我们感谢博士。 R.裘德Samulski和小肖重组AAV3和AAV - EGFP的质粒，分别和盖尔汤林森博士的那种慷慨地提供Hep293TT细胞的礼物。这项研究是支持公共卫生服务补助金补助R01 HL - 076901，R01 HL - 097088，和P01 DK - 058327从美国国立卫生研究院（项目1）（AS）的一部分。

1。重组腺相关病毒血清型3（rAAV3）载体的包装<ol><li>前往网站:" <a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a> "的DNA计算的金额。 </li><li>预混合腺病毒辅助质粒pHelper，pACG2c3（AAV的辅助）和pdsAAV - CB - EGFP在中期（rAAV载体）没有FBS和抗生素。总成交量应该是8750μL十个15厘米2板。 </li><li>添加到DNA在步骤1.2中的解决方案直接裴1250μL（0.1％M / V，Polysciences，CAT＃23966，pH值4.5）。旋涡几秒钟的DNA - PEI的解决方案。 </li><li>孵育DNA - PEI在室温（RT）5分钟，形成一个复杂的解决方案。 </li><li>加入1毫升的DNA - PEI解决方案，每15厘米2板。 </li><li> 4小时后取出的媒介，与25毫升新鲜的完全培养基代替10％胎牛血清（C型DMEM）。 </li><li> 72个小时，转染后，刮去的细胞，倒入250 mL锥形离心管。 </li><li>在3000转的自旋，在4 ° C为10分钟。倒掉上清液。 </li><li>重悬细胞沉淀在5毫升RB的TMS的缓冲液（50毫米的Tris - HCl，150 mM氯化钠，pH值8.0）。转移到一个新的15 mL锥形管。 </li><li>在干冰乙醇浴10分钟和解冻冻结在37 ° C为10分钟。重复三次。 </li><li>添加1μL4.8米氯化镁2和2 BenzonaseμL（25 U /μL，Novagen公司，卡特彼勒＃70664-3）。涡和孵化在37 ° C为40分钟。 </li><li>在4000转离心，在4 ° 40分钟C和收集上清（澄清裂解液）。 </li><li>在13 mL快速密封的离心管（美国贝克曼，CAT＃342413），负载碘克沙醇从上到下的渐变（OptiPrep，CAT＃1114542）:2毫升，15％，25％的2毫升，1.5毫升40％ 1.5毫升60％的碘克沙醇。碘克沙醇梯度顶部加载的上清（步骤1.12）。填写与Rb TMS的缓冲液管。 </li><li>在75000转速为1小时离心机在16℃使用贝克曼90Ti转子。 </li><li>通过插入一个注射器在40-60％的边界，收集40％的碘克沙醇分数。 </li><li>在一个50 mL锥形管传输40％的碘克沙醇的一小部分，并用缓冲液40毫升（20毫米的Tris，15 mM氯化钠，pH值8.5）。 </li><li>组装的过滤装置，使用HiTrap Q惠普柱（通用电气医疗集团，卡特彼勒＃17-1154-01）。以下缓冲区清洗列:25缓冲液（20毫米的Tris，15 mM氯化钠，pH值8.5）; 25毫升缓冲液B（10毫米的Tris，500 mM氯化钠，pH值8.5）; 50 mL缓冲液A中; 40毫升病毒样本的一个缓冲区中，缓冲区50毫升（20毫米三，15毫米氯化钠，pH 8.5）; 20毫升缓冲液C（20毫米，175毫米氯化钠，pH值8.5三）; </li><li>收集彗星在20毫升阿波罗集中（轨道Biosciences公司）的缓冲区。 4，在3000转离心° C为10分钟。 </li><li>丢弃的流量通过，并添加20毫升的冰鲜PBS离心3000转，在4 ° 10分钟的彗星。 </li><li>丢弃的流量通过，并添加500μL冰鲜PBS。洗净载体膜转移到硅处理的Eppendorf管中，并存储在小分装冷冻于-80 ° C。 </li></ol> 2。 rAAV3矢量滴度测定<ol><li>解冻10μL纯化的Eppendorf管中冰病毒。加入40μL2 O DDH </li><li>新增的Benzonase 0.2μL（25 U /μL，Novagen公司，CAT＃70664）和37 °为1小时。 </li><li>取50μL质粒在另一Eppendorf管（0.2纳克/μL）的标准。 </li><li>添加在每个管，并在65 ° C孵育30分钟的100毫米NaOH 50μL。 </li><li>冰寒立即在冰上至少5分钟。 </li><li>切割转印膜（Millipore公司，卡特彼勒＃INYC00010）根据生物点滤纸（BIO - RAD，CAT＃1620161）。在10X SSC冲洗20分钟三滤纸膜。 </li><li>设置狭缝杂交SF器械（生物点，CAT＃170-6542）。连接到一个保温瓶，并添加100μL10倍SSC狭缝杂交仪。 </li><li>每个步骤2.5样品中加入100μL20X SSC和1μL6X样染料。 </li><li>膜在狭缝杂交装置装到每个样品100μL。 </li><li>添加到每个样品100μL，10倍SSC的另一个。 </li><li>重复步骤2.9和2.10，直到所有的样品通过膜。 </li><li>拆解狭缝杂交仪器和删除膜。放入督察连接器SL - 1000型紫外交联剂。打开"能源"，"最佳交联"（显示1200），推动"开始"。 </li><li>煮沸1毫升（SS）的鲑鱼精子DNA（费舍尔，CAT＃NC9753983）为5分钟。冰浴至少5分钟。 </li><li>准备预杂交液混合DDH 2 O 15毫升27.5毫升20X SSC，50X登哈特的解决方案的5毫升1.25毫升的20％SDS，波利亚和0.25毫升，1毫升的SS DNA步骤2.13。 </li><li>暂停在预杂交液25毫升在上限的玻璃圆筒，在65 ° C过夜杂交室孵育膜。 </li><li>稀释10μLDDH 2 O的50纳克的DNA模板。熬在100 ° C下5分钟，冷却至少5分钟，立即在冰上。 </li><li>在1分钟在室温下3000 rpm的离心机。新增10毫米3 dNTPs 2μL的（缺乏dCTP），2μLHexanucleotide混合（罗氏，CAT＃11277081001），1μL的Klenow酶（罗氏，CAT＃11008404001）和5μLα-32 P - dCTP。在37 ° C下10分钟。 </li><li>离心2分钟3000转的G - 50柱（通用电气医疗集团，卡特彼勒＃27-5330-01）。步骤2.17负载探头入列。在2分钟3000转离心收集流过。 </li><li>计数放射性。在100℃，5分钟，立即冰浴至少5分钟，煮探头。 </li><li>添加到预杂交液与膜探针（6x10 5 CPM /毫升）。 </li><li>在65 ° C孵育过夜杂交室。 </li><li>倒掉杂交液和冲洗2X SSC +0.1％SDS的膜在室温为15分钟。 </li><li>丢弃的解决方案，并在65℃30分钟冲洗膜在0.1倍SSC 0.1％的SDS。 </li><li>弃掉的解决方案，在0.1倍SSC冲洗膜在室温下简要。 </li><li> 6小时暴露于-80 ° C的电影和发展电影。一个典型的结果是如图1所示。 </li></ol> 3。 rAAV3载体介导的转导人肝癌细胞的转基因表达<ol><li>种子1 × 10 4细胞（或者Huh7肝癌或Hep293TT）在每孔96孔板。在加湿的CO 2培养箱孵育至少18小时。 </li><li>从96孔板介质。 </li><li>洗涤细胞两次中等50μL没有FBS和抗生素，并删除。 </li><li>添加50μL介质没有在5000 VGS /细胞的FBS和抗生素和rAAV载体。 </li><li>板块中的CO 2培养箱孵育4小时。 </li><li>丢弃的介质。 50μL完全培养基洗涤细胞两次删除。 </li><li>每孔加入150μL的C - DMEM孵育板中的CO 2培养箱中培养72小时，并可视EGFP的表达，用荧光显微镜（DMI 4000B;莱卡微系统） 。 </li><li> ImageJ分析软件（美国国立卫生研究院，美国马里兰州贝塞斯达，的），从三个井病毒感染的细胞图像定量分析。 </li><li>转基因表达被评定为总面积的绿色荧光（像素2）每视野。一个典型的结果如图2所示。 </li><li>流式细胞仪检测转基因表达，也可以。 </li></ol> 4。代表性的成果: 上文所述的协议，可以有效地生成腺相关病毒血清型载体。典型的产量是500μL，含10月11日 〜VGS /μL，纯化载体的股票。矢量股票的纯度是由比较DNA定量插槽印迹杂交信号，并没有Benzonase消化。有效地净化rAAV3载体转导人类肝癌 - 肝母细胞瘤（HB）和肝细胞癌（HCC） - 细胞株。 <img src="/files/ftp_upload/2538/2538fig1.jpg" alt="图1" /> 图1。DNA定量吸入印迹分析确定rAAV3载体滴度。纯化病毒库存，消化与Benzonase（上排）的2倍稀释， 用 32 P标记EGFP特定DNA探针分析DNA定量槽杂交。 AAV3载体滴度由1纳克（中间行）或10毫微克（下排）装上膜的AAV - EGFP的质粒标准比较。这些数字对应的DNA拷贝。 <img src="/files/ftp_upload/2538/2538fig2.jpg" alt="图2" /> 图2重组腺相关病毒载体介导人肝癌细胞的转基因表达。 Hep293TT细胞，最近成立的人肝细胞线4条，无论是模拟感染（左图），或转5000 VGS /要么scAAV2 - EGFP或scAAV3 - EGFP载体的细胞在37 ° C， 2小时。转基因表达的转导后72小时可视化使用荧光显微镜。

