Name: cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro
Uploaded: 2014-03-18T14:30:00
Description: Watch this Scientific Journal Video about Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro at JoVE.com

作者要感谢biolitec研究有限公司（德国耶拿）对好心为我们提供了脂质体mTHPC配方，并特别感谢苏珊娜格拉夫和阿诺Wiehe工厂对他们的帮助和技术专长。我们还要感谢凯尔斯丁里迪，谁产生的结果有很大一部分，是coresponsible其出版11。 这项工作是由来自Schweizerischer社团组织Balgrist，苏黎世大学，苏黎世Krebsliga以及从沃尔特湖和约翰娜·沃尔夫基金会，瑞士苏黎世的赠款，并从EuroNanoMed ERA-NET / SNF瑞士拨款补助支持美国国家科学基金会31NM30-一分之十三万一千零四。这项工作也是由HSM的坎顿苏黎世的肌肉骨骼肿瘤（高度专业化医学）计划的支持。

1。 mTHPC在相应的低的吸收和高转移居屋和143B的OS细胞系的比较<ol><li>制备含有DMEM，含F12，并且在一个比4.5:4.5:1热灭活的胎牛血清的细胞培养基。 注意：在PS mTHPC的脂质体制剂溶解在水中以1.5毫克/毫升mTHPC的最终浓度。 </li><li>板0.2×10 6个居屋和143B细胞/孔在6孔板中（一式三份对各条件），并让细胞贴壁过夜。 注意：该数量必须调节到个体的细胞系。 8...

<ol>
	<li>Juarranz, A., Jaen, P., Sanz-Rodriguez, F., Cuevas, J., Gonzalez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+cancer.+Basic+principles+and+applications.">Photodynamic therapy of cancer. Basic principles and applications.</a> Clin. Transl. Oncol. 10, 148-154 (2008).</li><li>Agostinis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+cancer:+an+update.">Photodynamic therapy of cancer: an update.</a> CA Cancer. 61, 250-281 (2011).</li><li>D'Adamo, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appraising+the+current+role+of+chemotherapy+for+the+treatment+of+sarcoma.">Appraising the current role of chemotherapy for the treatment of sarcoma.</a> Semin. Oncol. 38 Suppl 3, 19-29 (2011).</li><li>Allison, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+meta-analysis+of+osteosarcoma+outcomes+in+the+modern+medical+era.">A meta-analysis of osteosarcoma outcomes in the modern medical era.</a> Sarcoma. , (2012).</li><li>Anninga, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+adjuvant+treatment+for+osteosarcoma:+where+do+we+stand.">Chemotherapeutic adjuvant treatment for osteosarcoma: where do we stand.</a> Eur. J. 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Cancer. 37, 32-38 (2001).</li><li>Briccoli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resection+of+recurrent+pulmonary+metastases+in+patients+with+osteosarcoma.">Resection of recurrent pulmonary metastases in patients with osteosarcoma.</a> Cancer. 104, 1721-1725 (2005).</li><li>Milla Sanabria, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+indirect+photodynamic+therapy+effects+on+the+cellular+and+molecular+components+of+the+tumor+microenvironment.">Direct and indirect photodynamic therapy effects on the cellular and molecular components of the tumor microenvironment.</a> Biochim. Biophys. Acta. 1835, 36-45 (2013).</li><li>Mitra, S., Foster, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photophysical+parameters,+photosensitizer+retention+and+tissue+optical+properties+completely+account+for+the+higher+photodynamic+efficacy+of+meso-tetra-hydroxyphenyl-chlorin+vs+Photofrin.">Photophysical parameters, photosensitizer retention and tissue optical properties completely account for the higher photodynamic efficacy of meso-tetra-hydroxyphenyl-chlorin vs Photofrin.</a> Photochem. Photobiol. 81, 849-859 (2005).</li><li>Ma, L., Moan, J., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+new+photosensitizer,+meso-tetra-hydroxyphenyl-chlorin,+for+use+in+photodynamic+therapy:+a+comparison+of+its+photobiological+properties+with+those+of+two+other+photosensitizers.">Evaluation of a new photosensitizer, meso-tetra-hydroxyphenyl-chlorin, for use in photodynamic therapy: a comparison of its photobiological properties with those of two other photosensitizers.</a> Int. J. Cancer. 57, 883-888 (1994).</li><li>Reidy, K., Campanile, C., Muff, R., Born, W., Fuchs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTHPC-mediated+photodynamic+therapy+is+effective+in+the+metastatic+human+143B+osteosarcoma+cells.">mTHPC-mediated photodynamic therapy is effective in the metastatic human 143B osteosarcoma cells.</a> Photochem. Photobiol. 88, 721-727 (2012).</li><li>Triesscheijn, M., Ruevekamp, M., Aalders, M., Baas, P., Stewart, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+sensitivity+of+microvascular+endothelial+cells,+fibroblasts+and+tumor+cells+after+in+vitro+photodynamic+therapy+with+meso-tetra-hydroxyphenyl-chlorin.">Comparative sensitivity of microvascular endothelial cells, fibroblasts and tumor cells after in vitro photodynamic therapy with meso-tetra-hydroxyphenyl-chlorin.</a> Photochem. Photobiol. 80, 236-241 (2004).</li><li>Triesscheijn, M., Baas, P., Schellens, J. H., Stewart, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+in+oncology.">Photodynamic therapy in oncology.</a> Oncologist. 11, 1034-1044 (2006).</li><li>Burch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+canine+osseous+tumors+with+photodynamic+therapy:+a+pilot+study.">Treatment of canine osseous tumors with photodynamic therapy: a pilot study.</a> Clin. Orthop. Relat. Res. 467, 1028-1034 (2009).</li></ol>

