Name: therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles
Uploaded: 2012-01-04T13:00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles at JoVE.com

这项研究是由美国国家癌症研究所的纳米技术联盟，为癌症的癌症纳米技术卓越中心（CCNE）授予U54 - CA151881的支持。

<ol>
	<li>Vimalachandran, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+prevention+of+pancreatic+cancer.">Genetics and prevention of pancreatic cancer.</a> Cancer. Control. 11, 6-14 (2004).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+evaluation+of+thiol-modified+gelatin+nanoparticles+for+intracellular+DNA+delivery+in+response+to+glutathione.">Preparation and evaluation of thiol-modified gelatin nanoparticles for intracellular DNA delivery in response to glutathione.</a> Bioconjug. Chem. 16, 1423-1432 (2005).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+glycol)-modified+thiolated+gelatin+nanoparticles+for+glutathione-responsive+intracellular+DNA+delivery.">Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery.</a> Nanomedicine. 3, 32-42 (2007).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenic+gene+therapy+with+systemically+administered+sFlt-1+plasmid+DNA+in+engineered+gelatin-based+nanovectors.">Antiangiogenic gene therapy with systemically administered sFlt-1 plasmid DNA in engineered gelatin-based nanovectors.</a> Cancer. Gene. Ther. 14, 488-498 (2007).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+and+pharmacokinetic+analysis+of+long-circulating+thiolated+gelatin+nanoparticles+following+systemic+administration+in+breast+cancer-bearing+mice.">Biodistribution and pharmacokinetic analysis of long-circulating thiolated gelatin nanoparticles following systemic administration in breast cancer-bearing mice.</a> J. Pharm. Sci. 96, 397-407 (2007).</li><li>Kaul, G., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-targeted+gene+delivery+using+poly(ethylene+glycol)-modified+gelatin+nanoparticles:+in+vitro+and+in+vivo+studies.">Tumor-targeted gene delivery using poly(ethylene glycol)-modified gelatin nanoparticles: in vitro and in vivo studies.</a> Pharm. Res. 22, 951-961 (2005).</li><li>Bardeesy, N., DePinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+biology+and+genetics.">Pancreatic cancer biology and genetics.</a> Nat. Rev. Cancer. 2, 897-909 (2002).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+peptide+ligand+of+epidermal+growth+factor+receptor+for+targeted+delivery+of+therapeutics.">Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics.</a> FASEB. J. 19, 1978-1985 (2005).</li><li>McCormick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+gene+therapy:+fringe+or+cutting+edge.">Cancer gene therapy: fringe or cutting edge.</a> Nat. Rev. Cancer. 1, 130-141 (2001).</li><li>Green, D. R., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+functions+of+the+tumour+suppressor+p53.">Cytoplasmic functions of the tumour suppressor p53.</a> Nature. 458, 1127-1130 (2009).</li><li>Barton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+of+the+p53+tumour+suppressor+gene+in+human+pancreatic+cancer.">Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer.</a> Br. J. Cancer. 64, 1076-1082 (1991).</li></ol>

