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

Denne undersøgelse blev støttet af National Cancer Institute Alliance i nanoteknologi for Cancer Center for Cancer Nanoteknologi Excellence (CCNE) indrømme U54-CA151881.

<ol>
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Kontrol og EGFR målrettede thiolated gelatine nanopartikler blev udarbejdet med effektive DNA indkapsling og stabilitet. De partikler af alle disse systemer var i intervallet 150-250 nm i diameter. Zeta potentiale har bevist, at dette system er en anelse negativ system. Med SEM analysen blev de størrelser af nanopartikler det samme med Zetasizer resultat. ESCA analyse kunne bekræfte PEG og peptid overflade modifikation. Western blot analyse viste, at Panc-1 celler havde en høj EGFR niveauer, og dette cellelinie blev anvendt til in vitro-undersøgelser. Både kontrol og overflade-modificerede nanopartikler var relativt mindre cytotoksiske i Panc-1 celler i forhold til PEI. Cell handel undersøgelser viste hurtige optagelse og plasmid frigivelse af EGFR-målrettede nanopartikler i Panc-1 celler. Levering af journalist plasmider DNA udtrykke med EGFR-målrettede nanopartikler resulterede i højeste niveauer afGFP udtryk i forhold til andre kontroller, herunder Lipofectin-kompleks DNA. Med det samme system, udløste transfektion med WT-p53 plasmid downstream-apoptotiske pathway og inducerede hurtige apoptose i Panc-1 celler. Disse foreløbige resultater tyder på, at EGFR-målrettede thiolated gelatine nanopartikler kan tjene som et sikkert og effektivt DNA levering system til genterapi som en behandling for kræft i bugspytkirtlen.

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Type B gelatine, Bloom 225 </td><td> Sigma-Aldrich </td><td> G9391 </td><td></td></tr><tr><td> 2-iminothiolane hydrochlorid </td><td> Sigma-Aldrich </td><td> I6256 </td><td></td></tr><tr><td> pEGFP-N1 plasmid </td><td> Elim Biopharm </td><td> N / A </td><td></td></tr><tr><td> pORF-hp53 E. coli </td><td> Invivogen </td><td> porf-hp53 </td><td></td></tr><tr><td> Glyoxal opløsning (40wt.% i H 2 O) </td><td> Sigma-Aldrich </td><td> 128465 </td><td></td></tr><tr><td> Glycine </td><td> Sigma-Aldrich </td><td> 410225-250G </td><td></td></tr><tr><td> QIA filter Plasmid Mega-kit </td><td> Qiagen </td><td> 12281 </td><td></td></tr><tr><td> Beckman LE 80K ultracentrifuge </td><td> Beckman </td><td> N / A </td><td></td></tr><tr><td> Freezone 6 liter Console Freeze Dry Systems </td><td> Labconco </td><td> 7753020 </td><td></td></tr><tr><td> MPEG-SCM, MW 2.000 Da </td><td> Laysan </td><td> MPEG-SCM-2K-1g </td><td></td></tr><tr><td> MAL-PEG-SCM, MW 2.000 Da </td><td> Jenkem Teknologi </td><td> A5001-1 </td><td></td></tr><tr><td> Zetasizer Nano </td><td> Malvern </td><td> Zetasizer Nano ZS </td><td></td></tr><tr><td> Hitachi 4800 felt emission scanning elektron mikroskop </td><td> Hitachi </td><td> S-4800 UHR FE-SEM </td><td></td></tr><tr><td> Quant-IT PicoGreen dsDNA Reagens og sortimenter </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> Lipofectin transfektion Reagens </td><td> Invitrogen </td><td> 18292011 </td><td></td></tr><tr><td> DMEM </td><td> Mediatech cellgro </td><td> 10 013 CM </td><td></td></tr><tr><td> RPMI </td><td> Mediatech cellgro </td><td> 50 020 PB </td><td></td></tr><tr><td> Pierce BCA Protein Assay Kit </td><td> Thermo Scientific </td><td> 23225 </td><td></td></tr><tr><td> iBlot Tør Blotting System </td><td> Invitrogen </td><td> IB1001 </td><td></td></tr><tr><td> XCell SureLock Mini-Cell og XCell II Blot Module Kit CE-mærke </td><td> Invitrogen </td><td> EI0002 </td><td></td></tr><tr><td> Pierce ECL Western Blotting Substrat </td><td> Thermo Scientific </td><td> 32109 </td><td></td></tr><tr><td> Kodak Digital X-ray Specimen (DXS) System </td><td> Kodak </td><td> N / A </td><td>; </td></tr><tr><td> CellTiter 96 AQ ueous én løsning celledeling Assay (MTS) </td><td> Promega </td><td> G3580 </td><td></td></tr><tr><td> BIOTEK SynergyHT pladelæser </td><td> BIOTEK </td><td> N / A </td><td></td></tr><tr><td> Nanodrop 2000 </td><td> Thermo Scientific </td><td> N / A </td><td></td></tr><tr><td> Et-trins RT-PCR kit </td><td> Qiagen </td><td> 210212 </td><td></td></tr><tr><td> Kromatin Kondens / membranpermeabilitet / Dead Cell Apoptose Kit </td><td> Invitrogen </td><td> V23201 </td><td></td></tr><tr><td> Apo-ONE Homogen caspase-07/03 Assay kit </td><td> Promega </td><td> G7790 </td><td></td></tr><tr><td> Hybaid PCR Sprint termisk cycler </td><td> Thermo Scientific </td><td> N / A </td><td></td></tr><tr><td> EGF-receptor antistof </td><td> Cellsignalering </td><td> 2232 </td><td></td></tr><tr><td> β-Actin Antibody </td><td> Celle signalering </td><td> 4967 </td><td></td></tr><tr><td> Anti-kanin IgG, HRP-linked Antibody </td><td> Celle signalering </td><td> 7074 </td><td></td></tr><tr><td> Monoklonalt GFP antistof </td><td> Novus Biologicals </td><td> NB600-597 </td><td></td></tr><tr><td> Ged Polyklonale GFP antistof (alkalinfosfatase) </td><td> Novus Biologicals </td><td> NB600-1502 </td><td></td></tr><tr><td> Phosphatasesubstrattabletter Kit </td><td> Thermo Scientific </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

