这项工作得到了 K12 HD068322 (SCM) 的支持;R01 CA179921 和 P51 OD011092 (AVM);并授予美国心脏协会 (SB) 14PRE20320014 奖。

注意: 本协议中描述的所有过程都应在 BSL-2 条件下执行. 
 1. KSHV 库存准备 <ol> <li> 准备缓冲区: 在 ddH 2 O 中溶解292.24 毫克 EDTA, 带到225毫升, 并调整到 pH 8。溶解605.7 毫克在 ddH 2 O, 带来225毫升, 并调整到 pH 值8。结合 EDTA 和三磷酸溶液, 加入4.38 克氯化钠, 调整最终体积到500毫升, 过滤杀菌, 并存储在4和 #176; C . </li>
	<li> 培养 KSHV 阳性、eb 病毒阴性的原发性渗出性淋巴瘤细胞系 BCBL-1 在37和 #176 的湿式恒温箱中; C 加 5% CO 2 在 RPMI 中补充10% 热灭活胎牛血清 (FBS) 和抗菌素, 以约1每毫升为 1.2 x 10 6 单元格. </li>
	<li> 诱导 KSHV 生产, 分裂培养1:2 与新鲜培养基和增加佛波 12-酸13乙酸 (PMA) 到最后集中 20 ng/毫升和孵育文化为5天。作为替代方法, 用丁酸钠 (0.1 毫米) 治疗细胞3天, 诱导裂解病毒复制. </li>
	<li> 要收获病毒微粒, 首先离心培养上清在 300 x g 为5分钟在室温下颗粒细胞和转移清到新鲜的管子. </li>
	<li> 进一步澄清上清, 以避免细胞碎片转移, 离心机在 2500 x g 为10分钟, 在4和 #176; C. 
	注: 作为一种替代的高速离心澄清文化清, 过滤通过无菌0.45 和 #181; m 过滤器可以执行。我们已经观察到使用过滤的滴度显著下降;因此, 我们通常执行第二个离心步骤. </li>
	<li> 下一步, 覆盖5毫升的25% 蔗糖溶液在缓冲区与约30毫升澄清培养上清在 6 ultracentrifuge 管. </li>
	<li> 离心平衡管在 7.5万 x g 为2小时在4和 #176; C 在真空之下. </li>
	<li> 醒酒培养上清液, 并将每根管子的边缘印迹, 以尽可能的去除上清和蔗糖溶液, 然后重150和 #181 中可能不可见的病毒颗粒. </li>
	<li> 池所有悬浮病毒, 混合良好, 并冻结25和 #181; 我等分在-80 和 #176; C . </li>
</ol> 2。ec 与 E6 和 E7 瘤病毒基因的转导 注意: 我们经常使用标准的组织培养烧瓶来生长初级 ec。但是, 如果获得 EC 的增长不理想, 则应考虑使用商业文化烧瓶. 
<ol> <li> 在 37 和 #176 中培养主要的人皮肤微血管或淋巴 ec; C 加 5% CO 2 在 ec 生长培养基中 (华; 含有 ec 基培养基 [循证医学] 辅以 EGM-2 BulletKit [含 FBS,氢化可的松, 人成纤维细胞生长因子 B, 血管 EC 生长因子, 胰岛素样生长 factor-1, 抗坏血酸, 人上皮生长因子, 抗生素和肝素) 在 T75 细胞培养瓶到大约50% 汇合. </li>
	<li> 为转导, 培养 PA317 LXSN 16E6E7 细胞 24 , 35 在一个湿恒温孵化器在 37 和 #176; C 加 5% CO 2 在 DMEM 补充 10% FBS 和抗菌素在T150 细胞培养烧瓶, 直到它们大约90% 汇合。然后在16毫升培养基中进行夜间孵育. </li>
	<li> 在室温下, 通过离心在 300 x g 中澄清 PA317 介质5分钟. </li>
	<li> 将华从 EC 培养物中移除, 并将12毫升的澄清 PA317 培养基和4小时孵育成覆盖细胞. 
	注: 离心16毫升的条件培养基在 300 x g, 5 分钟的结果在一个紧凑的颗粒, 不干扰的小心后去除12毫升澄清上清。然而, 如果转移 PA317 LXSN 16E6E7 细胞到初级 ec 文化是一个关注 (包装细胞线将很快超越 ec), 然后过滤的条件上清通过一个0.45 和 #181; m 过滤器在传输之前可以执行. </li>
	<li> 用新鲜华取代6毫升的 PA317 培养基, 并在夜间孵育. </li>
	<li> Refeed EC 与12毫升新鲜华和孵化进一步 48 h. </li>
