默克尔细胞多瘤病毒感染与检测
Summary
在这里, 我们提出了一个方案, 以感染原生真皮成纤维细胞与 mcpyv。该方案包括皮肤成纤维细胞的分离、mcpyv 病毒的制备、病毒感染、免疫荧光染色和荧光原位杂交。该协议可以扩展到描述 mcpyv-主机相互作用的特征, 并发现 mcpyv 注入的其他细胞类型。
Abstract
默克尔细胞多瘤病毒 (mcpyv) 感染可导致默克尔细胞癌 (mcc), 这是一种极具攻击性的皮肤癌。由于缺乏适当的细胞培养模型, 充分研究 mcpyv 分子生物学和致癌机制的力学研究受到了阻碍。在这里, 我们描述了一组执行和检测人类皮肤细胞 mcpyv 感染的协议。这些协议描述了人真皮成纤维细胞的分离、重组 mcpyv 病毒的制备, 以及免疫荧光 (if) 染色和原位 dna 杂交链反应 (hcr) 检测病毒感染的方法, 这是一种高度敏感的方法。荧光原位杂交 (fish) 方法。有兴趣的研究人员可以对本文中的协议进行调整, 以确定支持 mcpyv 感染的其他细胞类型或细胞系。所描述的 fish 方法也可以用来检测受感染的人类皮肤中存在的低病毒 dna 水平。
Introduction
默克尔细胞多瘤病毒 (mcpyv) 是一种小型双链 dna 病毒, 与罕见但具有攻击性的皮肤癌, 默克尔细胞癌 (mcc)1,2。mcc 的死亡率, 约 33%, 超过黑色素瘤 3,4。mcpyv 的圆形基因组约为 5 kb 1,5 被非编码调控区 (ncrr) 一分为二, 进入早期和后期编码区域1。ncrr 包含病毒复制 (ori) 的病毒来源和病毒转录的双向启动子 6,7。早期区域编码肿瘤抗原蛋白称为大 t (lt), 小 t (st), 57kt, 替代 lt orf (alto), 以及自动调节 mirna1,8, 9, 10.后期区域编码衣壳蛋白 vp1 和 vp211,12,13。lt 和 st 是研究最多的 mcpyv 蛋白, 已被证明支持病毒 dna 复制和 mcpyv 诱导的肿瘤发生 5。mcpyv dna 在宿主基因组中的克隆整合, 在多达80% 的 mcc 中被观察到, 很可能是病毒阳性肿瘤发生 14,15的一个因果因素。
在过去20年中, mcc 的发病率增加了两倍16。无症状 mcpyv 感染在一般人群中也很普遍,为 17,18,19。随着 mcc 诊断数量的增加和 mcpyv 感染的高流行率, 有必要提高我们对病毒及其致癌潜力的认识。然而, mcpyv 生物学和致癌机制的许多方面仍然鲜为人知20。这主要是因为 mcpyv 在现有细胞系11、12、21、22、23以及直到最近的皮肤细胞中复制得很差, 直到最近, 还有能够支持 mcpyv 的皮肤细胞感染没有被发现 22。由于缺乏传播病毒的细胞培养系统, 充分研究 mcpyv 及其与宿主细胞相互作用的力学研究受到了阻碍。
我们发现, 从新生儿包皮中分离出的原代人真皮成纤维细胞 (hdf)在体外和体外24支持强大的 mcpyv 感染。通过本研究, 建立了 mcpyv24的第一个细胞培养感染模型。在该模型系统的基础上, 我们发现, 通过 wnt/β-儿茶素信号转导途径和其他生长因子诱导基质金属蛋白酶 (mmp) 基因刺激 mcpyv 感染。此外, 我们还发现, fda 批准的 mek 拮抗剂曲美替尼有效地抑制 mcpyv 感染5,25。通过这些研究, 我们还建立了一套隔离人真皮成纤维细胞 24,25的协议, 准备 mcpyv 病毒 11,12, 对人类真皮成纤维细胞进行 mcpyv 感染24,25和 mcpyv 蛋白的 if 染色26。此外, 我们还采用原位 dna 杂交链反应 (hcr) 技术27 , 开发了一种高度敏感的 fish 技术 (hcr-dna fish), 用于检测受感染的人类皮肤细胞中的 mcpyv dna。这些新方法将有助于研究 mcpyv 的感染周期以及细胞对 mcpyv 感染的反应。维持 mcpyv 感染的天然宿主水库细胞和引起 mcc 肿瘤的细胞仍然是未知的。我们在这篇手稿中描述的技术可以用来检查各种类型的人体细胞, 以确定储集细胞和 mcc 肿瘤的起源。我们已建立的方法, 如 hcr-dna fish, 也可用于检测其他 dna 肿瘤病毒和宿主细胞相互作用的特征。
Protocol
Representative Results
Discussion
上述方法包括从人体皮肤组织中分离真皮成纤维细胞、制备重组 mcpyv 病毒、培养细胞感染、免疫荧光染色和采用 hcr 技术的敏感 fish 方法。应该使研究人员能够分析 mcpyv 感染 27.在体外实现 mcpyv 感染的最关键步骤之一是生产高滴度病毒制剂。利用本文所述的重组 mcpyv 病毒的制备方案, 我们实现了每毫升的 10, 12个基因组拷贝的病毒滴度, 共 11,<sup …
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢 meenhard herlyn 博士 (wistar 研究所) 和 m. celeste simon 博士 (宾夕法尼亚大学) 提供试剂和技术支持。我们还感谢我们实验室的成员进行了有益的讨论。这项工作得到了国家卫生研究院 (r01ca187718、r01ca147778 和 R01CA187718)、nci 癌症中心支助赠款 (nci p30 c12016520) 和 penn cfar 奖 (p30 ai 045008) 的支持。