<ol>
	<li>Zincarelli, C., Soltys, S., Rengo, G., Rabinowitz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+AAV+serotypes+1-9+mediated+gene+expression+and+tropism+in+mice+after+systemic+injection.">Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.</a> Mol Ther. 16, 1073-1080 (2008).</li><li>Glushakova, L. G., Lisankie, M. J., Eruslanov, E. B., Ojano-Dirain, C., Zolotukhin, I., Liu, C., Srivastava, A., Stacpoole, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV3-mediated+transfer+and+expression+of+the+pyruvate+dehydrogenase+E1+alpha+subunit+gene+causes+metabolic+remodeling+and+apoptosis+of+human+liver+cancer+cells.">AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells.</a> Mol Genet &#38; Metabol. 98, 289-299 (2009).</li><li>Ling, C., Lu, Y., Kalsi, J. K., Jayandharan, G. R., Li, B., Ma, W., Cheng, B., Gee, S. W., McGoogan, K. E., Govindasamy, L., Zhong, L., Agbandje-McKenna, M., Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatocyte+growth+factor+receptor+is+a+cellular+coreceptor+for+adeno-associated+virus+serotype+3.">Human hepatocyte growth factor receptor is a cellular coreceptor for adeno-associated virus serotype 3.</a> Hum Gene Ther. 21, 1741-1747 (2010).</li><li>Chen, T. T., Rakheja, D., Hung, J. Y., Hornsby, P. J., Tabaczewski, P., Malogolowkin, M., Feusner, J., Miskevich, F., Schultz, R., Tomlinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+cancer+cell+line+derived+from+an+aggressive+childhood+liver+tumor.">Establishment and characterization of a cancer cell line derived from an aggressive childhood liver tumor.</a> Pediatr Blood &#38; Cancer. 53, 1040-1047 (2009).</li><li>Daya, S., Berns, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+using+adeno-associated+virus+vectors.">Gene therapy using adeno-associated virus vectors.</a> Clin Microbiol Rev. 21, 583-593 (2008).</li><li>Zhong, L., Li, B., Mah, C. S., Govindasamy, L., Agbandje-McKenna, M., Cooper, M., Herzog, R. W., Zolotukhin, I., Warrington, K. H., Weigel-Van Aken, K. A., Hobbs, J. A., Zolotukhin, S., Muzyczka, N., Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+of+adeno-associated+virus+2+vectors:+point+mutations+in+tyrosines+lead+to+high-efficiency+transduction+at+lower+doses.">Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses.</a> Proc Natl Acad Sci USA. 105, 7827-7832 (2008).</li><li>Petrs-Silva, H., Dinculescu, A., Li, Q., Min, S. H., Chiodo, V., Pang, J. J., Zhong, L., Zolotukhin, S., Srivastava, A., Lewin, A. S., Hauswirth, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transduction+of+the+mouse+retina+by+tyrosine-mutant+AAV+serotype+vectors.">High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors.</a> Mol Ther. 17, 463-471 (2009).</li><li>Jayandharan, G. R., Zhong, L., Sack, B. K., Rivers, A. E., Li, M., Li, B., Herzog, R. W., Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+adeno-associated+virus+(AAV)-protein+phosphatase-5+helper+viruses+for+efficient+liver+transduction+by+single-stranded+AAV+vectors:+therapeutic+expression+of+factor+IX+at+reduced+vector+doses.">Optimized adeno-associated virus (AAV)-protein phosphatase-5 helper viruses for efficient liver transduction by single-stranded AAV vectors: therapeutic expression of factor IX at reduced vector doses.</a> Hum. Gene Ther. 21, 271-283 (2010).</li><li>Li, M., Jayandharan, G. R., Li, B., Ling, C., Ma, W., Srivastava, A., Zhong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transduction+of+fibroblasts+and+mesenchymal+stem+cells+by+tyrosine-mutant+AAV2+vectors+for+their+potential+use+in+cellular+therapy.">High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.</a> Hum Gene Ther. 21, 1527-1543 (2010).</li><li>Kauss, M. A., Smith, L. J., Zhong, L., Srivastava, A., Wong, K. K. J. r., Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+long-term+transduction+and+multilineage+engraftment+of+human+hematopoietic+stem+cells+transduced+with+tyrosine-modified+recombinant+adeno-associated+virus+serotype+2.">Enhanced long-term transduction and multilineage engraftment of human hematopoietic stem cells transduced with tyrosine-modified recombinant adeno-associated virus serotype 2.</a> Hum Gene Ther. 21, 1129-1136 (2010).</li><li>Qiao, C., Zhang, W., Yuan, Z., Shin, J. H., Li, J., Jayandharan, G. R., Zhong, L., Srivastava, A., Xiao, X., Duan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotype+6+capsid+tyrosine-to-phenylalanine+mutations+improve+gene+transfer+to+skeletal+muscle.">Adeno-associated virus serotype 6 capsid tyrosine-to-phenylalanine mutations improve gene transfer to skeletal muscle.</a> Hum Gene Ther. 21, 1343-1348 (2010).</li><li>Markusic, D. M., Herzog, R. W., Aslanidi, G. V., Hoffman, B. E., Li, B., Li, M., Jayandharan, G. R., Ling, C., Zolotukhin, I., Ma, W., Zolotukhin, S., Srivastava, A., Zhong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transduction+and+correction+of+murine+hemophilia+B+using+AAV2+vectors+devoid+of+multiple+surface-exposed+tyrosines.">High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.</a> Mol Ther. 18, 2048-2056 (2010).</li><li>Ojano-Dirain, C., Glushakova, L. G., Zhong, L., Zolotukhin, S., Muzyczka, N., Srivastava, A., Stacpoole, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+PDH+deficiency+using+AAV8-siRNA+vector-mediated+knockdown+of+pyruvate+dehydrogenase+E1&#945;.">An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1&#945;.</a> Mol Genet &#38; Metabol. 101, 183-191 (2010).</li><li>Petrs-Silva, H., Dinculescu, A., Li, Q., Deng, W. T., Pang, J. J., Min, S. H., Chiodo, V., Neeley, A. W., Govindasamy, L., Bennett, A., Agbandje-McKenna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+properties+of+tyrosine-mutant+AAV2+vectors+in+the+mouse+retina.">Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina.</a> Mol Ther. , (2011).</li></ol>