没有利益冲突的声明。

控制和表皮生长因子受体有针对性的硫醇盐明胶纳米粒子的制备与高效的DNA封装和稳定。所有这些系统的粒径在150-250 nm的范围内。 Zeta电位已经证明，这个系统是一个稍微负的系统。纳米粒子的大小随着扫描电镜分析，激光粒度仪的结果相同。 ESCA分析，可以确认PEG和肽表面改性。 Western blot分析表明，PANC - 1细胞有高EGFR表达水平，此细胞株在体外研究中使用。在控制和表面改性的纳米粒子在PANC - 1细胞相对较少的细胞毒性相比，爱德华王子岛。 细胞贩运研究表明针对性的纳米粒子在PANC - 1细胞表皮生长因子受体的质粒快速吸收和释放。报告质粒DNA与表皮生长因子受体针对性的纳米粒子表达的交付导致最高水平的GFP的表达相对其他控制系统，包括脂质体复合物的DNA。与同一个系统，与野生型p53质粒转染引发下游的细胞凋亡途径和诱导PANC - 1细胞快速凋亡。 这些初步结果表明，表皮生长因子受体有针对性的硫醇盐明胶纳米粒子可以作为一个安全，高效的DNA传递系统治疗胰腺癌的基因治疗。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> B型明胶，布卢姆225 </td><td> Sigma - Aldrich公司</td><td> G9391 </td><td></td></tr><tr><td> 2 iminothiolane盐酸</td><td> Sigma - Aldrich公司</td><td> I6256 </td><td></td></tr><tr><td>以pEGFP - N1质粒</td><td>琳生物制药</td><td> N / A </td><td></td></tr><tr><td> PORF - hp53大肠杆菌</td><td> Invivogen </td><td> PORF - hp53 </td><td></td></tr><tr><td>乙二醛溶液（40wt.％H 2 O） </td><td> Sigma - Aldrich公司</td><td> 128465 </td><td></td></tr><tr><td>甘氨酸</td><td> Sigma - Aldrich公司</td><td> 410225 - 250G </td><td></td></tr><tr><td> QIA过滤质粒兆丰套件</td><td> Qiageñ </td><td> 12281 </td><td></td></tr><tr><td>贝克曼乐80K超速离心机</td><td>贝克曼</td><td> N / A </td><td></td></tr><tr><td>自由贸易区6升控制台冷冻干燥系统</td><td> Labconco </td><td> 7753020 </td><td></td></tr><tr><td> MPEG - SCM，MW 2000大</td><td> Laysan </td><td> MPEG - SCM - 2K - 1G </td><td></td></tr><tr><td> MAL - PEG - SCM，分子量2000大</td><td> Jenkem技术</td><td> A5001 - 1 </td><td></td></tr><tr><td>纳米激光粒度仪</td><td>马尔文</td><td>纳米激光粒度仪安芷生</td><td></td></tr><tr><td>日立4800场发射扫描电子显微镜</td><td>日立</td><td> S - 4800 UHR FE - SEM </td><td></td></tr><tr><td> QUANT - IT PicoGreen dsDNA抗体试剂和试剂盒</td><td> Invitrogen公司</td><td> P7589 </td><td></td></tr><tr><td>脂质体转染试剂</td><td> Invitrogen公司</td><td> 18292011 </td><td></td></tr><tr><td> DMEM培养液</td><td> Mediatech cellgro </td><td> 10 013厘米</td><td></td></tr><tr><td>的RPMI </td><td> Mediatech cellgro </td><td> 50 020 PB </td><td></td></tr><tr><td>皮尔斯BCA蛋白检测试剂盒</td><td> Thermo Scientific的</td><td> 23225 </td><td></td></tr><tr><td> iBlot干杂交系统</td><td> Invitrogen公司</td><td> IB1001 </td><td></td></tr><tr><td> XCell SureLock迷你细胞和XCell第二杂交模块套件CE标志</td><td> Invitrogen公司</td><td> EI0002 </td><td></td></tr><tr><td>皮尔斯的ECL西方印迹基板</td><td> Thermo Scientific的</td><td> 32109 </td><td></td></tr><tr><td>柯达数字化X射线标本（DXS）系统</td><td>柯达</td><td> N / A </td><td>; </td></tr><tr><td> CellTiter 96 ueous AQ的一个解决方案细胞增殖实验（MTS） </td><td> Promega公司</td><td> G3580 </td><td></td></tr><tr><td>百特SynergyHT酶标仪</td><td>百特</td><td> N / A </td><td></td></tr><tr><td> Nanodrop 2000 </td><td> Thermo Scientific的</td><td> N / A </td><td></td></tr><tr><td>一步法RT - PCR试剂盒</td><td> Qiagen公司</td><td> 210212 </td><td></td></tr><tr><td>染色质凝聚/膜通透性/死细胞凋亡试剂盒</td><td> Invitrogen公司</td><td> V23201 </td><td></td></tr><tr><td> APO - ONE均质的caspase - 3 / 7检测试剂盒</td><td> Promega公司</td><td> G7790 </td><td></td></tr><tr><td> Hybaid PCR扩增Sprint的热循环仪</td><td> Thermo Scientific的</td><td> N / A </td><td></td></tr><tr><td>表皮生长因子受体抗体</td><td>细胞信号</td><td> 2232 </td><td></td></tr><tr><td> β-肌动蛋白抗体</td><td>细胞信号转导</td><td> 4967 </td><td></td></tr><tr><td>抗兔IgG，HRP联抗体</td><td>细胞信号转导</td><td> 7074 </td><td></td></tr><tr><td>鼠单克隆GFP抗体</td><td> Novus公司生物制品</td><td> NB600 - 597 </td><td></td></tr><tr><td>山羊多克隆GFP抗体（碱性磷酸酶） </td><td> Novus公司生物制品</td><td> NB600 - 1502 </td><td></td></tr><tr><td>磷酸酶底物包</td><td> Thermo Scientific的</td><td> 37620 </td><td></td></tr></tbody></table>

therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles

在美国每年超过32000例患者被诊断出患有胰腺癌和相关的疾病是具有非常高的死亡率 1的。存在迫切需要制定小说翻译临床治疗策略，可以提高胰腺癌患者的惨淡生存统计。虽然在癌症基因治疗已经显示出了巨大的承诺，面临的主要挑战是在安全和有效的传递系统，从而导致持续的转基因表达的发展。 明胶是一个最多才多艺的天然生物高分子，广泛应用于食品和医药产品。从我们的实验室以往的研究表明，B型明胶的物理封装的DNA，保存后，细胞内交货的质粒和提高转染效率的超螺旋结构。明胶硫醇，巯基可以被引入到聚合物中，并会fORM纳米粒子，从而稳定整个复杂，一旦二硫键内二硫键被打破，由于在细胞质中的谷胱甘肽存在，有效载荷将发布2-5。聚乙二醇（PEG）的修改氏族，进入体循环，提供长期流通的时间和优先目标，以肿块，由于超的新生血管通透性增强通透性和保留的效果6。有研究表明过度表达的表皮生长因子受体（EGFR）的PANC - 1人类胰腺癌细胞7。为了积极目标胰腺癌细胞株，表皮生长因子受体（EGFR）特异性多肽通过PEG间隔的粒子表面上的共轭。 大多数管理抑癌基因，如野生型p53（WT - P53），恢复在细胞 9的促凋亡的功能都集中在抗肿瘤基因疗法</SUP&gt;。 p53作为一个关键的信号通路在细胞生长，调节细胞凋亡，细胞周期阻滞，新陈代谢和其他进程 10的机制功能。在胰腺癌，大多数细胞p53蛋白的基因突变，从而导致凋亡活性的损失。引进野生型p53，细胞凋亡可被修复，并进一步触发癌细胞的细胞死亡 11 。 基于上述理由，我们设计了野生型p53基因传递和评估交付效率和PANC - 1细胞的转染表皮生长因子受体靶向肽修饰硫醇盐明胶纳米粒子。

Watch this Scientific Journal Video about Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles at JoVE.com

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

系统性基因传递和转染治疗胰腺癌的B型明胶为基础的设计nanovectors系统（氏族）的开发。他们可以通过与表皮生长因子受体（EGFR）的特异性多肽nanparticles表面改性，表皮生长因子受体受体目标和下降低细胞内谷胱甘肽的浓度高，如环境，释放质粒。

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

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