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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCBL-1 cells</td>
			<td>NIH AIDS Reagent Program</td>
			<td>3233</td>
		</tr>
		<tr>
			<td>PA317 cells</td>
			<td>ATCC</td>
			<td>CRL-2203</td>
		</tr>
		<tr>
			<td>Neonatal dermal microvascular endothelial cells</td>
			<td>Lonza</td>
			<td>CC-2505</td>
		</tr>
		<tr>
			<td>EBM-2 Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
		</tr>
		<tr>
			<td>EGM-2 BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
		</tr>
		<tr>
			<td>anti-KSHV LANA/ORF 73</td>
			<td>Advanced Biotechnologies</td>
			<td>13-210-100</td>
		</tr>
		<tr>
			<td>TrypLETMExpress, no phenol red</td>
			<td>ThermoFisher</td>
			<td>12604013</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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

Nanyang Technological University

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

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

科研

生物学

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3&#39;UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into &#62;75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Adenofection:用于研究分子伴侣在细胞地貌被耗尽，救援实验中的角色方法

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

使用 FluoZin-3 测定研究哺乳动物细胞中的锌转运机制

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

作者聚焦:通过 ZnT1 功能探索细胞锌调节 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...

一种用于研究卡波西肉瘤细胞转化的体外模型

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克里斯托弗休斯：在体外血管生成的研究模型（面试）

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在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

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一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

体外 E3泛素连接酶功能分析

使用体外锌转运测定表征哺乳动物锌转运蛋白

一种用于研究卡波西肉瘤细胞转化的体外模型

摘要

探索更多视频

此视频中的章节

克里斯托弗休斯：在体外血管生成的研究模型（面试）

微流体装置与Groove的模式，为研究细胞行为

研究有丝分裂的显微注射技术果蝇合胞体胚胎

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Adenofection:用于研究分子伴侣在细胞地貌被耗尽，救援实验中的角色方法

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

体外 E3泛素连接酶功能分析

使用 体外 锌转运测定表征哺乳动物锌转运蛋白

使用体外锌转运测定表征哺乳动物锌转运蛋白