CN111514460A - 大气压冷等离子体在抑制谷氨酰胺酶活性方面的用途及酶抑制剂 - Google Patents
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Abstract
本发明提出了大气压冷等离子体在抑制谷氨酰胺酶活性方面的用途及酶抑制剂。通过简便运行大气压冷等离子体装置,利用产生的大气压冷等离子体抑制谷氨酰胺酶活性。相比常规的谷氨酰胺酶抑制剂,利用大气压冷等离子体能够更高效地抑制谷氨酰胺酶活性,用时更短、程度可控。应用本发明对谷氨酰胺酶检测试剂盒产品进行质检,能够快速、准确地得出质检结论。
Description
技术领域
本发明涉及生物技术领域,特别涉及一种谷氨酰胺酶抑制剂。
背景技术
大气压冷等离子体是在常温大气压下通过放电产生的一种电离气体。大气压冷等离子体作为一种新技术,近年来在生物医学领域得到广泛应用,例如灭菌、皮肤病治疗、牙科疾病治疗、细胞转染、伤口愈合和癌症治疗等。与正常细胞相比癌细胞具有增殖快,易转移等特点。
临床上目前大多采用手术治疗,放疗和化疗等手段治疗癌症,但这些治疗手段往往会给病人带来很大的副作用,所以在癌症治疗领域还有很多需要解决的问题。
细胞代谢是一系列有序化学反应的总称,是维持细胞正常生长和繁殖的重要生理机制之一。然而癌细胞能够通过对代谢重新编程,进而促进癌细胞内碳同化为脂质、蛋白质和核酸等大分子,产生癌细胞生长和增殖所需的大量中间代谢物,从而实现快速和爆炸性的增殖。谷氨酰胺代谢在肿瘤细胞的生物合成、能量代谢和细胞稳态中起着重要作用,其促进了肿瘤的生长。此外,谷氨酰胺酶(GLS)在许多肿瘤细胞中过度表达,并将谷氨酰胺转化为谷氨酸,为癌细胞提供了营养,进而促进癌细胞快速生长和增值。
BPTES是谷氨酰胺酶(GLS)的一种有效选择性抑制剂,其IC50为0.16μM,Ki值大约为3μM。
发明内容
申请人通过研究大气压冷等离子体诱导癌细胞凋亡的机制,发现大气压冷等离子体至少可通过明显抑制谷氨酰胺酶活性,从而在癌细胞关键的代谢通路上抑制癌细胞的增殖,甚至导致其凋亡。进而,本申请提出以下应用技术方案:
第一方面,大气压冷等离子体在抑制谷氨酰胺酶活性方面的用途。
第二方面,大气压冷等离子体在制备谷氨酰胺酶抑制剂方面的用途。
第三方面,去离子水通入大气压冷等离子体形成的活化水作为谷氨酰胺酶抑制剂的用途。
第四方面,大气压冷等离子发生器在构建用于抑制谷氨酰胺酶活性的实验设备方面的用途。
第五方面,一种谷氨酰胺酶检测试剂盒的质检方法,包括:
取谷氨酰胺酶样本,并分为至少两组;所述谷氨酰胺酶样本已被验证具有活性;
取同批次的至少两个谷氨酰胺酶检测试剂盒;
其中一组谷氨酰胺酶样本经一个检测试剂盒进行检测;
使另一组谷氨酰胺酶样本首先被大气压冷等离子体处理,然后经另一个检测试剂盒进行检测;
若两组检测结果等同,则判定该批次的检测试剂盒不合格(或许该批次中有部分检测试剂盒实际是合格的,但从生产管理和流通市售的角度,则认定该批次产品不合格)。
本发明通过简便运行大气压冷等离子体装置,利用产生的大气压冷等离子体抑制谷氨酰胺酶活性,作用到与癌细胞生长繁殖有密切关系的代谢通路上,从而抑制癌细胞的活力。可以预期,本发明不局限于针对癌细胞,还可以拓展应用于其他通过抑制谷氨酰胺酶直接或间接产生生物、化学效果的场合。
相比常规的谷氨酰胺酶抑制剂,利用大气压冷等离子体能够更高效地抑制谷氨酰胺酶活性,用时更短、程度可控。
应用本发明对谷氨酰胺酶检测试剂盒产品进行质检,能够快速、准确地得出质检结论。
附图说明
图1大气压冷等离子体装置的结构示意图;
图2大气压冷等离子体处理之后,正常骨髓基质细胞和molm-13白血病细胞的细胞活力示意图;
图3大气压冷等离子体处理组和对照组癌细胞差异代谢物层次聚类分析结果的热图;
图4大气压冷等离子体处理后癌细胞谷氨酰胺酶活性变化示意图;
图5不同浓度谷氨酰胺酶抑制剂(BPTES)作用24h对癌细胞谷氨酰胺酶活性影响示意图;
图6不同浓度BPTES作用24h,48h,72h后,癌细胞活力变化示意图;
图7 20μM/LBPTES以及20μM/LBPTES和谷氨酸盐组合分别作用48h对癌细胞活力影响示意图。
具体实施方式
以下详细介绍本申请的相关实验及分析,发明人具体的研发过程不限于此。
