次数至少3次,每次10秒。
作为优选地,所述步骤4)中,冻融裂解为-20℃~-196℃温度条件下冻结,室温条件下融化,冻融操作重复至少3次。
本发明还提出了上述人体免疫复合因子作为免疫制剂在临床上提高机体免疫力和治疗衰老、肿瘤、自身免疫性疾病、神经性疾病、艾滋病的应用。
本发明的人体免疫复合因子可用于临床应用的范围包括:
抗衰老:美容、养颜、失眠症、脱发及白发(变黑)。
恶性肿瘤:各类肿瘤,包括实体瘤及血液恶性肿瘤如急性淋巴细胞白血病(ALL)。
自身免疫性疾病:类风湿关节炎、风湿性关节炎、类风湿性心脏病、系统性红斑狼疮、多发性硬化症、自身免疫性溶血性贫血、溃疡性结肠炎、甲状腺机能亢进、原免疫性疾病发性血小板紫癜。
神经性疾病:阿尔茨海默氏病(老年痴呆,AD)、脑性麻痹、缺氧缺血性脑病、自闭症、创伤性脑损伤、脊髓损伤、听力损失、肌萎缩性脊髓侧索硬化症、多发性神经炎、神经性皮炎、强直性脊柱炎。
其他疾病:II型糖尿病、艾滋病、多发性硬化症、皮肤病(如硬皮病、银屑病)、过敏性疾病(如过敏性哮喘、枯草热)、高尿酸血症、矽肺、慢性股骨头坏死、慢性肾炎性肾病、慢性肝病、慢性疲劳、肥胖症。临床上均可能获得一定程度的疗效。
作为优选地,应用的具体方法为:
a)将免疫制剂以1:20的比例加入生理盐水袋中,静脉滴注,3日1次,5次为一个疗程;
b)直接采用免疫制剂进行局部肌肉注射或者肿瘤局部注射,1-5mL/次。
与现有的临床生物学技术比较,本发明具有的有益效果为:本发明的作用机理是将外周血(包括脐血)中的单个核细胞(主要是淋巴细胞)培养扩增,诱导其产生有效的免疫复合因子,包括多肽及各种小分子物质,所以临床效果更明显,作用更全面。另外,由于排除了外源性细胞的输入,输入后不会引起发热、乏力、肌肉酸痛等副作用,所以安全性高,患者更容易接受,免疫疗效更全面。本发明制备的单核淋巴细胞冻融提取物,使用方便、易于保存和运输,可同时作为提高机体免疫力、抗衰老及治疗肿瘤及其它疾病的有效生物制剂。
具体实施方式
为让本领域的技术人员更加清晰直观的了解本发明,下面将对本发明作进一步的说明。
本发明提出的人体免疫复合因子的制备方法,包括如下步骤:
1)人外周血单个核细胞的分离:仪器单采或人工抽取人外周血(包括脐血),肝素抗凝处理及Ficoll密度梯度离心法分离得到单个核细胞;
2)人外周血单个核细胞的培养与诱导:将所得的单个核细胞重悬并置于AIM-V无血清培养基培养,调整细胞浓度为1×106个/mL,同时加入PHA-植物血凝素,使PHA在体系中所占含量为10-100ug/mL,继续置于37℃、5%的CO2孵箱中培养,以后隔日补加AIM-V无血清培养液,培养5-10天,使细胞浓度至少达到1×107个/mL,收获细胞;
3)细胞的纯化除杂:离心法收集细胞(4℃,2000rpm/min,5分钟),采用生理盐水洗涤细胞,除去培养液;按常规操作检测排除细菌、真菌、内毒素污染;
4)细胞破碎和产物收集:再次离心收集沉淀细胞(4℃,2000rpm/min,5分钟),加入生理盐水,使细胞稀释成2×106个/mL悬液,经超声破碎(15-25kHz,10秒/次,3-5次)及冻融裂解(-20℃~-196℃,冻融3次或多次),低温离心(4℃,2000rpm/min,5分钟)收集上清即为免疫复合因子制剂,冷冻保藏备用。本实施例中加入的PHA-植物血凝素在体系中所占含量为50ug/mL,并将制得的免疫复合因子制剂命名为DZX。