非致病性人类细小病毒，腺相关病毒（AAV）为基础的重组载体已发展起来的，目前在基因治疗临床试验5使用。此前，10个最常用的腺相关病毒血清型，一般的AAV3载体的转导效率已报道在体外 和体内1，要特别低。然而，我们最近的观察，AAV3载体转导人肝癌细胞系得非常好，决定由流式细胞仪 2定量，AAV3利用人肝细胞生长因子受体（hHGFR的细胞作为一个具有约束力，并在这些条目的共同受体）细胞3，强烈建议，特别是有选择性的AAV3组织，嗜在一般人的肝细胞，人肝癌细胞。然而，由于AAV3载体，也有效地转导人体正常肝细胞2， 其在体内的癌基因治疗中的应用潜力将受到负面影响。一个可能的策略来规避这种潜在的问题涉及转录针对癌细胞通过确定一个基因的产物，是有选择性的肿瘤产生，但不正常的肝细胞。例如，以往的研究表明，血清α-甲胎蛋白（AFP）作为一个跟踪的存在，进展和/或某些类型的肝癌再次发生的具体标记，因为正常的肝细胞产生极少量的一般这种蛋白质。事实上，我们已经使用了AAV3含AFP启动，目标在肝癌细胞中的转基因表达的载体，但不是在正常的肝细胞，研究目前正在进行中，测试了肝癌的小鼠移植瘤模型这种方法的疗效。在我们进一步的研究中，我们所观察到的表面暴露在6-14病毒衣壳蛋白的酪氨酸残基的定点突变，不同的腺相关病毒血清型载体转染效率可显着增强。自6 7表面暴露的酪氨酸也AAV3保守，我们这些残余物的定点诱变和酪氨酸突变AAV3载体的转导效率明显增强人肝癌细胞（未发表数据）。研究目前正在进行评价其安全性和酪氨酸突变AAV3载体在小鼠移植瘤模型，为人类肝母细胞瘤和肝细胞癌的疗效，如果成功的话，最佳的酪氨酸突变AAV3血清型载体可能被证明是针对人体肝脏为潜在的基因治疗的癌症。

