CN109420168A - 一种抑制wt1基因突变的生物制剂制备技术 - Google Patents
一种抑制wt1基因突变的生物制剂制备技术 Download PDFInfo
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- CN109420168A CN109420168A CN201710769228.0A CN201710769228A CN109420168A CN 109420168 A CN109420168 A CN 109420168A CN 201710769228 A CN201710769228 A CN 201710769228A CN 109420168 A CN109420168 A CN 109420168A
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Abstract
本申请公开了一种抑制WT1基因突变的生物制剂的制备方法，包括：从人体血液中分离出T淋巴细胞；将T淋巴细胞放置于培养液中进行扩增培养；将培养好的T淋巴细胞与细胞株放在培养瓶中进行驯化；将细胞株驯化好T淋巴细胞与癌细胞一起进行测试，当T淋巴细胞具有攻击癌细胞的能力后，进行分离；将分离出的T淋巴细胞与WT1基因进行混合测试，驯化后的T淋巴细胞具有抵制WT1基因突变的能力；再次将这些具备抵制攻击能力的T淋巴细胞从培养液中分离，与人体免疫球蛋白一起制成活性制剂，进行密封冷藏。本发明的优点是：从源头上抑制WT1基因的突变，阻止癌细胞的产生，可以有效预防癌症，预防效果达到95％以上，结果更加准确。
Description
技术领域
本发明涉及一种抑制WT1基因突变的生物制剂的制备方法。
背景技术
目前，中国癌症发病率和死亡率一直在上升，从2010年开始已经成为中国的一个主要公共卫生问题。2017年2月，国家癌症中心发布了最新癌症数据，汇总了全国347家癌症登记点的数据，数据显示：全国每天约1万人确诊癌症；肺癌为发病率、死亡率双率第一；甲状腺癌快速上升。同年，世界新发病例约1409万，中国新发癌症病例占世界的1/4。
科学研究显示，各种癌症其实都与人类代表肿瘤发生的基因WT1 基因有直接联系，不管是肺癌、胃癌、甲状腺癌或是其它种类的癌症都与这个基因突变有关。现有技术对癌症的防治有很多种药物，但是对于治疗癌症并无确切的预防效果，主要表现在：第一，对癌症的预防作用不明确，无法有效预防癌症；第二，药物对人体免疫系统的破坏力很大，部分患者无法忍受。
发明内容
本发明的目的在于克服上述不足，提供一种抑制WT1基因突变的生物制剂的制备方法，其能够对预防癌症产生确切的技术效果。
为了实现上述目的，本发明采用的技术方案为：一种抑制WT1基因突变的生物制剂的制备方法，其特征在于，包括以下步骤：从人体血液中分离出T淋巴细胞；将T淋巴细胞放置于培养液中进行扩增培养；将培养好的T淋巴细胞与细胞株放在培养瓶中进行驯化；将细胞株驯化好T淋巴细胞与癌细胞一起进行测试，当T淋巴细胞具有攻击癌细胞的能力后，进行分离；将分离出的T淋巴细胞与WT1基因进行混合测试，驯化后的T淋巴细胞具有抵制WT1基因突变的能力；再次将这些具备抵制攻击能力的T淋巴细胞从培养液中分离，与人体免疫球蛋白一起制成活性制剂，进行密封冷藏。
本发明的有益效果为：