为了实现在响应PDT最佳的细胞毒性，关键是要选择合适的激光设置和孵育时间。这里所描述的过程是一致的，高效的，确定的PS吸收和量化PDT诱导的细胞毒性体外 。使用PS mTHPC的特定吸收波长，蜂窝PS摄取可以以直接的方式来确定，并且PS可以被激活，以产生细胞毒性的活性氧物种。 利用这种体外设置中，所有的参数，如聚苯乙烯浓度，药物的光的时间间隔和激?...

骨肉瘤（OS）的艺术处理今天的状态，原发性骨肿瘤，包括为新辅助化疗和手术的组合。这个治疗方案显示增加的患者从使用化疗前约20％的局部疾病的存活率，到目前的60-70％3,4。然而，在过去的二十年中，操作系统患者局部病变的总生存期已趋于稳定4，5。此外，这些患者中30％-40％在确诊后3年内复发和转移性疾病继续有20-30％4一个贫穷的生存，6，7。为了改善这些患者的预后，新的治疗策略需要发展。 光动力疗法（PDT），一个相当新颖的抗癌疗法，采用特定波长光的一个photosensi的激励光敏剂（PS），其中其注射到血液中后积聚在肿瘤细胞。在PS的激光激发产生的氧自由基在氧的存在下，其诱导肿瘤细胞和细胞死亡的细胞毒反应。除了这个主要机制，另外两个PDT诱发的生物学过程有助于减少肿瘤的生长：PDT引起血管收缩和血栓形成的肿瘤微血管，因而局部缺氧及缺氧的肿瘤内，导致肿瘤梗死。最后，PDT受伤和死亡的肿瘤细胞引发局部免疫反应，一个很独特的PDT的功能。这涉及补体系统和抗原的活化呈递树突状细胞8。因此，条件是肿瘤抗原与淋巴样细胞，随后激活的演示文稿中创建，导致肿瘤特异性免疫。 到目前为止，PDT已被用于治疗多种类型的软组织肿瘤和hyperpl亚洲，如光化性角化病，Barrett食管，支气管内肿瘤，膀胱癌，基底细胞癌，和姑息治疗头颈部癌2。治疗是已知的诱导局部，大规模坏死，只有很少的副作用，因此具有选择性地根除肿瘤组织的潜力。尽管有这些优势，PDT的应用程序保持技术上比化疗药物的管理更加严格。为了达到最大的效力，在PS的浓度，光照射时间和总的光能量传递需要被优化。这可以在体内实验来完成，但是，由于相对大量的需要被优化的参数的初步确定体外最佳条件下，它是更有效的。 在下面描述的实验中，我们使用PS 5,10,15,20 -四（间-羟基苯基）叶绿素测试PDT的体外功效奥林简称mTHPC（ 图1A）。 mTHPC是在医药品Foscan，这是目前在临床上对姑息治疗头部和颈部癌症的使用的活性物质。它是最有效的PS中的一个，在低浓度下已经大规模诱导的细胞损伤，并且它被证明是优于其它PS在组织穿透9,10的条款。它的光吸收光谱（ 图1B）示出了两个显着的峰，一个在417 nm和第二在652纳米，这是用于积累PS的组织定位和对PDT诱导，分别。 目前，脂质体制剂用于mTHPC正在开发中。在这里，我们描述的程序来量化这种脂质体制剂的摄取，并且在两个人的OS细胞株进行PDT;低转移性居屋和高转移性143B细胞。一些这里介绍的数据已报告前面11。钍这里描述É方法让我们来研究PDT疗效转移表型的影响。 143B细胞原位注射到免疫缺陷的SCID小鼠的后肢引起intratibial转移性原发性肿瘤，模型密切模仿人类转移性疾病。因此，建议在体外实验中是完全合适的，以评估在体内实验中用于以后的最佳PDT设置。

