作者没有确认。

1. 细胞的制备
<ol><li>在生物安全柜中使用无菌技术, 将2毫升培养基中的2万细胞放入6井板中的每一个井中。</li>
	<li>放置6井板在一个湿润的孵化器 (37 °c, 5% CO2) 为 24 h 允许细胞坚持板材。</li>
</ol>2. 制备用于治疗细胞的光敏剂
<ol><li>在培养基中准备0.5、1和2毫米的 5-阿拉溶液。如果胎牛血清 (血清) 是培养基中的一个成分, 则在培养基溶液中制备和治疗 5-孵化的细胞, 0.1% 的血清。11,12在这种情况下, SCC-13 细胞不需要血清, 因此 5-阿拉氨酸直接添加到角质的无血清培养基补充牛垂体提取物和表皮生长因子。</li>
	<li>吸入培养基, 用至少2毫升磷酸盐缓冲生理盐水 (PBS) 冲洗细胞。</li>
	<li>吸入 PBS 并将2毫升的0、0.5、1或2毫米的溶液放入单独的板材或水井中。</li>
	<li>在5至36摄氏度加热块上孵育0、0.5、1或2毫米 37-阿拉的细胞, 以防止细胞在使用铝箔孵化过程中受到光的照射。</li>
	<li>吸入 0, 0.5, 1 和2毫米 5-阿拉的解决方案, 并清洗细胞与2毫升的 PBS。</li>
	<li>吸入 PBS 并加入2毫升的培养基进行蓝光照射。</li>
</ol>3. 蓝光光疗法
<ol><li>在照射电池之前, 打开蓝光装置 (例如, 发光二极管、荧光灯或卤素灯) 并运行一个周期 (1000 秒) 以预热设备。蓝光装置的输出波长为 417 +/-5 nm。在处理前允许设备运行一个周期, 以确保蓝光波长和辐照度在整个 photoactivation 阶段是一致的。</li>
	<li>使用光度计测量细胞表面的辐照度。蓝色光辐照在细胞表面应该是10兆瓦/cm2。光和细胞之间的距离可能需要调整, 以达到10兆瓦/厘米2的辐照度取决于光源的强度。</li>
	<li>在蓝色光源下的黑色表面放置0、0.5、1和2毫米的治疗组。用蓝光照射细胞1000秒 (16 分钟和四十年代), 总剂量为 10 J/厘米2。在蓝光 photoactivation 之后, 细胞就可以进行分析了。</li>
</ol>4. 收集和染色
<ol><li>根据制造商的指导方针 (见材料表) 准备流量缓冲器。</li>
	<li>吸入培养基, 用2毫升的 PBS 冲洗细胞。</li>
	<li>添加1毫升0.25% 胰蛋白酶-EDTA, 并允许细胞分离约3至5分钟。细胞可以在显微镜下检查以确认细胞脱离。</li>
	<li>添加1毫升培养基 (或 PBS) 与10% 胎牛血清, 以停用胰蛋白酶。</li>
	<li>收集细胞悬浮到标记的5毫升流动管。</li>
	<li>在离心机中旋转5毫升的流动管, 在 201 x g处进行5分钟的抽吸溶液, 不去除细胞颗粒。</li>
	<li>添加200µL 的流缓冲器与共轭蛋白抗体 (1 µL 蛋白 v 每39µL 的流量缓冲区) 的每个样本。</li>
	<li>并用重悬细胞和地方在孵化器 (37 °c, 5% CO2) 为20分钟允许蛋白-V 捆绑。</li>
	<li>向每个样品添加3µL 7-反倾销。室温下孵育5分钟。</li>
</ol>5. 流量细胞分析
<ol><li>遵循流式细胞仪制造商的设置指南 (见材料表)。</li>
	<li>用流式细胞仪采集和分析样品。13
</li>
	<li>采用方差分析14对治疗和对照组进行统计学比较。将治疗组的方法与控制的平均值进行比较, 用 Dunnett 的试验进行后分析。</li>
</ol>

