CN114990055A - 一种含有支化结构抗菌肽的新型细胞培养基及其制备方法 - Google Patents
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Abstract
本发明公开了一种含有支化结构抗菌肽的新型细胞培养基及其制备方法。所述支化结构抗菌肽是由L‑赖氨酸、D‑赖氨酸或上述两种赖氨酸或其盐酸盐聚合而成。将该支化结构抗菌多肽加入商用细胞培养基中，其可在低浓度下表现对细菌、真菌和支原体高效广谱抗菌效果，其降解产物为生物体必需的营养物质赖氨酸，对细胞无任何毒副作用。相较于现有的通过在商用培养基中额外加入双抗(链霉素和青霉素)的抗菌方法，含有支化结构抗菌肽的培养基具有优异的抗菌性能，不会产生任何耐药性，使用过程不需要额外加入抗菌剂，可用于细胞培养，是一种优异的新型细胞培养基。
Description
技术领域
本发明涉及一种含有支化结构抗菌肽的新型细胞培养基及其制备方法，属于生化制剂领域。
背景技术
细胞实验有助于科研人员在不牺牲动物的情况下，在体外模拟体内环境(无菌、适宜温度、酸碱度、含氧量、营养成分等)，从而研究细胞的增殖、分化、迁移、凋亡等一系列生理过程。在生物材料和药物研发过程中，体外细胞相关实验的结果也能在开展相关动物实验前，初步验证其急慢性生物毒性和有效性，防止不必要的动物牺牲。自1951年海拉肿瘤细胞系在体外成功培养以来，细胞培养技术不断深入发展，人们已经可以将不同来源、不同形态的肿瘤细胞和正常细胞在体外进行培养，并进行传代、迁移、分化等细胞行为调控。
但在细胞培养过程中，微生物污染一直是一个重大威胁。培养细胞过程中的物理、化学和生物因素都可能导致培养环境的污染。由于微生物污染在初始阶段往往难以发现，随着污染微生物的不断增殖和代谢，微生物不仅消耗细胞培养基中的养分，同时分泌多种代谢产物，包含多种对有毒的物质，所以在污染严重时往往已无法补救。因此，如何预防细胞培养过程中的微生物污染对细胞实验的稳定性和结果可靠性非常重要。
由于培养基中通常含有大量对热不稳定的的营养成分，无法用高压蒸汽法灭菌，因此目前通常使用直径为0.22μm的微孔滤膜进行过滤灭菌。为了保证培养过程中尽可能没有污染，在使用时经常会加入链霉素和青霉素杀灭微生物。上述常用做法在培养大部分细胞时能达到预防绝大部分微生物感染的效果，因而广泛用于现在的实验操作中。
然而通过文献查阅可以发现，首先支原体由于尺寸非常小，难以用常见微孔滤头(直径为0.22μm左右)以及空气过滤系统完全去除支原体。而抗生素加入培养基后同样难以杀灭支原体，而且对细胞培养及其一些特定的生理过程具有副作用。对干细胞来说，有研究表明在培养基中加入青霉素和链霉素对仓鼠胚胎细胞发育有显著影响，在体外胚胎发育过程中，加入青霉素和链霉素的培养基内“8细胞”胚胎和囊胚的占比显著低于对照组[1]；将人脐带组织源间充质干细胞在含有不同浓度青霉素和链霉素的培养基中培养，发现青霉素和链霉素在低浓度下可促进该细胞的增殖，但高浓度下会导致该细胞的分泌物表达水平下降[2]；炎症牙髓干细胞的研究显示抗生素的加入会导致其肿瘤坏死因子-α(TNF-α)表达水平下降[3]。抗生素的添加还会干扰心肌细胞的动作电位，影响细胞膜表面钙、钾离子通道电位的调节[4]。类似的，在培养基中添加抗生素也会影响神经元细胞的兴奋性，并改变神经元的电生理特性，这可能是通过改变神经元兴奋性的离子传导性来实现的[5]。
另一方面，抗生素的滥用导致了耐药微生物甚至多重耐药微生物的出现，即使不断有抗生素获得批准后投入使用，但相应的，耐药微生物也不断出现，例如耐甲氧西林金黄色葡萄球菌，导致现有抗生素在未来其广谱抗微生物能力将大幅下降。
参考文献：
[1]Zhou H,Mckiernan S H,Ji W,et al.Effect of antibiotics ondevelopment in vitro of hamster pronucleate ova[J].Theriogenology,2000,54(7):999-1006.
[2]李艳萍,时庆,邢晓,等.青霉素、链霉素体外对人脐带组织源间充质干细胞胞外分泌物基因表达的影响[J].中国实验血液学杂志,2011(01):163-168.
[3]王利娟,俞艳,曹灵,等.青霉素和链霉素对炎症牙髓干细胞的增殖,成牙成骨分化能力及TNF-α表达的影响[J].口腔生物医学,2014,5(1):4.
