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CN114908051A - 一种逆转生物年龄制剂及其制备方法 - Google Patents

一种逆转生物年龄制剂及其制备方法 Download PDF

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CN114908051A
CN114908051A CN202210514823.0A CN202210514823A CN114908051A CN 114908051 A CN114908051 A CN 114908051A CN 202210514823 A CN202210514823 A CN 202210514823A CN 114908051 A CN114908051 A CN 114908051A
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洪纪宪
刘华
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Abstract

本发明涉及一种制备逆转生物年龄制剂的方法，步骤包括：将细胞毒性T淋巴细胞与细胞有丝分裂原、细胞因子以及免疫佐剂置于培养容器内的液态细胞培养基中共同培养，以获得免疫细胞培养物，并从所述免疫细胞培养物中分离具有免疫活性的细胞群，即得所述逆转生物年龄制剂。使用本发明的逆转生物年龄制剂能够明显逆转生物年龄，含1010细胞毒性T淋巴细胞的逆转生物年龄制剂在一周内分三次使用，连续使用3个月后生物年龄逆转1.3年，连续使用9个月后生物年龄逆转4.5年。

Description

一种逆转生物年龄制剂及其制备方法
技术领域
本发明涉及生物制剂技术领域，尤其涉及一种逆转生物年龄制剂及其制备方法。
背景技术
目前，通过人生长激素(GH)或/和GH释放剂、脱氢表雄酮(DHEA)以及二甲双胍的组合可使得老年人的胸腺再生，以防止老年人的年龄相关免疫功能障碍(免疫衰老)或恢复免疫功能(免疫衰老的逆转)，这种免疫衰老的逆转通过基因芯片的测试，被定义为生物表观年龄(生物年龄)的逆转。但长期使用上述组合可能会对人体产生毒性，以及其他可能的副作用。
发明内容
本发明的目的是针对现有技术中的不足，提供一种逆转生物年龄制剂及其制备方法。
为实现上述目的，本发明采取的技术方案是：
本发明的第一方面是提供一种制备逆转生物年龄制剂的方法，步骤包括：
将细胞毒性T淋巴细胞与细胞有丝分裂原、细胞因子以及免疫佐剂置于培养容器内的液态细胞培养基中共同培养，以获得免疫细胞培养物，并从所述免疫细胞培养物中分离具有免疫活性的细胞群，即得所述逆转生物年龄制剂。
优选地，所述细胞毒性T淋巴细胞的浓度为1×103个/mL-1×1011个/mL。
优选地，所述细胞有丝分裂原选自刀豆素、植物血凝素、美洲商陆、脂多糖或葡聚糖中的至少一种。
优选地，所述细胞有丝分裂原的浓度为10万单位/L-1000万单位/L。
优选地，所述细胞有丝分裂原的浓度为0.1mg/L-10mg/L。
优选地，所述细胞因子选自淋巴因子、单核因子、激活炎症的细胞因子或刺激造血的细胞因子中的至少一种；所述淋巴因子来源于淋巴细胞、单核细胞或产生淋巴因子的细胞。
优选地，所述细胞因子选自白细胞介素、干扰素、集落刺激因子、趋化性细胞因子或转化生长因子中的至少一种。
优选地，所述细胞因子选自白细胞介素-2、白细胞介素-6、白细胞介素-15或干扰素中的至少一种。
优选地，所述细胞因子的浓度为20万单位/L-500万单位/L。
优选地，所述免疫佐剂选自生物性佐剂、无机佐剂、有机佐剂、合成佐剂、油剂或弗氏佐剂中的至少一种。
优选地，所述免疫佐剂的浓度为0.01mL/L-1mL/L。
优选地，所述培养容器为三维大体积高密度细胞培养容器。
优选地，所述共同培养的时间为3天-180天。
优选地，步骤还包括：以所述免疫细胞培养物，或所述细胞群为原料，在所述液态细胞培养基中进行克隆，以获得具有免疫活性的细胞株。
优选地，所述克隆选自间歇循环刺激法或连续刺激法中的任意一种。
本发明的第二方面是提供一种如上所述方法制得的逆转生物年龄制剂。
本发明的第三方面是提供一种检测如上所述逆转生物年龄制剂对基因甲基化表达水平变化的方法，所述方法包括：采用基因甲基化芯片进行检测。
本发明采用以上技术方案，与现有技术相比，具有如下技术效果：
使用本发明的逆转生物年龄制剂能够明显逆转生物年龄，含1010细胞毒性T淋巴细胞的逆转生物年龄制剂在一周内分三次使用，连续使用3个月后生物年龄逆转1.3年，连续使用9个月后生物年龄逆转4.5年。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作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。
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
下面结合具体实施例对本发明作进一步说明，但不作为本发明的限定。
实施例1
本实施例提供一种制备逆转生物年龄制剂的方法，步骤包括：
将细胞毒性T淋巴细胞与刀豆素、白细胞介素-2以及5％吐温-80置于三维大体积高密度细胞培养容器内的液态细胞培养基中共同培养30天，以获得免疫细胞培养物，并从所述免疫细胞培养物中分离具有免疫活性的细胞群，即得所述逆转生物年龄制剂；
其中，所述刀豆素在所述液态细胞培养基中的浓度为10万单位/L；所述白细胞介素-2在所述液态细胞培养基中的浓度为50万单位/L；所述5％吐温-80在所述液态细胞培养基中的浓度为0.5mL/L。
实施例2
将细胞毒性T淋巴细胞与植物血凝素、干扰素以及5％吐温-80置于三维大体积高密度细胞培养容器内的液态细胞培养基中共同培养30天，以获得免疫细胞培养物，并从所述免疫细胞培养物中分离具有免疫活性的细胞群，即得所述逆转生物年龄制剂；
其中，所述植物血凝素在所述液态细胞培养基中的浓度为0.5mg/L；所述干扰素在所述液态细胞培养基中的浓度为500万单位/L；所述5％吐温-80在所述液态细胞培养基中的浓度为0.1mL/L。
实施例3
将细胞毒性T淋巴细胞与刀豆素、白细胞介素-2以及5％吐温-80置于三维大体积高密度细胞培养容器内的液态细胞培养基中共同培养45天，以获得免疫细胞培养物，并从所述免疫细胞培养物中分离具有免疫活性的细胞群，即得所述逆转生物年龄制剂；
其中，所述刀豆素在所述液态细胞培养基中的浓度为100万单位/L；所述白细胞介素-2在所述液态细胞培养基中的浓度为100万单位/L；所述5％吐温-80在所述液态细胞培养基中的浓度为0.1mL/L。
实施例4
将细胞毒性T淋巴细胞与植物血凝素、干扰素以及5％吐温-80置于三维大体积高密度细胞培养容器内的液态细胞培养基中共同培养60天，以获得免疫细胞培养物，并从所述免疫细胞培养物中分离具有免疫活性的细胞群，即得所述逆转生物年龄制剂；
其中，所述植物血凝素在所述液态细胞培养基中的浓度为1mg/L；所述干扰素在所述液态细胞培养基中的浓度为300万单位/L；所述5％吐温-80在所述液态细胞培养基中的浓度为0.5mL/L。
检测实施例
随着年龄的增加，基因被复制次数逐渐增多，基因甲基化水平的变化也相应增高，表达结果会出现的偏差增多；基于此，通过基因芯片illumina 850K可以用来测试基因甲基化随着年龄老化的变化。
含1010细胞毒性T淋巴细胞的逆转生物年龄制剂在一周内分三次使用，在连续使用3个月后，通过基因芯片illumina 850K测试基因的甲基化变化，并经非线性回归分析所得的生物年龄为66；
继续连续使用6个月后，通过基因芯片illumina 850K测试80多万基因的甲基化变化，部分数据如下表所示。
表1
Figure BDA0003641061340000041
Figure BDA0003641061340000051
如表1所示的数据经非线性回归分析所得的生物年龄如表2所示。
表2
Figure BDA0003641061340000052
Figure BDA0003641061340000061
由于Horvath算法为目前世界公认为最精准的，故采用Horvath算法计算获得的生物年龄。
综上所述，通过芯片illumina 850K和ISCAN仪器检测可知，含1010细胞毒性T淋巴细胞的逆转生物年龄制剂在一周内分三次使用，连续使用3个月后生物年龄逆转1.3年，连续使用9个月后生物年龄逆转4.5年。
以上所述仅为本发明较佳的实施例，并非因此限制本发明的实施方式及保护范围，对于本领域技术人员而言，应当能够意识到凡运用本发明说明书内容所作出的等同替换和显而易见的变化所得到的方案，均应当包含在本发明的保护范围内。