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DUSA/太阳制药提供了 5-阿拉和蓝光光设备。这份手稿由巴拉特·贾格迪奥博士的剩余资金支持。这些内容不代表美国退伍军人事务部或美国政府的意见。

30分钟孵化、1、2毫米 5-SCC-13 细胞凋亡显著增加, 其次为1000秒蓝光。这些结果与我们以前发表的研究相一致, 证明 5-孵化30分钟后蓝色光激活导致成纤维细胞在细胞凋亡12的百分比增加, 14。
该实验方法对 PDT 的研究具有优势和局限性。所描述的方法符合临床实践作为一个商业上可用的 PS 和光源使用。研究人员可以自定义不同细胞类型、PSs、光源和辐照参数的方法。然而, 这种方法有局限性, 因为这个协议是为在单层培养的黏附细胞设计的。在临床实践中, 组织结构或疾病病理学 (即底角化过度或纤维化) 的差异可能会降低 PS 吸收或光穿透15。因此, 治疗剂量和实验结果可能与临床实践没有直接的对应。以球形肿瘤模型和微流控芯片为方法, 对肿瘤微环境16、17、18进行了较严密的复制。球体有内部和外部细胞的利基, 可能差异纳入光敏和代表异构的肿瘤结构。其他研究人员使用微流控芯片筛选治疗条件和光敏的血管输送。然而, 对于不熟悉该技术的研究人员来说, 微流控芯片的开发或难于实现的成本可能很高。此外, 球体和微流控芯片可能无法反映光敏直接应用于肿瘤表面而无血管传播的皮肤癌的临床治疗。研究人员可能需要评估单层培养、球形肿瘤模型和微流控芯片在不同疾病系统中研究 PDT 的利弊。由于没有免疫细胞或细胞外基质, 因此不可能确定 PDT 如何影响复杂的细胞间相互作用。研究人员可能需要用动物模型和临床试验证实体外实验结果。在体外光动力实验中, 保持一致的孵化和光照参数, 对于提高检测的有效性具有重要意义。
已知温度改变了 PDT19的功效。一项研究表明, 细胞死亡, 5 的摄取, PP IX 的形成, 和细胞因子释放可能会加强孵化细胞与 5-阿拉在温度高达44摄氏度19。44摄氏度以上的潜伏期可能导致热诱导细胞死亡, 孵化温度低于20摄氏度可能不会导致显著的 5-阿拉的摄取和积累19。我们建议研究人员在恒温温度可调的加热块上执行 PS 孵化, 以控制潜在的混杂热效应。此外, 重要的是要孵化的光敏剂足够的时间, 以允许转化为 5-阿拉的 PP ix。在该协议中, SCC-13 细胞以 5-亚拉巴马州为30分钟的孵化, 成功诱导细胞凋亡。我们以前已经证明, 5-阿拉的10分钟孵化并没有显著增加成纤维细胞凋亡与未经处理的控制成纤维细胞11,14。PP-IX 开始积累在小鼠皮肤 5-阿拉孵化六分钟37摄氏度。因此, 经过十分钟的5的孵化, 可能没有足够的积累, PP IX 诱导细胞凋亡20。我们建议的最低潜伏期20至30分钟的 5-阿拉的基础上, 我们以前的研究11,14。研究人员可能需要根据实验室设置、感兴趣的光敏剂、细胞类型和临床指示来优化潜伏期。抗体滴定和优化实验可以执行的最佳结果使用制造商的指导方针。其他的兴趣分析可以执行后, 蓝色光 photoactivation 包括 dihydroethidium 流细胞定量的 ROS。14
在 photoactivation 阶段, 每单位表面积 (即辐照度) 和总辐照剂量 (即通量) 的光含量变化可能会影响 ROS 的形成和细胞凋亡21。通量、辐照度和时间的关系可以用以下等式描述:
剂量 (J/厘米2) = 辐照度 (W/厘米2) x 时间 (s)
由于剂量依赖于时间, 延长或缩短 photoactivation 阶段可能改变治疗效果。辐照度与光源和靶组织的距离平方成正比。因此, 如果光线太远, 可能没有足够的光能传递给目标组织来激发 PS. 或者, 辐照度可以通过反射周围表面的光线来增加。因此, 我们建议执行光辐照与放置在黑色表面的细胞培养板, 以防止光反射。研究人员可能获得商用或 FDA 批准的蓝光发光二极管和荧光器件用于 PDT 实验, 但这些器件可能有不同的功率输出和光场均匀性。每个实验前, 应使用波长特定的光度计测量光场在细胞表面的辐照度和均匀度, 以求得一致的结果。如果有必要, 可使用商用扩散器来提高场的均匀性。
总之, 我们描述了一种体外方法, 通过详述理论、实验方法、局限性、潜在缺陷和优化建议来调查 PDT。pdt 是一个有用的临床程序, 并在体外研究可以为新的 PSs 的发展, 协议的优化, 和新的迹象, pdt。