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<td>DMEM</td>
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<td>PEI</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>23966</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70664-3</td>
<td>&nbsp;</td>
<tr>
<td>HiTrap Q HP column</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>17-1154-01</td>
<td>&nbsp;</td>
<tr>
<td>Salmon sperm DNA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9753983</td>
<td>&nbsp;</td>
<tr>
<td>Iodixanol gradient</td>
<td>&nbsp;</td>
<td>OptiPrep</td>
<td>1114542</td>
<td>&nbsp;</td>
<tr>
<td>G-50 Columns</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>27-5330-01</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors

重组载体的非致病性的人类细小病毒，腺相关病毒（AAV2）已经开发，目前正在使用的基因治疗临床试验。最近，一些额外的腺相关病毒血清型也被孤立，这已显示出各种小型和大型的动物模型 1的选择性组织向性。 10个最常用的腺相关病毒血清型，AAV3是目前效率最低的，在体外以及体内转导的细胞和组织。 然而，我们最近发表的研究报告，我们已经证明AAV3载体转导人肝癌 - 肝母细胞瘤（HB）和肝细胞癌（HCC） - 细胞株非常有效，因为AAV3利用人肝细胞生长因子受体细胞作为有约束力的共同受体并在这些细胞中的条目 2,3 。 在这篇文章中，我们描述，以实现高效率的重组AAV3携带报告基因的载体转导人肝癌细胞所需的步骤。重组AAV3携带治疗基因的载体的使用，最终可能导致潜在的人类基因治疗肝癌。

Watch this Scientific Journal Video about High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors at JoVE.com

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

在这篇文章中，我们描述了鉴定的腺相关病毒血清型3（AAV3）针对人肝癌细胞的最有效的载体。

高效率转导肝癌细胞的重组腺相关病毒血清型3载体

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

high-efficiency-transduction-liver-cancer-cells-recombinant-adeno

Universidad San Sebastian

Research

JoVE Journal

Medicine

13.2K 次观看.  University of Florida. 在这篇文章中，我们描述了鉴定的腺相关病毒血清型3（AAV3）针对人肝癌细胞的最有效的载体。

视频: High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

一个实时的基于电阻抗技术测量癌细胞入侵内皮细胞单层

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

科研

医学

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

三维胶原基质支架植入作为肝脏再生策略

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...