	<li> 对 EC 进行亚文化化, 去除培养基, 用12毫升无阳离子 PBS 洗涤, 并在37和 #176 上添加3毫升的商业酶解离液 (, 如 , TrypLE), 并孵育在 3 min. </li>
	<li> 将电池悬浮液转移至15毫升锥形管, 在室温下 300 x g 离心机5分钟。重的结果细胞团在新鲜的华和分裂均匀成 3 x T75 烧瓶, 最后体积12毫升华每瓶. </li>
	<li> 要选择 ec 转与 E6 和 E7, 添加 G418 最终浓度为200和 #181; 两个通道的 g/毫升, 之后, 转 ec 可以被镀为感染或液氮冷冻. </li>
</ol> 3。转 ec 与 KSHV 感染的研究 <ol> <li> 在感染前的前一天, 用酶消化的商业酶解离溶液, 如上所述。重细胞颗粒在1毫升的华。使用5和 #181; L 细胞准备1/10 稀释的台盼蓝。使用例和种子 2.5 x 10 5 活细胞计数2毫升的华每井在6井板. </li>
	<li> 在感染的当天, 用3毫升的 PBS 去除华和洗涤细胞, 每井有钙和镁, 然后加入2毫升循证医学. 
	注意: 在感染过程中, 必须使用循证医学而非华, 因为华中的肝素将抑制病毒微粒与靶 EC 的随后感染. </li>
	<li> 用 KSHV 感染细胞, 增加5到20和 #181; L 病毒储存到每个井和漩涡板混合。对于模拟感染的细胞, 在每个井中添加等量的缓冲液. </li>
	<li> 在室温下离心板在 400 x g 处30分钟, 然后在37和 #176 上孵育板; C 为 90 min. </li>
	<li> 如果研究的目的是要调查需要同步病毒感染的早期事件, 例如 , 在 中的早期事件, 请在此时删除病毒接种。用2毫升新鲜华冲洗和 refeed 细胞。否则, 添加2毫升华和孵化文化过夜. </li>
	<li> Refeed 细胞2毫升华感染后的一天, 然后每隔一天. </li>
	<li> 当细胞大约90% 汇合时, 通过酶消化的方法, 利用商业酶解离溶液, 然后将三口井中的细胞汇集成 T75, 同时注意总通道数和通道感染. </li>
	<li> 扩大模拟和 KSHV 感染 EC 的1:3 分裂至少两个通道的时间细胞可以用于实验或冷冻液氮. 
	注: 随着 EC 转与 E6 和 E7 的分裂, 模拟和 KSHV 感染细胞的维护培养应在到达汇合之前分裂 (〜85到 90%). </li> </ol>
 4. 用免疫荧光法确认感染与 KSHV 的关系 注意: 检测 KSHV 潜伏期相关核抗原 (LANA-1/ORF73) 提供了一个可靠的定量测量的感染。抗拉娜抗体是商用的. 
<ol> <li> 在染色前的前一天, 在1毫升华中, 每一个井板上都有模拟或 KSHV 感染 EC, 在 1 x 10 5 单元中, 每口都有一个24孔, 并在夜间孵育. </li>
	<li> 用500和 #181 清洗细胞两次; PBS 加钙和 magniseum。在一个摇摆的平台上洗三十年代. </li>
	<li> 在外部 ventillated 通风罩中, 用500和 #181 固定电池, 室温下为15分钟, 4% 甲醛. </li>
	<li> 用500和 #181 清洗细胞三次; 洗涤缓冲液 (0.1% 卫 X-100 加0.02% 山羊血清在 PBS 和阳离子中). </li>
	<li> 用500和 #181 在洗涤缓冲液中用2% 只山羊血清对细胞进行30分钟的室温分组. </li>
	<li> 用500和 #181 清洗电池三次; 洗涤缓冲器的 L. </li>
	<li> 使用200和 #181 对模拟或 KSHV 感染的 EC 进行标记; 主要抗体稀释1:100在洗涤缓冲器为30分钟在室温在一个摇摆物。在200和 #181 中孵育余下的两口井; 只在洗涤缓冲器的 L. </li>
	<li> 用500和 #181 清洗电池三次; 洗涤缓冲器的 L. </li>
	<li> 将所有四口井都贴上200和 #181 的标签; 二级抗体的 L 在洗涤缓冲液中稀释 1:100, 在一个摇杆上的室温为30分钟. </li>
	<li> 用500和 #181 清洗电池三次; 洗涤缓冲器的 L. </li>
	<li> 对每一个井应用20和 #181; L 安装培养基含有 DAPI 和片, 并用倒置荧光显微镜对染色进行评价. </li>
	<li> 通过计算至少200个细胞中的拉娜阳性细胞核数, 确定感染 KSHV 的细胞的百分比。感染重组 KSHV-Bac16 也可以通过观察的文化表达的 GFP. </li>
</ol>