制备方法简单，从人体血液中分离出T淋巴细胞，并进行扩增培养，驯化、分离，混合测试的步骤制得活性制剂，让人体产生抑制 WT1基因突变的记忆性T细胞。从源头上控制WT1基因，不让它突变，阻止癌细胞的产生，可以有效预防癌症，预防效果达到95％以上，结果更加准确。
具体实施方式
说明书后续描述为实施本申请的较佳实施方式，然所述描述乃以说明本申请的一般原则为目的，并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
本发明的抑制WT1基因突变的生物制剂的制备方法，包括以下步骤：从人体血液中分离出T淋巴细胞；将T淋巴细胞放置于培养液中进行扩增培养；将培养好的T淋巴细胞与细胞株放在培养瓶中进行驯化；将细胞株驯化好T淋巴细胞与癌细胞一起进行测试，当T淋巴细胞具有攻击癌细胞的能力后，进行分离；将分离出的T淋巴细胞与 WT1基因进行混合测试，驯化后的T淋巴细胞具有抵制WT1基因突变的能力；再次将这些具备抵制攻击能力的T淋巴细胞从培养液中分离，与人体免疫球蛋白一起制成活性制剂，进行密封冷藏。
所述从人体血液中分离出T淋巴细胞的步骤包括在GMP万级生物洁净室的百级生物洁净台操作，操作温度为常温。
优选地，所述将T淋巴细胞放置于培养液中进行扩增培养的步骤包括：在KRA细胞培养液中进行扩增培养，所述KRA细胞培养液成分质量百分比为：
优选地，所述扩增培养的环境条件是二氧化碳培养箱温度37度扩增培养。
优选地，所述将培养好的T淋巴细胞与细胞株放在培养瓶中进行驯化包括：使得T淋巴细胞表面形成膜分子，参与细胞识别抗原，细胞活化、增殖、分化；并采用选择法或克隆形成法从原代培养物或细胞系中获得具有特殊性质或标志物的细胞株。
优选地，所述驯化的环境是无毒、无菌，温度为37度。
优选地，所述将细胞株驯化好T淋巴细胞与癌细胞一起进行测试的步骤，所述测试的条件是在37度无菌环境下进行，并采用免疫磁珠方式进行分离。
优选地，将分离出的T淋巴细胞与WT1基因进行混合测试的步骤，所述混合测试的条件是在37度无菌环境下进行。
优选地，所述混合溶液的配方选自以下成分的一种或几种：氯化钙，硝酸钙，硫酸镁，五水硫酸钙，七水硫酸亚铁，七水硫酸锌，L -丙胺酸，L-天(门)冬酰胺-水合物，L-天冬氨酸，L-半胱氨酸- 盐酸盐-水合物，L-胱氨酸-二盐酸盐，L-谷氨酸，甘氨酸，L-组氨酸-盐酸盐-水合物，L-羟脯氨酸，L-异亮氨酸，L-亮氨酸，L -赖氨酸-盐酸盐，L-蛋氨酸，谷光苷肽还原型，抗坏血酸，维生素 H，维生素B12，D-泛酸盐钙，四甲烯二胺(腐胺)-二盐酸盐，氯化胆碱，叶酸，无旋光肌醇，烟酰胺，吡哆醛-盐酸盐，维生素B6 -盐酸盐，维生素B2，硫胺-盐酸盐，硫辛酸，对-氨基苯甲酸，腺苷，尿核甙，胞啶，鸟苷。
优选地，所述培养瓶的条件是具有良好的透明度和无毒、无菌外，还需经表面改性处理使之能够贴壁、分裂和生长。
优选地，所述将这些具备抵制攻击能力的T淋巴细胞从培养液中分离，与人体免疫球蛋白一起制成活性制剂的步骤包括：分离的条件是常温离心即可，活性制剂的制备条件是在GMP万级生物洁净室的百级生物洁净台操作，操作温度为常温。
试验效果：
将本发明的生物制剂与现有技术的中药内服预防癌症组和西药制剂预防癌症组进行比较如下：
上述制备方法简单，从人体血液中分离出T淋巴细胞，并进行扩增培养，驯化、分离，混合测试的步骤制得活性制剂，让人体产生抑制WT1基因突变的记忆性T细胞。从源头上控制WT1基因，不让它突变，阻止癌细胞的产生，可以有效预防癌症，预防效果达到95％以上，结果更加准确。
