基于原代小脑颗粒细胞的A型肉毒毒素活性分析法的建立
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摘要
目的分离、培养SD大鼠原代小脑颗粒细胞,用于评价A型肉毒毒素活性。方法将出生后6~8 d的SD大鼠小脑颗粒细胞培养5~7 d后用A型肉毒毒素处理,采用免疫荧光法进行检测并评价毒素的活性,Western印迹法分析毒素活性与剂量关系。结果与结论成功培养了大鼠小脑颗粒细胞,并在原代神经细胞水平建立了A型肉毒毒素活性分析法,测定了毒素活性剂量关系,为进一步解析肉毒毒素作用的生化机制提供了技术手段。
Objective To isolate and culture the primary cerebellar granulosa cells(CGNs) of SD rats and evaluate the activity of botulinum neurotoxin A(Bo NT/A) based on CGNs. Methods CGNs of 6-to 8-days-old SD rats were isolated and cultured. After 5-7 d,the cells were treated with Bo NT/A. The activity of the toxin was evaluated with immunofluorescence,and the relationships between the activity and dose of the toxin were analyzed with Western blotting. Results and Conclusion CGNs Of SD rats were successfully cultured,a method for evaluating the activity of Bo NT/A was established at the level of primary nerve cells,and the relationships between the activity and dose of toxin were analyzed. This study provides a tool for further detailing the biochemical mechanism of Bo NT/A.
Objective To isolate and culture the primary cerebellar granulosa cells(CGNs) of SD rats and evaluate the activity of botulinum neurotoxin A(Bo NT/A) based on CGNs. Methods CGNs of 6-to 8-days-old SD rats were isolated and cultured. After 5-7 d,the cells were treated with Bo NT/A. The activity of the toxin was evaluated with immunofluorescence,and the relationships between the activity and dose of the toxin were analyzed with Western blotting. Results and Conclusion CGNs Of SD rats were successfully cultured,a method for evaluating the activity of Bo NT/A was established at the level of primary nerve cells,and the relationships between the activity and dose of toxin were analyzed. This study provides a tool for further detailing the biochemical mechanism of Bo NT/A.
引文
[1]朱力,王恒樑,黄留玉.肉毒毒素研究进展[J].生物技术通讯,2005,16(2):186-190.
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[4]Schiavo G,Matteoli M,Montecucco C.Neurotoxins affecting neuroexocytosis[J].Physiol Rev,2000,80(2):717-766.
[5]Pirazzini M,Rossetto O.Challenges in searching for therapeutics against botulinum neurotoxins[J].Expert Opin Drug Discov,2017,12(5):497-510.
[6]Turton K,Chaddock JA,Acharya KR.Botulinum and tetanus neurotoxins:structure,function and therapeutic utility[J].Trends Biochem Sci,2002,27(11):552-558.
[7]Lamanna C,Spero L,Schantz EJ.Dependence of time to death on molecular size of botulinum toxin[J].Infect Immun,1970,1(4):423-424.
[8]Dressler D,Lange M,Bigalke H.Mouse diaphragm assay for detection of antibodies against botulinum toxin type B[J].Mov Disord,2005,20(12):1617-1619.
[9]Schmidt JJ,Stafford RG,Millard CB.High-throughput assays for botulinum neurotoxin proteolytic activity:serotypes A,B,D and F[J].Anal Biochem,2001,296(1):130-137.
[10]Eubanks LM,Hixon MS,Jin W,et al.An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists[J].Proc Natl Acad Sci USA,2007,104(8):2602-2607.
[11]Burnett JC,Schmidt JJ,Stafford RG,et al.Novel small molecule inhibitors of botulinum neurotoxin A metalloprotease activity[J].Biochem Biophys Res Commun,2003,310(1):84-93.
[12]Simpson LL.The origin,structure,and pharmacological activity of botulinum toxin[J].Pharmacol Rev,1981,33(3):155-188.
[2]余云舟,孙志伟,郑涛,等.肉毒毒素防治药物的研究进展[J].军事医学,2012,36(12):954-958.
[3]Smith LA,Rusnak JM.Botulinum neurotoxin vaccines:past,present,and future[J].Crit Rev Immunol,2007,27(4):303-318.
[4]Schiavo G,Matteoli M,Montecucco C.Neurotoxins affecting neuroexocytosis[J].Physiol Rev,2000,80(2):717-766.
[5]Pirazzini M,Rossetto O.Challenges in searching for therapeutics against botulinum neurotoxins[J].Expert Opin Drug Discov,2017,12(5):497-510.
[6]Turton K,Chaddock JA,Acharya KR.Botulinum and tetanus neurotoxins:structure,function and therapeutic utility[J].Trends Biochem Sci,2002,27(11):552-558.
[7]Lamanna C,Spero L,Schantz EJ.Dependence of time to death on molecular size of botulinum toxin[J].Infect Immun,1970,1(4):423-424.
[8]Dressler D,Lange M,Bigalke H.Mouse diaphragm assay for detection of antibodies against botulinum toxin type B[J].Mov Disord,2005,20(12):1617-1619.
[9]Schmidt JJ,Stafford RG,Millard CB.High-throughput assays for botulinum neurotoxin proteolytic activity:serotypes A,B,D and F[J].Anal Biochem,2001,296(1):130-137.
[10]Eubanks LM,Hixon MS,Jin W,et al.An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists[J].Proc Natl Acad Sci USA,2007,104(8):2602-2607.
[11]Burnett JC,Schmidt JJ,Stafford RG,et al.Novel small molecule inhibitors of botulinum neurotoxin A metalloprotease activity[J].Biochem Biophys Res Commun,2003,310(1):84-93.
[12]Simpson LL.The origin,structure,and pharmacological activity of botulinum toxin[J].Pharmacol Rev,1981,33(3):155-188.