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3H-Penciclovir (3H-PCV) Uptake Assay
3H-喷昔洛韦(3H-PCV) 摄取试验   

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Abstract

Thymidine Kinase from human Herpes simplex virus type 1 (HSV1-TK) in combination with specific substrate prodrug nucleotide analogue ganciclovir (GCV) has been widely used as suicidal therapeutic gene for cancer gene therapy. HSV1, and its mutant (HSV1-sr39TK) with improved substrate specificity, were used as reporter genes for PET-imaging of various biological functions in small animals, by combining with radiolabeled substrates such as 18F-FHBG and 124I-FIAU. 3H-Penciclovir (PCV) uptake assay is a method of choice used to determine the expression level of HSV1-TK in mammalian cells and tissues. HSV1-TK phosphorylate PCV and result in the formation of penciclovir monophosphate, and its subsequent phopsphorylation by cellular TK lead to the formation of penciclovir triphosphate, which is trapped selectively in cells express HSV-TK. 3H-Penciclovir enables the detection of penciclovir uptake of mammalian cells and tissues by radioactive procedures such as scintillation counting. Here we describe the protocol to carry out 3H-Penciclovir uptakes in mammalian cells.

Materials and Reagents

  1. Control and experimental cells (HEK293T and HEK293T-sr39TK)
  2. Dulbecco's Modified Eagle Medium (DMEM) (Life technologies, catalog number: 11965-084 )
  3. 3H-Penciclovir (specific activity 14.9 ci mmol-1) (Moravek Biochemicals, La Brea, CA, USA)
  4. Sodium hydroxide (NaOH) (0.1 N)
  5. Phosphate buffered saline (PBS) (pH 7.4) (Life technologies, catalog number: 10010-056 )
  6. Glass Scintillation vials
  7. Cytoscint Scintillation fluid (Acros Organics, Geel, Belgium)
  8. Cell culture plates (Greiner Bio-One, USA)
  9. Bradford protein assay reagent (Bio-Rad Laboratories, Hercules, CA)
  10. BSA (Sigma-Aldrich, catalog number: A9418 )
  11. Wash solution (Ice cold PBS) (pH 7.4)

Equipment

  1. 37 °C 5% CO2 Cell culture incubator (Themo Fisher Scientific, model: Napco 8000)
  2. Scintillation counter (Beckman Coulter, Brea, CA)
  3. Shaker (Boekel scientific, Model: 260350 )
  4. Radioactive Chemical hood (Hamilton, Two Rivers, WI)
  5. Pipettes (Gilson, Middleton, WI)
  6. Disposal (As per Environment and Health safety regulation)
  7. Spectrophotometer (Varian Inc, model: Varian Cary 50 )

Procedure

  1. Mammalian HEK293T and HEK293T cells stably expressing sr39-HSV1-TK (HEK293T-sr39TK) should be plated at a confluency of 60-80% in 12 well culture plates (150,000 cells/well in 1 ml DMEM). Incubate at 37 °C with 5% CO2 for 24 h.
  2. Twenty-four hours after initial plating remove the medium and add 0.5 μl of 3H-Penciclovir/well (0.5 mCi/ml of 8-3H-Penciclovir; specific activity 14.9 ci mmol-1) in 0.5 ml of respective culture medium.
  3. Incubate at 37 °C for 3 h. Maintain the incubation time consistent between samples.
  4. Keep the same cells (HEK293T) plated in equal number express no HSV1-TK as control.
  5. After 3 hours of incubation remove the medium carefully without disturbing cells, and wash the plates twice with 0.5 ml each of ice cold PBS. Pool the medium and wash solutions from respective well to measure the remaining total activities.
  6. Add 0.5 ml of 0.1 N NaOH to each well and leave it for 10 min at room temperature in a shaker to lyse the cells.
  7. Transfer 20 μl of homogenous cell lysate to measure total protein concentration by using Bradford protein assay reagent. In brief, to each 20 μl of cell lysate add 1 ml of 1x Bradford protein assay reagent and read the absorbance at 595 nm (A595) 5 min after incubation at room temperature in a spectrophotometer. Use standard graph prepared with the standards (BSA) diluted in 0.1 N NaOH to calculate the total protein present in 0.5 ml of cell lysate.
  8. Transfer the remaining cell lysate (480 μl) to scintillation vial and 5 ml of biodegradable scintillation fluid (Cytoscint fluid). Cells lacking HSV1-TK expression exposed to 3H-Penciclovir serve as negative control.
  9. The total remaining activity from each sample should be measured by using 480 μl of solution taken from the pooled medium and the wash solution, and also measured from 0.5 μl of 3H-Penciclovir diluted in 480 μl of medium.
  10. Measure the radioactivity from different samples in a Scintillation counter by taking an average reading/min by reading for 5 min. Normalize the results with protein concentration and relate with total activity.
  11. Results should be expressed as the percentage conversion of 3H-PCV per milligram of protein/total count, or as the conversion of 3H -PCV in moles per milligram of protein/total activity (Radioactivity within cells/[Radioactivity remained in medium + Radioactivity within cells]).

Acknowledgments

We thank Dr Sanjiv Sam Gambhir, Chairman, Department of Radiology, Stanford University for providing helpful support and facility for conducting this research. We thank the Department of Radiology, Stanford University for funding support (R. Paulmurguan), and thank the Canary Center at Stanford for providing the facilities. We also thank NIH-NCI RO1CA161091 (R. Paulmurugan) for partial funding support.

References

  1. Sekar, T. V., Foygel, K., Willmann, J. K. and Paulmurugan, R. (2013). Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging. Gene Ther 20(5): 529-537.