光动力疗法 (PDT) 是一个医疗程序, 涉及孵化外部应用光敏剂 (PS) 后, 可见光 photoactivation 诱导细胞凋亡。联邦药物管理局 (FDA) 批准了 pdt 治疗日光脂溢性 (AK), 临床指南建议 pdt 作为治疗某些非黑色素瘤皮肤癌和寻常痤疮1,2。新出现的非皮肤病疗法包括胃肠道、前列腺癌和妇科癌症的治疗3。PDT 是一种有利的治疗方式, 因为它是低成本, 无创, 并与最小的不良事件和疤痕2。
在 PDT 的第一步, 应用 PS, 并允许积累 intracellularly2。在美国, 局部 5-氨基乙酰丙酸 (5-阿拉) 通常用于治疗 AKs。在孵化阶段, 5-亚拉巴马州通过在合并细胞中的血红素生物合成途径转化为原卟啉 ix (PP ix)3。癌症和癌前细胞减少了 ferrochelatase 活性, 这将原卟啉 ix (PP ix) 转化为最终产物, 血红素。结果, 与正常细胞4,5相比, 癌细胞积累了 PP IX。这种积累的 PP IX 在癌症和前癌细胞相对于周围组织允许 PDT 是一个有针对性的方法, 最小的不良事件。当氧接受来自 PP ix 的激发电子时, 光辐照会导致活性氧 (ROS) 的产生。PP-IX 在紫外光谱中具有峰值吸收, 在可见光谱中有较小的峰值, 包括蓝色和红光2。ROS 的形成可能最终导致细胞凋亡, 膜破坏, 线粒体损伤, 免疫调节, 角质细胞增殖, 胶原蛋白周转6,7,8,9,10。
PDT 是在二十世纪七十年代发展起来的, 是一种进化的治疗方式3。FDA 批准的治疗 AKs 建议 5-14–18 h 的皮肤孵化, 但1至 2 h 孵化通常用于临床实践2。在体外, 我们已经表明, 较短的15分钟 5-阿拉孵化可能会增加成纤维细胞凋亡相比, 未经处理的成纤维细胞11,12。此外, 研究了新的氯、酞菁和纳米粒子的 PSs, 以提高 PDT 功效3。在此, 我们提出了一种体外方法来研究 PDT 在黏附鳞状细胞癌细胞。在本协议中, SCC-13 细胞以0、0.5、1.0 和2毫米 5-阿拉的孵化为30分钟和光活化作用, 417 nm 蓝色光为 s。主要结果是凋亡和坏死, 蛋白和 7-aminoactinomycin D (7) 流式细胞术测量。该治疗协议旨在模拟 pdt, 并可调整以研究使用 pdt 与其他细胞系, 光敏, 孵化温度, 或 photoactivation 波长。可能需要准备额外的控制组, 包括无瑕, 单荧光染色, 阴性, 和积极的控制流式细胞术。对凋亡/坏死流式细胞术的理论、规程、实验设计和门控进行了全面的回顾, 可在别处找到13。