实验中所使用的大气冷等离子体装置如图1所示,等离子体的介质阻挡放电结构由高压电极1、接地电极2和夹在两个电极之间的1mm厚的介质板3(聚四氟乙烯材料)组成,接地电极为网格状结构以实现沿面放电,在接地电极下方设置等离子体腔室4。放电电压峰峰值和频率分别是5KV和10KHz,放电所采用的气体是环境空气。
细胞的培养和处理方式:本实验使用的癌细胞是molm-13白血病细胞。将细胞种植在24孔细胞培养板中,每个孔含有3×105个细胞(300μl RPMI 1640培养基)。处理后,将细胞放入细胞培养箱培养24h。
处理细胞的两种具体操作:
a.将待处理的细胞放在等离子体腔室内,其中细胞培养板的上沿距离沿面1cm,然后依据实验条件放电不同时间。
b.将待处理的去离子水放在等离子体腔室中,其盛有去离子水的器皿上沿距离沿面1cm,依据实验条件放电不同时间,然后取部分等离子体活化水,将待处理细胞置于等离子体活化水中。
实验一 大气压冷等离子体处理正常细胞/癌细胞的对照实验
用上述大气压冷等离子体装置分别处理正常骨髓基质细胞和molm-13白血病细胞不同时间(20s,40s,60,120s,180s),两种细胞的活性变化结果如图2所示。由结果可以看出,随着大气压冷等离子体处理时间的延长,molm-13细胞株的细胞活力明显降低,而正常骨髓基质细胞来源的正常细胞株的细胞活力略有下降。
这验证了大气压冷等离子体对白血病细胞的生长和繁殖有很好的抑制作用。因为细胞代谢是维持正常生长和繁殖的重要生理机制,癌细胞能够对细胞代谢过程重新编程,产生大量利于其生长和繁殖的中间代谢物。
实验二 癌细胞代谢物的对照分析
对大气压冷等离子体处理组和对照组癌细胞(自然生长)的代谢物进行分析。白血病癌细胞样品提前24h种在24孔细胞培养板,每孔300μl RPMI 1640培养基含有3×105个细胞,处理组和对照组各有5个重复样品。处理后,将癌细胞放入细胞培养箱培养24h。
大气压冷等离子体处理组和对照组癌细胞的差异代谢物层次聚类分析结果以热图的形式展示在图3中,由图3所示结果可以看出,等离子体处理组癌细胞的谷氨酰胺明显上调(箭头已标出)。
因此,我们推测等离子体处理的白血病细胞的谷氨酰胺酶活性降低,由于谷氨酰胺不能正常代谢并转化为谷氨酸,从而抑制白血病细胞的增殖,甚至导致白血病细胞凋亡。
为了验证以上推测,我们检测了大气压冷等离子体处理白血病细胞20s、40s、180s之后谷氨酰胺酶的活性变化,结果如图4所示。由图4可以看出,等离子体处理白血病细胞后的谷氨酰胺酶活性明显降低,且随着处理时间的延长,酶活性降低的越明显。
实验三 现有酶抑制剂(BPTES)对谷氨酰胺酶活性及细胞活力的影响
图5是谷氨酰胺酶抑制剂(BPTES)对谷氨酰胺酶活性的影响,结果显示BPTES能够抑制谷氨酰胺酶的活性,且40μM/L浓度比20μM/L BPTES抑制程度更大。
图6是不同浓度BPTES分别作用与白血病细胞24h,48h,72h后,癌细胞活力变化,可以看出BPTES可以降低癌细胞的活力,且40μM/L浓度比20μM/L BPTES降低程度更大。
图7是20μM/L BPTES以及20μM/L BPTES与谷氨酸钠组合分别作用48h对癌细胞活力的影响,可以看,当我们在含有20μM/L BPTES的实验组中加入谷氨酸盐时,相对细胞活力有一定的增加。
综合以上,大气压冷等离子体治疗可以通过抑制白血病细胞的谷氨酰胺酶活性,使癌细胞内谷氨酰胺不能正常代谢产生谷氨酸,从而导致积累。谷氨酰胺的积累可能导致白血病细胞因为缺乏所需的营养物质、无法对抗酸性环境而死亡。
同时,利用大气压冷等离子体抑制癌细胞中的谷氨酰胺酶,相比现有的酶抑制剂(BPTES)更为高效,前者3分钟的处理就可达到后者72小时作用的效果,且前者调节更为灵活。
Claims (5)
1.大气压冷等离子体在抑制谷氨酰胺酶活性方面的用途。
2.大气压冷等离子体在制备谷氨酰胺酶抑制剂方面的用途。
3.去离子水通入大气压冷等离子体形成的活化水作为谷氨酰胺酶抑制剂的用途。
4.大气压冷等离子发生器在构建用于抑制谷氨酰胺酶活性的实验设备方面的用途。
5.一种谷氨酰胺酶检测试剂盒的质检方法,包括:
取谷氨酰胺酶样本,并分为至少两组;所述谷氨酰胺酶样本已被验证具有活性;
取同批次的至少两个谷氨酰胺酶检测试剂盒;
其中一组谷氨酰胺酶样本经一个检测试剂盒进行检测;
使另一组谷氨酰胺酶样本首先被大气压冷等离子体处理,然后经另一个检测试剂盒进行检测;
若两组检测结果等同,则判定该批次的检测试剂盒不合格。
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