简介

来自人单纯疱疹病毒1型(HSV1-TK)的胸苷激酶与特定底物前药核苷酸类似物更昔洛韦(GCV)的组合已广泛用作癌症基因治疗的自杀性治疗基因。将具有改善的底物特异性的HSV1和其突变体( HSV1-sr39TK )用作小动物中各种生物学功能的PET成像的报告基因,通过与放射性标记的底物例如18 F-FHBG和124 I-FIAU。 H-Penciclovir(PCV)摄取试验是一种选择用于测定哺乳动物细胞和组织中HSV1-TK表达水平的方法。 HSV1-TK磷酸化PCV并导致形成penciclovir单磷酸,并且其随后通过细胞TK的磷酸化导致形成选择性地捕获在表达HSV-TK的细胞中的三磷酸喷昔洛韦。通过诸如闪烁计数的放射性方法,可以检测到更昔洛韦对哺乳动物细胞和组织的摄取。在这里我们描述在哺乳动物细胞中进行 H - Penciclovir摄取的方案。

材料和试剂

  1. 对照和实验细胞(HEK293T和HEK293T-sr39TK)
  2. Dulbecco改良的Eagle培养基(DMEM)(Life technologies,目录号:11965-084)
  3. (Moravek Biochemicals,La Brea,CA,USA)的3 H - 比昔洛韦(比活性14.9 ci mmol -1 )。
  4. 氢氧化钠(NaOH)(0.1N)
  5. 磷酸盐缓冲盐水(PBS)(pH 7.4)(Life technologies,目录号:10010-056)
  6. 玻璃闪烁瓶
  7. Cytoscint闪烁液(Acros Organics,Geel,Belgium)
  8. 细胞培养板(Greiner Bio-One,USA)
  9. Bradford蛋白测定试剂(Bio-Rad Laboratories,Hercules,CA)
  10. BSA(Sigma-Aldrich,目录号:A9418)
  11. 洗涤溶液(冰冷PBS)(pH 7.4)

设备

  1. 37℃5%CO 2细胞培养箱(Themo Fisher Scientific,型号:Napco 8000)
  2. 闪烁计数器(Beckman Coulter,Brea,CA)
  3. Shaker(Boekel scientific,型号:260350)
  4. 放射性化学罩(Hamilton,Two Rivers,WI)
  5. 移液管(Gilson,Middleton,WI)
  6. 处置(根据环境和健康安全规定)
  7. 分光光度计(Varian Inc,型号:Varian Cary 50)

程序

  1. 哺乳动物   HEK293T和HEK293T细胞稳定表达sr39-HSV1-TK (HEK293T-sr39TK)应在60- 80%的汇合度在12孔中接种 培养板(150,000个细胞/孔,在1ml DMEM中)。 37℃孵育   5%CO 2小时,持续24小时
  2. 在初始铺板后24小时,除去培养基,并加入0.5μl的3 H - 喷昔洛韦/孔(0.5mCi/ml的8-羟色胺 - 比昔洛韦;比活性 14.9 ci mmol -1
  3. 在37℃孵育3小时。 保持样品之间的孵育时间一致。
  4. 保持相同数量的相同细胞(HEK293T)不表达HSV1-TK作为对照
  5. 后   3小时的孵育小心地移除培养基而不打扰 细胞,并用0.5ml冰冷的PBS洗涤板两次。 游泳池 将来自相应孔的介质和洗涤溶液测量 剩余总活动
  6. 向每个孔中加入0.5ml的0.1N NaOH,并在室温下在振荡器中保持10分钟以裂解细胞。
  7. 转让   20μl均质细胞裂解液,测量总蛋白浓度 通过使用Bradford蛋白测定试剂。 简而言之,对每20微升细胞   裂解物加入1ml 1x Bradford蛋白测定试剂并读取 在室温下孵育5分钟后在595nm(A <595)的吸光度 在分光光度计中。 使用标准准备的标准图 (BSA)稀释在0.1N NaOH中,计算存在的总蛋白 0.5ml细胞裂解物。
  8. 转移剩余的细胞裂解液 (480μl)至闪烁瓶和5ml可生物降解的闪烁 流体(Cytoscint流体)。 缺乏暴露于 3的H-Penciclovir的HSV1-TK表达的细胞作为阴性对照。
  9. 总数 每个样品的剩余活性应使用480μl测量 来自合并的培养基和洗涤溶液的溶液,以及 从稀释在480μl培养基中的0.5μl的3 H-泛昔洛韦测量。
  10. 测量   闪烁计数器中不同样品的放射性 取平均读数/min读5分钟。 归一化 结果与蛋白质浓度和与总活动有关
  11. 结果应表示为每毫克蛋白质/总计数的 H-PCV的转化百分比,或者表示为 H   -PCV,以每毫克蛋白质的摩尔数/总活性(放射性 在细胞内/[放射性保持在培养基+放射性 单元格])。

致谢

我们感谢斯坦福大学放射学系主任Sanjiv Sam Gambhir博士为进行这项研究提供有用的支持和便利。 我们感谢斯坦福大学放射科的资金支持(R. Paulmurguan),并感谢斯坦福的加那利中心提供的设施。 我们还感谢NIH-NCI RO1CA161091(R. Paulmurugan)的部分资助支持。

参考文献

  1. Sekar,T.V.,Foygel,K.,Willmann,J.K.and Paulmurugan,R。(2013)。 双重治疗性报告基因融合用于增强癌症基因治疗和成像。 Gene Ther 20(5):529-537。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sekar, T. V. and Paulmurugan, R. (2013). 3H-Penciclovir (3H-PCV) Uptake Assay. Bio-protocol 3(17): e887. DOI: 10.21769/BioProtoc.887.
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