<table border="0" cellpadding="0" cellspacing="0" width="1259">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Keratinocyte serum free medium&#160;</td>
			<td style="width:211px;">Thermofisher</td>
			<td style="width:204px;">17005042</td>
			<td style="width:623px;">Culture medium for SSC-13 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM low glucose glutamax</td>
			<td>Thermofisher</td>
			<td>10567022</td>
			<td>Possible culture medium for other cell types</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">6-well culture plates</td>
			<td>Thermofisher</td>
			<td>720083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Thermofisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA</td>
			<td>Thermofisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td>Thermofisher</td>
			<td>15250061</td>
			<td>For cell counting and plating&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BLU-U light&#160;</td>
			<td>DUSA</td>
			<td>Request from manufacturer</td>
			<td>Other blue LED, flourescent, or halogen lights may used&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-ALA</td>
			<td>DUSA</td>
			<td>Request from manufacturer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Atlanta Biologicals</td>
			<td>C17032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowCellect Annexin Red Kit</td>
			<td>Millipore Sigma&#160;</td>
			<td>FCCH100108&#160;</td>
			<td>Propidium iodide may replace 7-AAD. Comes with annexin-V, 7-AAD, and flow buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCanto II</td>
			<td>BD</td>
			<td>Request from manufacturer</td>
			<td>Any flow cytometer may be used&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Counting Chamber</td>
			<td>Hausser</td>
			<td>3120</td>
			<td>For cell counting and plating&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo</td>
			<td>FlowJo</td>
			<td>Request from manufacturer</td>
			<td>Flow cytometry analysis software</td>
		</tr>
	</tbody>
</table>

an in vitro approach to photodynamic therapy

光动力疗法 (PDT) 是一个医疗程序, 涉及孵化外部应用光敏剂 (PS) 后, 可见光 photoactivation 诱导细胞凋亡。联邦药物管理局已批准 pdt 治疗日光脂溢性, 临床指南建议 pdt 作为治疗某些非黑色素瘤皮肤癌和寻常痤疮。PDT 是一种有利的治疗方式, 因为它是低成本, 无侵入性, 并与最小的不良事件和惊吓有关。在 PDT 的第一步, 应用 PS, 并允许积累 intracellularly。随后的光照射诱导活性氧的形成, 这可能最终导致细胞凋亡, 膜的破坏, 线粒体损伤, 免疫调节, 角质细胞增殖, 胶原蛋白的更替。在此, 我们提出了一种体外方法来研究 PDT 在黏附细胞系。该治疗协议旨在模拟 pdt, 并可调整以研究使用的 pdt 与各种细胞系, 光敏, 孵化温度, 或 photoactivation 波长。鳞状细胞癌细胞以0、0.5、1.0 和2毫米 5-氨基乙酰丙酸 (5-亚拉巴马州) 孵化, 30 分钟和光活化作用417纳米蓝光。以蛋白和 7-aminoactinomycin D 流式细胞术检测为主要结局指标为凋亡和坏死。在 5-阿拉三十分钟的孵化后, 细胞凋亡的剂量依赖性增加。在体外光动力实验中, 保持一致的孵化和光照参数, 对于提高检测的有效性具有重要意义。pdt 是一个有用的临床程序, 并在体外研究可以为新的 PSs 的发展, 协议的优化, 和新的迹象, pdt。