发生是一个多步骤的过程, 它绕过了一个有机体36中的重要安全措施。由于 KS 病变存在于慢性炎症的频谱, 以真正的肉瘤, 阐明某些病理生理学过程介导的 KSHV 要求, 一些研究在细胞培养模式, 支持转换9。应该指出的是, 接触抑制的丧失和锚定依赖性生长, 表型指示细胞转化, 不容易发展继发于原 ec 或 ec 的外源表达的端粒酶的感染。因此, 虽然在这里描述的 ks 的体外模型并没有重述说明 ks 细胞的 ks 肿瘤微环境或行为的所有方面, 但它特别适合于研究感染诱导的宿主细胞基因表达的变化。KSHV, 有助于 KS 肿瘤的发生。
第一次报告的基因表达谱分析研究使用的文化系统描述的受体酪氨酸激酶 c 试剂盒作为一个贡献的转化表型的 KSHV 感染的细胞25。使用显性负结构或酪氨酸激酶抑制剂 siRNA 或抑制 c-试剂盒信号的 c 试剂盒的表达式. STI 571 (伊马替尼) 通过长时间的病灶形成, 干扰了 KSHV 明显的转化能力感染细胞的培养。伊马替尼的临床评价证实了这种药物在流行性 KS 患者中的有效性47,48,49。其他潜在的治疗目标也已经确定, 包括血红素 oxygenase-126,50, CXCR727, 和β肾上腺素受体28, 所有这些都在生长或转化中发挥作用EC体外.确认这些基因表达模式在原 EC 以及 ks 活检组织证实了该系统的生理相关性, 并强调其作为一个临床前模型的 ks 疗法的价值25,26, 51,52,53,54,55。
E6/E7 转导系统的另一个优点是, 与原 EC 相比, 永生的延长寿命能够长期维持年龄和通道匹配的模拟感染控制细胞, 以便对 KSHV 的结果进行比较评价。感染.重要的是要注意, 为了最大限度地增加转与 E6 和 E7 的通道数, 这是至关重要的分裂文化之前达到汇合 (〜85至 90%)。此外, 这些细胞在长时间内耐受无血清条件。初级 EC 在生长因子提取后迅速进行程序性细胞死亡, 因此不能长时间培养, 没有血清或重组生长因子56,57。这种依赖性使得分泌信号通路的感染与 KSHV 难以识别和研究, 因为在充分转变之前 KSHV 感染细胞也将经历程序性细胞死亡后, 生长因子退出 (未发表观察)。然而, E6 和 E7 蛋白在这个模型中的存在, 防止了在生长因子提取后的程序性细胞死亡, 并允许长时间 (例如, 48 h) 培养在低血清或血清自由培养基上25,26,28。
KS 是一种复杂和不寻常的疾病, 具有慢性炎症和细胞转化的特点。新的治疗策略的临床需要是巨大的, 并且这里描述的细胞培养模型有独特的物产允许评估候选药物目标。