Mere end 32.000 patienter diagnosticeret med kræft i bugspytkirtlen i USA om året, og sygdommen er forbundet med meget høj dødelighed 1. Presserende behov for at udvikle nye klinisk-oversætbare terapeutiske strategier, der kan forbedre den triste overlevelse statistik over kræft i bugspytkirtlen patienter. Selv om genterapi i kræft har vist en enorm løfte, den største udfordring ligger i udviklingen af ​​sikre og effektive levering system, hvilket kan føre til en vedvarende transgenet udtryk. Gelatine er et af de mest alsidige naturlige biopolymer, i vid udstrækning anvendes i fødevarer og farmaceutiske produkter. Tidligere undersøgelser fra vores laboratorium har vist, at type B gelatine kunne fysisk indkapsle DNA, der bevarede den supercoiled struktur plasmid og forbedret transfektion effektivitet ved intracellulære levering. Ved thiolation af gelatine, kunne sulfhydryl grupper indføres i polymer og ville fOrm disulfid-bro inden for nanopartikler, der stabiliserer hele komplekset, og når disulfid-bro er brudt på grund af tilstedeværelsen af glutathion i cytosolen, ville nyttelast blive frigivet 2-5. Poly (ethylen glycol) (PEG)-modificeret Gens, når det administreres i det systemiske kredsløb, giver lang-cirkulation gange og fortrinsvis mål til svulsten masse på grund af den hyper-gennemtrængelighed af de nydannede kar gennem den øgede permeabilitet og fastholdelse virkning 6. Undersøgelser har vist, overekspression af den epidermale vækstfaktor receptor (EGFR) på Panc-1 menneskets bugspytkirtel adenocarcinom celler 7. For aktivt at målrette kræft i bugspytkirtlen cellelinie, EGFR specifikke peptid blev konjugeret på partikel overfladen gennem en PEG spacer. 8 De fleste anti-tumor genterapier er fokuseret på administration af tumor suppressor gener, såsom vildtype p53 (WT-p53), for at genoprette den pro-apoptotiske funktion i cellerne 9 </sup&gt;. P53 mekanisme fungerer som en kritisk signalvej i celle vækst, som regulerer apoptose, cellecyklusstop, stofskifte og andre processer 10. Inden for bugspytkirtelkræft, har de fleste celler mutationer i p53-protein, der forårsager tab af apoptotiske aktivitet. Med introduktionen af WT-p53, kunne apoptose repareres og yderligere udløser celledød i kræftceller 11. Baseret på ovenstående grundlag, har vi designet EGFR målrettet peptid-modificerede thiolated gelatine nanopartikler til WT-p53 genet levering og evalueret levering effektivitet og transfektion i Panc-1 celler.

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

Type B gelatine-baserede manipuleret nanovectors system (gensen) blev udviklet til systemisk gen levering og transfektion i behandlingen af ​​kræft i bugspytkirtlen. Ved ændring med epidermal vækstfaktor receptor (EGFR) specifikke peptid på overfladen af ​​nanparticles, kunne de mål på EGFR receptor og frigivelse plasmid under reducerende miljø, såsom høj intracellulær glutathion koncentrationer.

Gene Levering og transfektion i Human bugspytkirtelkræftceller hjælp epidermal vækstfaktor receptor-målrettede Gelatine-baseret Engineered Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

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