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刊物信息

期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

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U-2 OS细胞/CCK-8法测定1型单纯疱疹病毒溶瘤活性

An U-2 OS cells/CCK-8 method for oncolytic activity determination of herpes simplex virus type 1

分类号:R917
出版年·卷·期(页码):2020,40 (1):31-36
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的: 建立U-2 OS细胞/CCK-8比色法,用于测定1型单纯疱疹病毒(herpes simplex virus type 1,HSV-1)溶瘤活性。方法: 选择3株对HSV-1敏感的的细胞株,在细胞培养板上以梯度浓度的HSV-1感染一定时间后,加入CCK-8进行显色,用酶标仪检测后采用四参数曲线法进行量效关系拟合。在选择出量效关系最好的细胞株基础上,对方法的各项实验条件进行优化,并采用活性测定标准品对结果进行校正分析。对建立的方法初步验证准确性和精密度,并用上述方法对3批样品的溶瘤活性进行检测。结果: 优化后的实验采用U-2 OS细胞株,1:4的样品系列稀释比例,5×104 PFU·mL-1的样品系列稀释初始浓度,每孔2.5×104个细胞的铺板数量,72 h感染时间和4 h CCK-8试剂反应时间等条件进行样品测定,得到的四参数方程拟合的量效关系曲线R2大于0.99,信噪比较高,初步验证结果显示该方法的回收率为113.4%,日间精密度分析的几何变异系数(GCV)为21.8%。3批测试样品的溶瘤活性分别为3.52×103、3.93×104和1.61×105 U·mL-1结论: 建立的U-2 OS细胞/CCK-8法可用于以HSV-1为载体的基因治疗药物溶瘤活性测定。

-----英文摘要:---------------------------------------------------------------------------------------

Objective: To develop an U-2 OS cells/CCK-8 method for oncolytic activity determination of herpes simplex virus 1(HSV-1). Methods: Three candidate cell lines sensitive to HSV-1 were seeded in 96-well cell plates and infected with serial dilutions of HSV-1 for a certain period of time. Then CCK-8 was added and the cell plates were detected on a microplate reader. The four-parameter model was used for dose-effect curve fitting. The cell line showing the best reactivity was selected, and on this basis, the experimental conditions were optimized and the results were calibrated using a reference standard for activity determination. The accuracy and precision of the established method was preliminarily validated. The oncolytic activity of 3 batches of test samples was determinated using the developed method. Results: The optimized method used U-2 OS as test cell line, 5×104 PFU·mL-1 as initial concentration of test sample and 1:4 as dilution ratio, 2.5×104 cells per well as cell number, 72 h as infection time and 4 h as reaction time of CCK-8. The dose-response curve fitted the four-parameter model with R2 being above 0.99 and the signal-to-noise ratio was high. The results showed that the recovery rate was 113.4%, and the inter-day precision was 21.8%. The oncolytic activities of 3 batches of test samples were 3.52×103 U·mL-1, 3.93×104 U·mL-1 and 1.61×105 U·mL-1, respectively. Conclusion: The developed U-2 OS cell/CCK-8 method can be used to determine the oncolytic activity of HSV-1gene therapy products.

-----参考文献:---------------------------------------------------------------------------------------

[1] ZEYAULLAH M, PATRO M, AHMAD I, et al.Oncolytic viruses in the treatment of cancer:a review of current atrategies[J].Pathol Oncol Res, 2012, 18(4):771
[2] DAI MH, ZAMARIN D, GAO SP, et al.Synergistic action of oncolytic herpes simplex virus and radiotherapy in pancreatic cancer cell lines[J].Br J Surg, 2010, 97(9):1385
[3] WATANABE D.Medical application of herpes simplex virus[J].J Dermatol Sci, 2010, 57(2):75
[4] COFFIN R.Interview with Robert Coffin, inventor of T-VEC:the first oncolytic immunotherapy approved for the treatment of cancer[J].Immunotherapy, 2016, 8(2):103
[5] MATTILA RK, HARILA K, KANGAS SM, et al.An investigation of herpes simplex virus type 1 latency in a novel mouse dorsal root ganglion model suggests a role for ICP34.5 in reactivation[J].J Gen Virol, 2015, 96(8):2304
[6] LIPSON EJ, SHARFMAN WH, CHEN S, et al.Safety and immunologic correlates of Melanoma GVAX, a GM-CSF secreting allogeneic melanoma cell vaccine administered in the adjuvant setting[J].J Transl Med, 2015, 13(1):214
[7] KOHLHAPP FJ, KAUFMAN HL.Molecular pathway:mechanism of action for talimogene laherparepvec, a new oncolytic virus immunotherapy[J].Clin Cancer Res, 2016, 22(5):1048
[8] SHERIDAN C.First oncolytic virus edges towards approval in surprise vote[J].Nat Biotechnol, 2015, 33(6):569
[9] 李永红.基因治疗药物的质量控制[C]//2016年生物技术药物理化特性分析与质量研究技术研讨会资料汇编.北京:中国药学会, 2016:50 LI YH.Quality control of gene therapy drugs[C]//Data Collection of 2016 Technical Seminar on Analysis of Physical and Chemical Properties and Quality Research of Biotechnology Drugs.Beijing:Chinese Pharmaceutical Association, 2016:50
[10] 伍立群.单纯疱疹病毒进入细胞机制的研究进展[J].国际病毒学杂志, 2010, 17(5):143 WU LQ.Advances in the mechanism of herpes simplex virus entry into cells[J].Int J Virol, 2010, 17(5):143
[11] 段徐华, 吴利红, 董闪闪, 等.不同质量标准中生物学活性测定四参数回归计算法的比较[J].中国医药工业杂志, 2018, 49(10):1428 DUAN XH, WU LH, DONG SS, et al.Comparison of four parameter regression calculation methods for biological activity determination in different quality standards[J].Chin J Pharm, 2018, 49(10):1428
[12] 高凯, 付志浩, 李永红, 等.重组复制型溶瘤单纯疱疹病毒人细胞巨噬细胞集落刺激因子的质量研究[J].中国药学杂志, 2011, 46(19):1520 GAO K, FU ZH, LI YH, et al.Quality control research on recombinant oncolytic herpes simplex virus serotype 1 encoding human GM-CSF gene[J].Chin Pharm J, 2011, 46(19):1520
[13] CAREW JF, KOOBY DA, HALTERMAN MW, et al.A novel approach to cancel therapy using an oncolytic herpes virus to package amplicons contain cytokine genes[J].Mol Ther, 2001, 4(3):250

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