在 SCC-13 细胞孵化30分钟与 0, 0.5, 1, 2 毫米 5-阿拉和辐照 1000 s 蓝光, 有剂量依赖性增加的细胞凋亡。总凋亡 (平均标准误差平均值) 为3.94 秒 0.34, 7.90 @ 0.52, 23.86 @ 0.52, 和 38.33, 1.81 分钟后, 30, 0, 0.5, 1 毫米的孵化, 分别和2秒钟的蓝色光 (图 5)。我们用方差分析的方法比较了0、0.5、1和2毫米 5-阿拉的凋亡细胞中的平均百分率。1和2毫米 5-亚拉巴马州的细胞凋亡与0毫米 5-阿拉巴马治疗细胞相比显著增加。图 2A显示了具有代表性的流细胞浇口 (FSC) 与侧向散射 (SSC) 的 SCC-13 细胞孵化与 0, 0.5, 1, 2 毫米 5-阿拉巴马州。圆形浇口包围 SCC-13 细胞群。在这个实验中, 收集了7500个事件, SCC-13 细胞种群门捕获了至少90% 事件。图 2B显示了 y 轴上 x 轴和蛋白上的 7-反的代表图。细胞群被封闭成四象限 (Q1 到 Q4), 以区分凋亡细胞。Q1 (高蛋白, 低 7) 代表细胞凋亡早期。Q2 (高蛋白 v, 高 7) 代表细胞在晚期凋亡或坏死。Q3 (低蛋白, 高 7) 代表细胞正在坏死。Q4 (低蛋白, 低 7) 代表细胞不发生凋亡或坏死。为了计算细胞凋亡的百分比, 我们增加了 Q1 和 Q2 细胞的百分比。5-photoactivation 潜伏期长度、潜伏期温度或辐照剂量的不一致可能改变 PDT 疗效。图 3展示了一个代表性的蛋白和 7-细胞的流动, 当孵化温度变化 (即27, 36, 或42°c)。细胞凋亡的温度依赖性增加。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58190/58190fig1.jpg"> 图 1: 三十分钟孵化 5-亚拉巴马州后细胞凋亡的剂量依赖性增加.5-阿拉在36摄氏度孵化三十分钟后, SCC-13 细胞中1000秒的蓝光。条形表示每个治疗和控制组中蛋白阳性细胞的平均百分比。实验以三项技术进行。统计学意义由方差分析确定, p < 0.05 用星号 (*) 表示。误差线表示平均值的平均标准误差。<a href="https://www.jove.com/files/ftp_upload/58190/58190fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58190/58190fig2.jpg"> 图 2: 代表性流式细胞仪地块.(A) 代表在0、0.5、1和2毫米 5-阿拉孵化细胞中的任意单位 (AU) 中有代表性的向前散射与侧散布式细胞仪地块。在这个实验中, 收集了7500个事件, SCC-13 细胞种群门捕获了90% 事件。(B) 代表蛋白与 7 -反流式细胞术在 AU 为 x 和 y 轴 0, 0.5, 1 和2毫米的孵化细胞。Q1: 早期凋亡细胞, Q2: 晚期凋亡/坏死细胞, Q3: 坏死细胞和 Q4: 存活细胞。<a href="https://www.jove.com/files/ftp_upload/58190/58190fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58190/58190fig3.jpg"> 图 3: 在不同温度下孵化 5-阿拉可改变 PDT 功效.代表蛋白与 7 -反流式细胞仪在 AU 为 x 和 y 轴的 SCC-13 细胞孵化与0.5 毫米 5-亚拉巴马州在 27, 36 和42°c。Q1: 早期凋亡细胞, Q2: 晚期凋亡/坏死细胞, Q3: 坏死细胞和 Q4: 存活细胞。<a href="https://www.jove.com/files/ftp_upload/58190/58190fig3large.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 Approach to Photodynamic Therapy at JoVE.com

An In Vitro Approach to Photodynamic Therapy

光动力疗法 (PDT) 是一个医疗程序, 涉及孵化外部应用光敏剂 (PS) 后, 可见光 photoactivation 诱导细胞凋亡。我们提出了一种体外pdt 协议, 旨在模拟 PDT, 可用于研究不同的 PS 孵化和光处理参数。