Claims (10)

1.一种制备逆转生物年龄制剂的方法，其特征在于，步骤包括：
将细胞毒性T淋巴细胞与细胞有丝分裂原、细胞因子以及免疫佐剂置于培养容器内的液态细胞培养基中共同培养，以获得免疫细胞培养物，并从所述免疫细胞培养物中分离具有免疫活性的细胞群，即得所述逆转生物年龄制剂。
2.根据权利要求1所述的方法，其特征在于，所述细胞有丝分裂原选自刀豆素、植物血凝素、美洲商陆、脂多糖或葡聚糖中的至少一种。
3.根据权利要求1所述的方法，其特征在于，所述细胞因子选自淋巴因子、单核因子、激活炎症的细胞因子或刺激造血的细胞因子中的至少一种；所述淋巴因子来源于淋巴细胞、单核细胞或产生淋巴因子的细胞。
4.根据权利要求3所述的方法，其特征在于，所述细胞因子选自白细胞介素、干扰素、集落刺激因子、趋化性细胞因子或转化生长因子中的至少一种。
5.根据权利要求4所述的方法，其特征在于，所述细胞因子选自白细胞介素-2、白细胞介素-6、白细胞介素-15或干扰素中的至少一种。
6.根据权利要求1所述的方法，其特征在于，所述免疫佐剂选自生物性佐剂、无机佐剂、有机佐剂、合成佐剂、油剂或弗氏佐剂中的至少一种。
7.根据权利要求1所述的方法，其特征在于，步骤还包括：以所述免疫细胞培养物，或所述细胞群为原料，在所述液态细胞培养基中进行克隆，以获得具有免疫活性的细胞株。
8.根据权利要求7所述的方法，其特征在于，所述克隆选自间歇循环刺激法或连续刺激法中的任意一种。
9.一种如权利要求1-8任一项所述方法制得的逆转生物年龄制剂。
10.一种检测如权利要求9所述逆转生物年龄制剂对基因甲基化表达水平变化的方法，其特征在于，所述方法包括：采用基因甲基化芯片进行检测。
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