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:251px;">PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-883</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HAM F12</td>
			<td>PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">heat-inactivated fetal calf serum</td>
			<td>GIBCO, Basel, Switzerland</td>
			<td style="width:127px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:291px;">mTHPC</td>
			<td style="width:251px;">biolitec research GmbH, Jena, Germany</td>
			<td style="width:127px;"></td>
			<td style="width:287px;">As this liposomal formulation is originating from R &#38; D 
			no catalogue number is available at the moment. Stock: 1.5mg/ml; provided in a 9:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG; &#62;99% purity)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectramax Gemini XS plate reader</td>
			<td style="width:251px;">Molecular Devices, Sunnyvale, CA</td>
			<td style="width:127px;">ID# 861</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Microscope Zeiss Observer.Z1</td>
			<td style="width:251px;">Axio Observer, Axio Vision Release 4.6.3 SP1, Jena Germany</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ceralas PDT 652 nm Laser</td>
			<td style="width:251px;">Biolitec AG, Jena, Germany</td>
			<td style="width:127px;">LD652nm2W400u</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Water-soluble tetrazolium (WST) reagent</td>
			<td style="width:251px;">Roche Diagnostics AG, Rotkreuz, Switzerland</td>
			<td style="width:127px;">1644807</td>
			<td></td>
		</tr>
	</tbody>
</table>

cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

近年来，出现了在寻找更有效的治疗对癌症具有较少的全身性副作用的困难。因此，光动力疗法是一种新的方法对多种肿瘤有选择性的治疗。 光动力疗法（PDT），使一个无毒的光敏剂（PS），其中，经活化的光在氧的存在下的特定波长的光，产生氧自由基，引发细胞毒性反应1的使用。尽管其批准几乎是二十年前由美国FDA，PDT是时下唯一用于治疗癌症类型（皮肤，膀胱）和nononcological疾病（牛皮癣，光化性角化病）2的数量有限。 利用光动力疗法的主要优点是能够执行一个局部治疗，以防止全身性副作用的能力。此外，它可以让肿瘤在细腻的部位治疗（ 如周围神经或血管）。这里，一个intraoperatPDT的IVE应用被认为是在骨肉瘤（OS），肿瘤的骨，针对原发肿瘤的卫星遗留在肿瘤外科手术切除肿瘤后，周围的组织。治疗的目标是减少复发的数量以及减少为（术后）转移的风险。 在本研究中，我们提出在体外 PDT程序以建立有效的治疗方法，用于重现人类疾病中确立intratibial OS的小鼠模型中广泛使用的操作系统的细胞系的最优PDT设置。在PS mTHPC的吸收进行了检查用分光光度计和光毒性被挑起与mTHPC的激光激发在652 nm到诱导细胞死亡评估用WST-1法和存活细胞计数。所建立的技术使我们能够确定最佳PDT设置在动物模型中未来的研究。他们是一个简单而快速的工具，用于PDT的疗效评价<em&gt;在体外体内应用程序之前。

与此报告的技术，我们研究mTHPC为基础的PDT在人类OS细胞。首先，mTHPC的时间和剂量依赖性摄取研究在低转移性居屋和在高转移性143B OS细胞系。 mTHPC摄取可以通过测量mTHPC的荧光用荧光分光光度计（ 图2中 ，再现从雷迪等人 11的许可）进行评估。 图2A示出mTHPC的时间依赖性摄取。将细胞悬浮液的荧光强度表示mTHPC的胞内水平。 图2B示出了剂量依赖?...

Watch this Scientific Journal Video about Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro at JoVE.com

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

这里所报告的协议允许光动力疗法（PDT）中的细胞系和的PDT设置施加治疗在动物模型中之前，优化的效果进行评价。

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