卡波西肉瘤 (KS) 是一种多的 angioproliferative 肿瘤, 影响真皮, 黏膜, 和内脏的网站, 发展最常见的设置先进的免疫抑制1。四流行病学形式被描述了: 典型, 懒惰形式典型地影响地中海和中东遗产的老年人;医源性, 由器官移植后免疫抑制剂治疗所致;流行病, 一种艾滋病定义的癌症;和地方性流行病, 是非洲流行地区儿童常见的一种艾滋病毒独立形式。随着有效的抗逆转录病毒药物治疗方案的问世, 艾滋病毒的流行在发展中国家被普遍诊断为不多。然而, 在许多非洲国家, 临床上侵袭性流行和流行的形式仍然是最常见的诊断癌症2,3,4。因此, 确定有效的病机-靶向药物治疗 KS 是一个研究的重点。
组织学上, KS 病变的特点是广泛的, 但不正常的新生血管, 即纺锤细胞的 EC 起源形成不连续的维管网络5。这些异常血管 ("血管狭缝") 允许红细胞外渗, 从而使病灶具有其特有的颜色。此外, 病灶中含有大量的白细胞, 它们是慢性炎症的特征 (如淋巴细胞、巨噬细胞和血浆电池)。免疫重建后 ks 病变的回归已被描述, 这表明 ks 具有超增生性病变和真正的肿瘤的特点6,7,8,9。
ks 疱疹病毒 (KSHV), 致病剂 ks, 被确定在 1994年10。自那时以来, 许多体外细胞培养模型已经开发, 以使发病机制的研究, 包括细胞说明从肿瘤活检材料和主要或端粒酶表达 EC 感染 KSHV体外11,12,13,14,15,16,17,18. 目前尚无可用的模型完全重述了 KS 肿瘤微环境, 但都为我们了解 KSHV 感染的病理提供了宝贵的知识。不像其他已知的瘤人类疱疹病毒-巴尔 (eb), KSHV 不容易转换在文化中的细胞后de 从头感染19,20,21, 22. 然而, 这一限制已经克服了换初级人类 EC 的混合微血管或淋巴来源与 E6 和 E7 基因的人 hpv 类型16之前感染 KSHV23,24.这些外源癌基因的表达显著增加了 KSHV 的转化潜能在体外部分通过进一步抑制视网膜母细胞瘤蛋白和 p5323,24。这种 EC 转导方法允许多个实验室识别 KSHV 感染诱发的宿主细胞基因表达的关键改变, 并显示出促进 KS 细胞存活和增殖的作用25,26,27,28,29,30,31,32. 本文所述的协议是直接的和高度可重现的, 并将导致产生年龄和通道匹配的 KSHV 感染 EC 和模拟感染的控制, 可以培养远长于原细胞, 并将允许调查 KSHV 所采用的致癌机制。虽然该协议包括一种从原发性渗出性淋巴瘤细胞系 BCBL-1 中生产野生型 KSHV 的方法, 但 E6/E7-immortalized EC 也很容易感染重组 BACmid 源性 KSHV-BAC1630。在其他地方33,34中描述了用于准备 BAC16 的协议。

<table><tbody><tr><td>BCBL-1 cells</td><td>NIH AIDS Reagent Program</td><td>3233</td><td></td></tr><tr><td>PA317 cells</td><td>ATCC</td><td>CRL-2203</td><td></td></tr><tr><td>Neonatal dermal microvascular endothelial cells</td><td>Lonza</td><td>CC-2505</td><td></td></tr><tr><td>EBM-2 Basal Medium</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>EGM-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>anti-KSHV LANA/ORF 73</td><td>Advanced Biotechnologies</td><td>13-210-100</td><td></td></tr><tr><td>TrypLE&lt;sup&gt;TM&lt;/sup&gt;Express, no phenol red</td><td>ThermoFisher</td><td>12604013</td><td></td></tr><tr><td>RPMI</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td></td><td></td><td></td></tr><tr><td>PBS with calcium and magnesium</td><td></td><td></td><td></td></tr></tbody></table>

an in vitro model for studying cellular transformation by kaposi sarcoma herpesvirus