DZX制剂动物模型治疗试验:
一、老年小鼠的治疗研究:
选取健康C57BL/6小鼠,不分雌雄。实验分成2组,每组10只:DZX治疗组、生理盐水对照组,在小鼠12月龄时开始治疗。DZX治疗组腹腔注射DZX,0.5ml/次,1次/周,共治疗3个月;生理盐水对照组以生理盐水代替DZX注射。在小鼠12、15、18、21月龄时进行行为学检测,包括避暗实验、Morris水迷宫实验、旷场迷宫实验及高架十字迷宫实验。行为学结果显示:Morris水迷宫实验中,定位航行实验说明,生理盐水对照组小鼠表现出逃避潜伏期较长;空间探索实验说明,生理盐水对照组小鼠在平台象限路程百分比,时间百分比均减少,与DZX治疗组比较有显著性差异。采用行为学对其焦虑指标进行检测,由旷场迷宫实验结果显示,与DZX治疗组小鼠相比,生理盐水对照组小鼠在中央区域路程百分比明显减少,二者有显著性差异。由高架十字迷宫实验结果显示,与DZX治疗组小鼠相比,生理盐水对照组在进入开臂区域路程百分比、进入开臂次数百分比减少明显,二者有显著性差异。避暗实验结果表明生理盐水对照组与DZX治疗组相比,错误次数更多,潜伏期更短。HE染色结果显示:DZX治疗组与健康12月龄小鼠比较接近,脑部细胞数目较多,排列整齐,神经元细胞完整;而生理盐水对照组小鼠出现明显的神经元空泡样变性,细胞排列疏松。
二、小鼠乳腺癌的治疗研究:
将高度转移性小鼠乳腺癌细胞株4T1移植在6周龄BALB/C小鼠乳房垫下,成瘤后作为小鼠乳腺癌模型。实验分成4组,每组8只:正常对照组、模型对照组、本发明方法提取物DZX治疗组、Gemcitabine与CD40抗体联合治疗组。DZX治疗组腹腔注射DZX,0.5ml/次,1次/2天,共治疗8周。模型对照组腹腔注射等量的生理盐水。与对照组及Gemcitabine治疗组比较,DZX治疗组肺部未见转移灶,而对照组肺部均见转移灶,Gemcitabine治疗组发现部分转移灶;治疗组生存期比其他各组显著延长。
三、大鼠原位膀胱癌的治疗研究:
采用N-甲基亚硝基脲(MNU)诱导构建大鼠原位膀胱癌模型。实验分成4组,每组8只:正常对照组、模型对照组、本发明方法提取物DZX治疗组、Gemcitabine与CD40抗体联合治疗组。DZX治疗组腹腔注射DZX,0.5ml/次,1次/2天,共治疗8周。模型对照组腹腔注射等量的生理盐水。在DZX治疗组,肿瘤大小平均减少了80%,在持续治疗30天后,这组小鼠的肿瘤不再继续生长。DZX治疗组中,有3只小鼠的肿瘤完全消失,而且没有见到肿瘤复发。
此外,从治疗结果还提示,DZX没有明显的副作用,DZX对其他组织和器官无损伤,实验小鼠也没有体重减轻现象。DZX治疗组明显优于各对照组(包括Gemcitabine与CD40抗体治疗组)。
四、小鼠白血病的治疗研究:
采用人B淋巴白血病细胞NALM-6细胞尾静脉注射移植到高度免疫缺陷NCG小鼠体内,构建小鼠白血病模型。实验分成4组,每组8只:正常对照组、模型对照组、本发明方法提取物DZX治疗组、Gemcitabine与CD40抗体联合治疗组。DZX治疗组腹腔注射DZX,0.5ml/次,1次/2天,共治疗6周。模型对照组腹腔注射等量的生理盐水。与其他各组比较,DZX治疗组体重、生存状态及生存期均优于其它各组;NALM-6细胞数量显著下降,流式细胞术检测T细胞表面标志,提示治疗组T效应细胞免疫功能显著优于各对照组。