说明示出并描述了本申请的若干优选实施例，但如前所述，应当理解本申请并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述申请构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本申请的精神和范围，则都应在本申请所附权利要求的保护范围内。
Claims (9)
1.一种抑制WT1基因突变的生物制剂的制备方法，其特征在于，包括以下步骤：
从人体血液中分离出T淋巴细胞；
将T淋巴细胞放置于培养液中进行扩增培养；
将培养好的T淋巴细胞与细胞株放在培养瓶中进行驯化；
将细胞株驯化好T淋巴细胞与癌细胞一起进行测试，当T淋巴细胞具有攻击癌细胞的能力后，进行分离；
将分离出的T淋巴细胞与WT1基因进行混合测试，驯化后的T淋巴细胞具有抵制WT1基因突变的能力；
再次将这些具备抵制攻击能力的T淋巴细胞从培养液中分离，与人体免疫球蛋白一起制成活性制剂，进行密封冷藏。
2.根据权利要求1所述的抑制WT1基因突变的生物制剂的制备方法，其特征在于，所述从人体血液中分离出T淋巴细胞的步骤包括在6MP万级生物洁净室的百级生物洁净台操作，操作温度为常温。
3.根据权利要求2所述的抑制WT1基因突变的生物制剂的制备方法，其特征在于，所述将T淋巴细胞放置于培养液中进行扩增培养的步骤包括：在KRA细胞培养液中进行扩增培养，所述KRA细胞培养液成分质量百分比为：
4.根据权利要求3所述的抑制WT1基因突变的生物制剂的制备方法，其特征在于，所述扩增培养的环境条件是二氧化碳培养箱温度37度扩增培养。
5.根据权利要求1所述的抑制WT1基因突变的生物制剂的制备方法，其特征在于，所述将培养好的T淋巴细胞与细胞株放在培养瓶中进行驯化包括：使得T淋巴细胞表面形成膜分子，参与细胞识别抗原，细胞活化、增殖、分化；并采用选择法或克隆形成法从原代培养物或细胞系中获得具有特殊性质或标志物的细胞株。
6.根据权利要求5所述的抑制WT1基因突变的生物制剂的制备方法，其特征在于，所述驯化的环境是无毒、无菌，温度为37度。
7.根据权利要求6所述的抑制WT1基因突变的生物制剂的制备方法，其特征在于，所述将细胞株驯化好T淋巴细胞与癌细胞一起进行测试的步骤，所述测试的条件是在37度无菌环境下进行，并采用免疫磁珠方式进行分离。
8.根据权利要求6所述的抑制WT1基因突变的生物制剂的制备方法，其特征在于，将分离出的T淋巴细胞与WT1基因进行混合测试的步骤，所述混合测试的条件是在37度无菌环境下进行。
9.根据权利要求3所述的抑制WT1基因突变的生物制剂的制备方法，其特征在于，所述混合溶液的配方选自以下成分的一种或几种：
氯化钙，硝酸钙，硫酸镁，五水硫酸钙，七水硫酸亚铁，七水硫酸锌，L-丙胺酸，L-天(门)冬酰胺-水合物，L-天冬氨酸，L-半胱氨酸-盐酸盐-水合物，L-胱氨酸-二盐酸盐，L-谷氨酸，甘氨酸，L-组氨酸-盐酸盐-水合物，L-羟脯氨酸，L-异亮氨酸，L-亮氨酸，L-赖氨酸-盐酸盐，L-蛋氨酸，谷光苷肽还原型，抗坏血酸，维生素H，维生素B12，D-泛酸盐钙，四甲烯二胺(腐胺)-二盐酸盐，氯化胆碱，叶酸，无旋光肌醇，烟酰胺，吡哆醛-盐酸盐，维生素B6-盐酸盐，维生素B2，硫胺-盐酸盐，硫辛酸，对-氨基苯甲酸，腺苷，尿核甙，胞啶，鸟苷。
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