卡波西肉瘤 (KS) 是一种不寻常的肿瘤, 由内皮细胞 (EC) 与 KSHV 感染引起的梭形细胞组成, 在免疫抑制的设置中最常见。尽管进行了几十年的研究, 但对 KS 的最佳治疗仍未明确, 临床结果在资源有限的环境中尤其不利。KS 病变是由病理血管生成, 慢性炎症, 和发生, 和各种体外细胞培养模型的发展, 以研究这些过程。KS 产生于 KSHV 感染的内皮细胞, 因此 EC 谱系细胞提供最合适的体外的纺锤细胞前体的代孕。然而, 由于 EC 有一个有限的体外寿命, 而且由于 KSHV 所采用的致癌机制比其他瘤病毒的效率低, 因此很难评估初级或端粒酶-永生 EC。因此, 建立了一种新的 EC-based 培养模型, 可随时支持 KSHV 感染后的转化。人 E6 和 E7 基因的异位表达16型, 允许延长年龄和通道匹配的模拟和 KSHV 感染 EC 的培养, 并支持在受感染细胞培养中真正转变的 (即瘤) 表型的发展.这种易于处理和高度重现的 KS 模型, 有助于发现一些重要的信号通路, 具有很高的潜力, 翻译成临床设置。

原 EC 的形态学被经典地描述为 "鹅卵石", 这种形态学不被 E6 和 E7 基因 (图 1A) 的表达所改变。单 E6 和 E7 基因的表达不诱导转化表型;因此, 细胞易于接触抑制, 并将停止分裂后, 达到融合的文化。然而, 细胞将增殖和再生的汇合后, trypsinization 和 replating 在较低的密度, 允许维护年龄和通道匹配的文化, 作为模拟感染的控制使用。
E6/E7-transduced EC 与 KSHV 的感染导致了文化的戏剧性的形态学变化, 使人联想到 KS 病变中观察到的 "纺锤细胞" 表型 (图 1B)。一个转化的表型也诱导感染与 KSHV, 这是显着的失去接触抑制时, 细胞被培养 post-confluence (图 1C) 和锚地独立生长软琼脂 (图 2)。在发生过程中, 接触抑制和外基质相互作用 (锚地) 的损失是细胞转换的两个标志,36。重要的是要注意, 初级 ec 也容易感染的 KSHV 和初级 ec 系统是非常宝贵的, 以阐明不同方面的病毒宿主相互作用, 及其后果。此类研究在文献中遍布, 并列举了几个例子37、38、39、40、41、42、43 ,44。
荧光染色表明, KSHV 感染细胞的纺锤体形态与病毒蛋白 LANA-1/ORF73 (图 3A和3B, 红色) 的表达有关。由于病毒在不同的制剂中的浓度会有所不同, 所以每井的病毒添加量应该调整, 以便 KSHV 感染的文化达到和 #62; 90% 感染在一两个段落。随着串行通道感染的百分比将接近 100%, 并将保持在文化的持续时间 (约20通道)。如果对分泌影响的研究是有意的, 或者如果相邻未感染的细胞作为对照 (例如, 用于涉及其他感兴趣的蛋白质的免疫荧光研究), 则可以使用较低数量的病毒。受感染的文化也支持溶解性病毒蛋白的表达, 但是, 正如在 KS 病变中所观察到的, 只有少数的细胞在文化中会自发地进行溶解活化 (图 3B, 绿色)。细胞感染 KSHV-BAC16, 这是标签与 GFP, 也开发一个纺锤形形态 (图 3C和3D)。当使用 KSHV-BAC16 时, 病毒滴可以通过使用流式细胞仪30在 48 h 后感染的细胞中感染具有连续稀释的浓病毒储存和评价细胞的 GFP 表达而获得。
对于重组和病毒的基因组, 也可以通过从受感染细胞中纯化 dna, 然后通过 PCR 定量化 KSHV dna 水平来测定砧木制剂中的病毒。然而, 你不应该假设所有的基因组都是传染性的45。如果对 KSHV 溶出周期的研究是有意的, 可以通过刺激 EC 与上述 BCBL-1 细胞的诱导剂来达到病毒的活化作用, 尽管重新激活的效率是较低的23。在 KSHV 反蛋白 ORF5046的情况下, EC 的转导也可诱发溶解循环。与其他细胞培养模型的发现一致, E6/E7-expressing EC KSHV 溶解基因的自发表达和化学诱导都随着时间的推移而减少, 尽管有稳定的潜伏感染的维护11,16.
<img alt="Figure 1" src="/files/ftp_upload/54828/54828fig1.jpg"> 