一种体外光动力治疗方法的研究

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

an-in-vitro-approach-to-photodynamic-therapy

Research

JoVE Journal

Medicine

An In Vitro Approach to Photodynamic Therapy

8.6K 次观看.  University of California, Davis. 光动力疗法 (PDT) 是一个医疗程序, 涉及孵化外部应用光敏剂 (PS) 后, 可见光 photoactivation 诱导细胞凋亡。我们提出了一种体外pdt 协议, 旨在模拟 PDT, 可用于研究不同的 PS 孵化和光处理参数。

视频: An In Vitro Approach to Photodynamic Therapy

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

科研

医学

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

光动力疗法治疗骨肉瘤细胞的细胞毒功效<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

A Multi-Modal Approach to Assessing Recovery in Youth Athletes Following Concussion

Concussion is one of the most commonly reported injuries amongst children and youth involved in sport participation. Following a concussion, youth can experience a range of short and long term neurobehavioral symptoms (somatic, cognitive and emotional/behavioral) that can have a significant impact on one&#8217;s participation in daily activities and pursuits of interest (e.g., school, sports, work, family/social life, etc.). Despite this, there remains a paucity in clinically driven research aimed specifically at exploring concussion within the youth sport population, and more specifically, multi-modal approaches to measuring recovery. This article provides an overview of a novel and multi-modal approach to measuring recovery amongst youth athletes following concussion. The presented approach involves the use of both pre-injury/baseline testing and post-injury/follow-up testing to assess performance across a wide variety of domains (post-concussion symptoms, cognition, balance, strength, agility/motor skills and resting state heart rate variability). The goal of this research is to gain a more objective and accurate understanding of recovery following concussion in youth athletes (ages 10-18 years). Findings from this research can help to inform the development and use of improved approaches to concussion management and rehabilitation specific to the youth sport community.

This article provides an overview of a multi-modal approach to assessing recovery following concussion in youth athletes. The described protocol uses pre- and post-concussion assessment of performance across a wide variety of domains and can inform the development of improved concussion rehabilitation protocols specific to the youth sport community.

多模式的方法来评估恢​​复的青少年运动员脑震荡之后

Concussion is one of the most commonly reported injuries amongst children and youth involved in sport participation. Following a concussion, youth can ...

A Neuroscientific Approach to the Examination of Concussions in Student-Athletes

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Concussion as defined by the 2013 Concussion Consensus Statement &#8220;may be caused either by a direct blow to the head, face, neck or elsewhere on the body with an &#8216;impulsive&#8217; force transmitted to the head.&#8221; Concussions leave the individual with both short- and long-term effects. The short-term effects of sport related concussions may include changes in playing ability, confusion, memory disturbance, the loss of consciousness, slowing of reaction time, loss of coordination, headaches, dizziness, vomiting, changes in sleep patterns and mood changes. These symptoms typically resolve in a matter of days. However, while some individuals recover from a single concussion rather quickly, many experience lingering effects that can last for weeks or months. The factors related to concussion susceptibility and the subsequent recovery times are not well known or understood at this time. Several factors have been suggested and they include the individual&#8217;s concussion history, the severity of the initial injury, history of migraines, history of learning disabilities, history of psychiatric comorbidities, and possibly, genetic factors. Many studies have individually investigated certain factors both the short-term and long-term effects of concussions, recovery time course, susceptibility and recovery. What has not been clearly established is an effective multifaceted approach to concussion evaluation that would yield valuable information related to the etiology, functional changes, and recovery. The purpose of this manuscript is to show one such multifaceted approached which examines concussions using computerized neurocognitive testing, event related potentials, somatosensory perceptual responses, balance assessment, gait assessment and genetic testing.

There is great variability in an individual&#8217;s risk for concussion and their corresponding recovery. A multifaceted approach to concussion evaluation is warranted; including baseline testing of athletes before participation in sport and timely evaluation post injury. The goal of this protocol is to provide an appropriate multifaceted approach to examine concussions.

一个神经科学方法脑震荡的学生运动员的考试

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

一种使用加速度计量化上肢表现在日常生活的方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

用内部方法设计上颌重建的 cad/cam 手术指南

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...