[4]The effects of gentamicin and penicillin/streptomycin on theelectrophysiology of human induced pluripotent stem cell-derivedcardiomyocytes in manual patch clamp and multi-electrode array system
[5]Bahrami F,Janahmadi M.Antibiotic Supplements AffectElectrophysiological Properties and Excitability of Rat Hippocampal PyramidalNeurons in Primary Culture[J].Iranian Biomedical Journal,2013,17(2):101.
发明内容
本发明的目的是针对现有技术的不足，提供一种含有支化结构抗菌肽的新型细胞培养基。
本发明的新型细胞培养基包括细胞培养基本体、胎牛血清和支化结构抗菌肽；每1L含有支化结构抗菌肽的新型细胞培养基由以下成分组成：
胎牛血清100-150mL
支化结构抗菌肽5mg–20g
细胞培养基本体余量。
本发明中涉及支化结构抗菌肽全由赖氨酸构成，赖氨酸数目＜100，其分子结构为支化、超支化或者树枝状结构，其数均分子量范围在3000-10000Da。该支化结构多肽无需考虑合成时控制氨基酸序列和手性结构，可大规模进行化学合成并提纯，相较于生物发酵成本较低。其次，该支化结构多肽不仅具有良好的生物相容性，包括良好的细胞相容性、组织相容性以及血液相容性，而且在低浓度下即具有高效的抗菌性能，其降解产物为天然氨基酸之一的赖氨酸，可以发挥抗菌功能后降解，为细胞或生物组织提供氨基酸营养来源。不同于传统青霉素和链霉素的联用抗菌法，含有该多肽的细胞培养基能够在不影响细胞的正常生理过程下，抵御普通微生物和耐药微生物感染的同时不产生耐药性。
所述的支化结构抗菌肽，该多肽由L-赖氨酸、D-赖氨酸或者上述两种氨基酸或其盐酸盐作为原料，脱水缩合聚合而成，其合成示意图如如1所示。
本发明中所述的支化结构抗菌肽，其在细胞培养基溶液环境下，对细菌、真菌和支原体均具有优越的杀灭能力，包括：(1)以金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌为例的革兰氏阳性菌及其耐药型变种菌株；(2)以大肠杆菌和铜绿假单胞菌为例的革兰氏阴性菌及其耐药型变种细菌株；(3)以酵母菌和白色念珠菌为例的酵母菌类真菌及其耐药变种真菌株；(4)以黑曲霉菌和黄曲霉菌等霉菌类真菌及其耐药变种真菌株；(5)支原体及其耐药性变种；
进一步的，所述新型细胞培养基中支化结构抗菌肽的浓度应不低于5μg/mL，可具有优异的杀菌作用，也不应高于20mg/mL，以保证培养基具有良好的生物相容性和细胞相容性，更进一步的，所述的培养基中支化结构抗菌肽的浓度范围为10-1000μg/mL，在该范围内效果最佳。
进一步的，所述的细胞培养基本体为Dulbecco改良依格尔培养基(DMEM)、最低必需培养基(MEM)、RPMI 1640培养基或其他可商业购买的细胞培养基，或者基于上述培养基成分衍生的改良版培养基。
进一步的，所述新型培养基的制备方法如下：在无菌环境下，将相应质量的支化结构抗菌肽溶于细胞培养基本体中，经0.22μm滤头过滤灭菌后，加入相应体积的胎牛血清，混合均匀后即得到新型细胞培养基。
本发明的有益效果是：
本发明所述培养基中的支化结构抗菌肽，具有优异的生物相容性，包括细胞相容性、血液相容性以及组织相容性，对细胞正常的增殖、迁移和分化等细胞行为无负面影响；
所述支化结构抗菌肽，可被细胞降解为生命体所必须的营养物质赖氨酸，参与生命体正常的生理活动；
所述培养基在细胞培养过程不会出现传统方法易出现的耐药性问题。
附图说明
图1支化结构抗菌肽对108CFU/mL的金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌和白色念珠菌的杀灭效果；
图2不同浓度的支化结构抗菌肽培养成纤维细胞后的细胞活性结果；
图3本发明制备的含有支化结构抗菌肽的细胞培养基和基于链霉素青霉素联用法的培养基的抗菌浓度对比。
具体实施方式
下面通过实施例对本发明进行具体描述。下述实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，该领域的技术熟练人员可以根据以上发明的内容做出一些非本质的改进和调整。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。
实施例1