Materials
| Fetal calf serum | HyClone | SH30071.03 | |
| MEM Non-Essential Amino Acids Solution, 100X | Thermo Fisher Scientific | 11140050 | |
| GLUTAMAX I, 100X | Thermo Fisher Scientific | 35050061 | L-Glutamine |
| DPBS, no calcium, no magnesium | Thermo Fisher Scientific | 14190136 | |
| 0.05% Trypsin-EDTA | Thermo Fisher Scientific | 25300-054 | |
| DMEM/F12 medium | Thermo Fisher Scientific | 11330-032 | |
| Recombinant Human EGF Protein, CF | R&D systems | 236-EG-200 | Store at -80 degree celsius |
| CHIR99021 | Cayman Chemical | 13122 | Store at -80 degree celsius |
| CHIR99021 | Sigma | SML1046 | Store at -80 degree celsius |
| Collagenase type IV | Thermo Fisher Scientific | 17104019 | |
| Dispase II | Roche | 4942078001 | |
| Antibiotic-Antimycotic | Thermo Fisher Scientific | 15240-062 | Protect from light |
| DMEM medium | Thermo Fisher Scientific | 11965084 | |
| Alexa Fluor 594 goat anti-mouse IgG | Thermo Fisher Scientific | A11032 | Protect from light |
| Alexa Fluor 488 goat anti-rabbit IgG | Thermo Fisher Scientific | A11034 | Protect from light |
| OptiPrep Density Gradient Medium | Sigma | D1556 | Protect from light |
| Paraformaldehyde | Sigma | P6148 | |
| anti-MCPyV LT (CM2B4) | Santa Cruz | sc-136172 | Lot # B2717 |
| MCV VP1 rabbit | Rabbit polyclonal serum #10965 | https://home.ccr.cancer.gov/lco/BuckLabAntibodies.htm | |
| Hygromycin | Roche | 10843555001 | |
| Basic Fibroblast Growth Factors (bFGF), Human Recombinant | Corning | 354060 | Store at -80 degree celsius |
| Benzonase Nuclease | Sigma | E8263 | |
| Plasmid-Safe ATP-Dependent DNase | EPICENTRE | E3101K | |
| Probe hybridization buffer | Molecular technologies | ||
| Probe wash buffer | Molecular technologies | ||
| Amplification buffer | Molecular technologies | ||
| Alexa 594-labeled hairpins | Molecular technologies | B4 | Protect from light |
| Triton X-100 | Sigma | X100 | |
| Quant-iT PicoGreen dsDNA Reagent | Thermo Fisher Scientific | P7581 | |
| BamHI-HF | NEB | R3136 | |
| Buffer PB | Qiagen | 19066 | |
| blue miniprep spin column | Qiagen | 27104 | |
| 50mL Conical Centrifuge Tubes | Corning | 352070 | |
| T4 ligase | NEB | M0202T | |
| MagicMark XP | Thermo Fisher Scientific | LC5602 |
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