图 1: 模拟和 KSHV 感染 EC 的形态学.(A) 模拟感染 EC 单分子膜展示了经典的卵石外观的相衬生命显微镜。如果没有 KSHV 基因的作用, 这些细胞就不会被转化, 因此会停止分裂, 并在达到文化融合时表现出接触抑制。(B) KSHV 感染的细胞, 相反, 形成一个细长的 "纺锤细胞" 形态, 使人联想到 KS 纺锤细胞。该小组显示了5天的 KSHV 感染细胞在到达汇合处时的形态学。(C) 在 KSHV 感染的细胞中, 宿主细胞基因表达的广泛变化导致细胞转化, 当 KSHV 感染的细胞被培养而不经过传代时就变得明显。在这样的条件下, KSHV 感染的细胞即使在达到融合后也会继续增殖, 导致细胞的多层次病灶的形成。该小组显示14天 PI KSHV 感染细胞的形态学, 培养9天 post-confluence。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54828/54828fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" src="/files/ftp_upload/54828/54828fig2.jpg"> 
图 2: 锚固独立生长.未细胞通常经历凋亡细胞死亡后, 失去接触的基板, 一个过程称为亡。(A) 当 E6/E7-transduced 模拟感染 EC, 这是没有转化, 被悬浮在软琼脂和培养了两个星期, 他们不形成殖民地。(B) KSHV 感染的 EC, 病毒转化, 是抗亡, 并将形成在软琼脂的殖民地。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54828/54828fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" src="/files/ftp_upload/54828/54828fig3.jpg"> 
图 3: 病毒蛋白表达的演示.(A) 在感染 KSHV (40X 放大倍数) 后, 纺锤体形态明显。(B) 在 (A) 中显示的同一字段的荧光染色表明, 纺锤体形态与病毒潜伏期蛋白 ORF73 (red) 的表达有关。受感染的细胞支持裂解病毒复制以及, 显示的小百分比的细胞表达病毒临cessivity 因子 ORF59 (绿色)。(A 和 B 从引用26中修改了。KSHV-BAC16 病毒 (C) 感染的细胞的纺锤体形态与 GFP (D) (20X 放大倍率) 的表达有关。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54828/54828fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus at JoVE.com

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

卡波西肉瘤 (KS) 是一种由感染致癌病毒的人 herpesvirus-8/KS 疱疹 (HHV-8/KSHV) 引起的肿瘤。这里描述的内皮细胞培养模型是唯一适合研究 KSHV 转化宿主细胞的机制。

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

an-vitro-model-for-studying-cellular-transformation-kaposi-sarcoma

Universidad San Sebastian

Research

JoVE Journal

Biology

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

8.2K 次观看.  University of Minnesota Medical School. 卡波西肉瘤 (KS) 是一种由感染致癌病毒的人 herpesvirus-8/KS 疱疹 (HHV-8/KSHV) 引起的肿瘤。这里描述的内皮细胞培养模型是唯一适合研究 KSHV 转化宿主细胞的机制。

视频: An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

确定失调卡波西氏肉瘤相关疱疹病毒（KSHV）诱导的基因

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

科研

免疫与感染

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

瞬时转染293T细胞复制缺陷型逆转录病毒生产

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

生物学

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

神经科学

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

癌症研究

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

肿瘤进展和血管生成的双生物发光成像

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...