DZX临床适用范围:
抗衰老:美容、养颜、失眠症、脱发及白发(变黑)。
恶性肿瘤:各类肿瘤,包括实体瘤及血液恶性肿瘤如急性淋巴细胞白血病(ALL)。
自体免疫性疾病:类风湿关节炎、风湿性关节炎、类风湿性心脏病、红斑狼疮、多发性硬化症、自身免疫性溶血性贫血、溃疡性结肠炎、甲状腺机能亢进、原免疫性疾病发性血小板紫癜。
神经性疾病:脑性麻痹、缺氧缺血性脑病、自闭症、创伤性脑损伤、脊髓损伤、听力损失、肌萎缩性脊髓侧索硬化症、阿尔茨海默氏病(老年痴呆,AD)、多发性神经炎、神经性皮炎、强直性脊柱炎。
其他疾病:
II型糖尿病、艾滋病、多发性硬化症、皮肤病(如硬皮病、银屑病)、过敏性疾病(如过敏性哮喘、枯草热)、高尿酸血症、矽肺、慢性股骨头坏死、慢性肾炎性肾病、慢性肝病、慢性疲劳、肥胖症。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种人体免疫复合因子的制备方法,其特征在于,包括如下步骤:
1)人外周血单个核细胞的分离:通过仪器单采或人工抽取人外周血,肝素抗凝处理及Ficoll密度梯度离心法分离得到单个核细胞;
2)人外周血单个核细胞的培养与诱导:将所得的单个核细胞重悬并置于AIM-V无血清培养基培养,调整细胞浓度为1×106个/mL,同时加入植物血凝素使得植物血凝素在体系中所占含量为10-100ug/mL,继续置于37℃、5%的CO2孵箱中培养,以后隔日补加AIM-V无血清培养液,培养5-10天,使细胞浓度至少达到1×107个/mL,收获细胞;
3)细胞的纯化除杂:将收获的细胞用生理盐水洗涤三次,以除去培养液中所有成分;
4)细胞破碎和产物收集:在收集的细胞中加入生理盐水,将细胞稀释成2×106个/mL悬液,经超声粉碎并重复冻融裂解处理后,离心收集上清即为免疫复合因子,冷冻保藏即可。
2.根据权利要求1所述的制备方法,其特征在于,所述外周血包括脐血。
3.根据权利要求1所述的制备方法,其特征在于,所述步骤2)和步骤4)中离心操作的条件为4℃、2000rpm/min,离心时间为5分钟。
4.根据权利要求1所述的制备方法,其特征在于,所述步骤2)中,加入的植物血凝素在体系中所占含量为50ug/mL。
5.根据权利要求1所述的制备方法,其特征在于,所述步骤3)中,用生理盐水洗涤后的细胞需检测以排除细菌、真菌、内毒素污染。
6.根据权利要求1所述的制备方法,其特征在于,所述步骤4)中,超声粉碎的条件为15-25kHz,次数至少3次,每次10秒。
7.根据权利要求1所述的制备方法,其特征在于,所述步骤4)中,冻融裂解为-20℃~-196℃温度条件下冻结,室温条件下融化,冻融操作重复至少3次。
8.根据权利要求1~7任一项所述的制备方法制备得到的人体免疫复合因子作为免疫制剂在临床上提高机体免疫力和治疗衰老、肿瘤、自身免疫性疾病、神经性疾病、艾滋病的应用。
9.根据权利要求8所述的应用,其特征在于,所述应用的具体方法为:
a)将免疫制剂以1:20的比例加入生理盐水袋中,静脉滴注,3日1次,5次为一个疗程;
b)直接采用免疫制剂进行局部肌肉注射或者肿瘤局部注射,1-5mL/次。
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