一种含有支化结构抗菌肽的新型培养基，其制备过程如下：
在无菌环境下，将1g支化结构抗菌肽溶于900mL DMEM培养基中，经0.22μm孔径的滤膜过滤后，再加入100mL胎牛血清，混合均匀，即得到1L含有支化结构抗菌肽的DMEM细胞培养基。
实施例2
一种含有支化结构抗菌肽的新型培养基，其制备过程如下：
在无菌环境下，将0.5g支化结构抗菌肽溶于850mL 1640培养基中，经0.22μm孔径的滤膜过滤后，再加入150mL胎牛血清，混合均匀，即得到1L含有支化结构抗菌肽的1640细胞培养基。
实施例3
一种含有支化结构抗菌肽的新型细胞培养基，其抗菌性能测定如下：基于实施例1中配制方法，分别将不同质量的支化结构抗菌多肽溶于900mL DMEM培养基中，经0.22μm孔径的滤膜过滤后，再加入100mL胎牛血清，得到含有不同浓度支化结构抗菌多肽的DMEM培养基。将金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌和白色念珠菌按最终浓度为107CFU/mL分别加入到上述培养基中，在37℃下共培养24h。发现含有不同浓度多肽的DMEM培养基对上述细菌和真菌的抗菌效果均为100％，该抗菌实验中菌液浓度高于所有国家抗菌标准文件中所建议的抗菌浓度，可认为该多肽具有极其高效的抗菌效果，其结果如图1所示。
实施例4
一种含有支化结构抗菌肽的新型细胞培养基，其细胞相容性测定如下：基于实施例1，配制含不同浓度支化结构多肽的DMEM培养基，将其用于培养成纤维细胞。按每孔1万个细胞将细胞种在96孔板上，将其与上述含有不同浓度多肽的培养基在37℃下进行培养，48h后用Cell Counting Kit-8法测定细胞活性。结果如图2，含有不同浓度多肽的DMEM培养基均具有优异的细胞相容性。
实施例5
基于实施例2，配制含有不同浓度支化结构多肽的1640培养基。以不含多肽的组别作为空白组，以加入青霉素(100U/mL)和链霉素(100mg/mL)的1640培养基作为双抗组进行对照。每组培养基中加入金黄色葡萄球菌(最终浓度为107CFU/mL)，发现在含有不同支化结构的多肽浓度下，DMEM培养基对金黄色葡萄球菌的抗菌效果均非常显著。在浓度低于双抗组时，含多肽的培养基抗菌结果依然十分显著。空白组则无任何抗菌效果。结果如图3所示。
Claims (8)
1.一种含有支化结构抗菌肽的新型细胞培养基，其特征在于，包括细胞培养基本体、胎牛血清和支化结构抗菌肽；每1L含有支化结构抗菌肽的新型细胞培养基由以下成分组成：
胎牛血清100-150ml
支化结构抗菌肽5mg–20g
细胞培养基本体余量。
2.根据权利要求1所述一种含有支化结构抗菌肽的新型细胞培养基，其特征在于，所述的支化结构抗菌肽由L-赖氨酸、D-赖氨酸或上述两种赖氨酸或其盐酸盐作为原料，脱水缩合聚合而成，其分子量范围为3-10kDa，其结构式如下：
3.根据权利要求1所述的一种含有支化结构抗菌肽的新型细胞培养基，其特征在于，所述培养基中支化结构抗菌肽的浓度为不低于5μg/mL。
4.根据权利要求1所述的一种含有支化结构抗菌肽的新型细胞培养基，其特征在于，所述抗菌肽可杀灭细菌、真菌和支原体，包括：(1)金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌等革兰氏阳性菌及其耐药型变种菌株；(2)大肠杆菌、铜绿假单胞菌等革兰氏阴性菌及其耐药型变种菌株(3)酵母菌、白色念珠菌等酵母菌类真菌及其耐药变种菌株；(4)黑曲霉菌、黄曲霉菌等霉菌类真菌及其耐药变种菌株；(5)支原体及其耐药性变种。
5.根据权利要求1所述的一种含有支化结构抗菌肽的新型细胞培养基，其特征在于，所述培养基中的支化结构抗菌肽的浓度不高于20mg/mL，以保证培养基具有细胞相容性和生物相容性。
6.根据权利要求1所述的一种含有支化结构抗菌肽的新型细胞培养基，其特征在于，在细胞培养过程中所述支化结构抗菌肽可在发挥抗菌效果后被降解为赖氨酸，作为细胞或生物组织的赖氨酸营养补充。
7.根据权利要求1所述的一种含有支化结构抗菌肽的新型细胞培养基，其特征在于，所述的细胞培养基本体为Dulbecco改良依格尔培养基(DMEM)、最低必需培养基(MEM)、RPMIMedium 1640培养基或其他可商业购买的细胞培养基，或者基于上述培养基成分衍生的改良版培养基。
8.根据权利要求1所述的一种含有支化结构抗菌肽的新型细胞培养基，其特征在于，所述新型培养基的制备方法如下：将相应质量的支化结构抗菌肽溶于细胞培养基本体中，经0.22μm滤头过滤灭菌后，加入相应体积的胎牛血清，混合均匀后即得到新型细胞培养基。
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