不同类型细胞对A型肉毒毒素效价检测的可行性分析  

Feasibility analysis of detecting the titer of botulinum toxin type A by different types of cells

作　　者：杨克娜 李小娟 张华捷 马霄 朱衍志 杨英超 YANG Kena;LI Xiaojuan;ZHANG Huajie;MA Xiao;ZHU Yanzhi;YANG Yingchao(Quality Control Department,Lanzhou Biotechnique Development Co.,Ltd.,Lanzhou 730046,Gansu Province,China;不详)

机构地区：[1]兰州生物技术开发有限公司质量检定室,甘肃兰州730046 [2]中国食品药品检定研究院生物制品检定所百白破疫苗与毒素室,北京102629

出　　处：《微生物学免疫学进展》2024年第2期28-34,共7页Progress In Microbiology and Immunology

基　　金：国家重点研发计划(2022YFF0711104)。

摘　　要：目的 分析4种连续细胞和1种人神经元细胞对A型肉毒毒素(botulinum neurotoxin type A,BoNT/A)效价检测的适用性,为高敏感性BoNT/A细胞检测方法的建立提供依据。方法 采用实时荧光定量逆转录聚合酶链式反应(quantitative reverse transcriptase chain reaction, qRT-PCR)对4种连续细胞中BoNTs毒性相关蛋白受体和N-乙基马来酰亚胺敏感因子附着蛋白受体(soluble N-ethylmaleimide-sensitive factor attachment protein receptor, SNARE)进行定量检测,并用Western blot评估4种连续细胞和1种人神经元细胞对BoNT/A的敏感性。结果 qRT-PCR结果显示,4种连续细胞中除突触囊泡蛋白2B(synaptic vesicle protein 2B,SV2B)不表达或表达量较低外,其他BoNTs相关蛋白受体和SNARE蛋白均有较高的表达。Western blot结果显示,4种连续细胞对BoNT/A的敏感性依次为人神经母细胞瘤细胞(SK-N-SH)、大鼠嗜铬细胞瘤细胞(PC12)、人神经母细胞瘤细胞(SH-5Y5Y)、人神经母细胞瘤细胞[BE(2)-M17]。在SK-N-SH中,孵育0.1 mg/mL的三唾液神经节苷脂GT1b(Trisialoganglioside GT1b, GT1b),5 000 U/mL BoNT/A可切割45%突触小体相关蛋白25(synaptosome-associated protein of 25 000,SNAP25)。Western blot检测发现,1种人神经元细胞对BoNT/A较敏感,在约100 U/mL时达到50%的SNAP25裂解。结论 人神经元细胞敏感性优于其他4种连续细胞,在建立BoNT/A细胞学检测方法中具有一定的潜力。Objective To analyze the applicability of four continuous cells and one human neuron cell for detection of botulinum neurotoxin A(BoNT/A) potency, and to provide a basis for the establishment of a highly sensitive BoNT/A cell-based assay. Methods The BoNTs toxicity-related protein receptor and N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE) were quantitatively detected by real-time fluorescent quantitative reverse transcriptase polymerase chain reaction(qRT-PCR) in four continuous cells, and the BoNT/A potency was evaluated by Western blot in four continuous cells and one human neuron cell. Results The qRT-PCR results showed that in all BoNTs related protein receptors and SNARE proteins were highly expressed in the four continuous cells, except for the vesicle protein 2B(SV2B),which was not express or express at a low level. The Western blot results showed that the sensitivity of four continuous cells to BoNT/A was human neuroblastoma cells(SK-N-SH),rat pheochromocytoma(PC12),human neuroblastoma cells(SH-5Y5Y) and human neuroblastoma cells [BE(2)-M17]. When incubated with 0.1 mg/mL Trisialoganglioside GT1b in SK-N-SH,5 000 U/mL BoNT/A can cleave 45% of synaptosome-associated protein of 25 000(SNAP25). Conclusion The sensitivity of human neurons is better than that of other four continuous cells, and have some potential in establishing BoNT/A cell based detection methods.

关 键 词：肉毒毒素 连续细胞 人神经元细胞 突触小体相关蛋白25 

分 类 号：Q2[生物学—细胞生物学]