Name: repression of multiple myeloma cell growth in vivo by single-wall carbon nanotube (swcnt)-delivered malat1 antisense oligos
Uploaded: 2018-12-13T13:00:00
Description: Watch this Scientific Journal Video about Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos at JoVE.com

Forfatterne takke Lerner forskningsinstitut proteom, genomisk, og billedbehandling kerner for deres hjælp og støtte. Finansiering: Dette værk blev økonomisk støttet af NIH/NCI grant R00 CA172292 (til J.J.Z.) og start-up midler (til J.J.Z.) og klinisk og translationel Science Collaborative (CTSC) af Case Western Reserve University Core udnyttelse Pilot Grant (til J.J.Z.). Dette arbejde udnyttes Leica SP8 Konfokal mikroskop, som blev købt med støtte fra nationale institutter for sundhed SIGNA grant 1S10OD019972-01.

Alle eksperimenter, der involverer dyr var forhåndsgodkendt af Cleveland Clinic IACUC (institutionelle Animal Care og brug Udvalget).
1. Sammenfatning af Functionalized SWCNTs
<ol><li>Mix 1 mg af SWCNTs, 5 mg af DSPE-PEG2000-Amin og 5 mL steriliseret nukleasen-frit vand i et glas scintillation hætteglas (20 mL). Ryst det godt til at opløse alle reagenser helt.</li>
	<li>Læg instrumenterne i ultralydsbad i hætteglasset i et vand bad sonikator ved et strømniveau på 40 W i 1 time ved stu...

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	<li>Jiang, X., 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=RNase+non-sensitive+and+endocytosis+independent+siRNA+delivery+system:+delivery+of+siRNA+into+tumor+cells+and+high+efficiency+induction+of+apoptosis.">RNase non-sensitive and endocytosis independent siRNA delivery system: delivery of siRNA into tumor cells and high efficiency induction of apoptosis.</a> Nanoscale. 5 (16), 7256-7264 (2013).</li><li>Murakami, T., 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=Water-dispersed+single-wall+carbon+nanohorns+as+drug+carriers+for+local+cancer+chemotherapy.">Water-dispersed single-wall carbon nanohorns as drug carriers for local cancer chemotherapy.</a> Nanomedicine (Lond). 3 (4), 453-463 (2008).</li><li>Kam, N. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bright+side+of+dark+matter:+lncRNAs+in+cancer.">The bright side of dark matter: lncRNAs in cancer.</a> Journal of Clinical Investigation. 126 (8), 2775-2782 (2016).</li><li>Ronchetti, D., 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=Distinct+lncRNA+transcriptional+fingerprints+characterize+progressive+stages+of+multiple+myeloma.">Distinct lncRNA transcriptional fingerprints characterize progressive stages of multiple myeloma.</a> Oncotarget. 7 (12), 14814-14830 (2016).</li><li>Wong, K. Y., 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=Epigenetic+silencing+of+a+long+non-coding+RNA+KIAA0495+in+multiple+myeloma.">Epigenetic silencing of a long non-coding RNA KIAA0495 in multiple myeloma.</a> Molecular Cancer. 14, 175 (2015).</li><li>Schmidt, L. H., 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=The+long+noncoding+MALAT-1+RNA+indicates+a+poor+prognosis+in+non-small+cell+lung+cancer+and+induces+migration+and+tumor+growth.">The long noncoding MALAT-1 RNA indicates a poor prognosis in non-small cell lung cancer and induces migration and tumor growth.</a> Journal of Thoracic Oncology. 6 (12), 1984-1992 (2011).</li><li>Ji, 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=MALAT-1,+a+novel+noncoding+RNA,+and+thymosin+beta4+predict+metastasis+and+survival+in+early-stage+non-small+cell+lung+cancer.">MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer.</a> Oncogene. 22 (39), 8031-8041 (2003).</li><li>Luo, J. H., 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=Transcriptomic+and+genomic+analysis+of+human+hepatocellular+carcinomas+and+hepatoblastomas.">Transcriptomic and genomic analysis of human hepatocellular carcinomas and hepatoblastomas.</a> Hepatology. 44 (4), 1012-1024 (2006).</li><li>Guffanti, 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=A+transcriptional+sketch+of+a+primary+human+breast+cancer+by+454+deep+sequencing.">A transcriptional sketch of a primary human breast cancer by 454 deep sequencing.</a> BMC Genomics. 10, 163 (2009).</li><li>Cho, S. F., 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=MALAT1+long+non-coding+RNA+is+overexpressed+in+multiple+myeloma+and+may+serve+as+a+marker+to+predict+disease+progression.">MALAT1 long non-coding RNA is overexpressed in multiple myeloma and may serve as a marker to predict disease progression.</a> BMC Cancer. 14, 809 (2014).</li><li>Handa, H., 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=Long+non-coding+RNA+MALAT1+is+an+inducible+stress+response+gene+associated+with+extramedullary+spread+and+poor+prognosis+of+multiple+myeloma.">Long non-coding RNA MALAT1 is an inducible stress response gene associated with extramedullary spread and poor prognosis of multiple myeloma.</a> British Journal of Haematology. 179 (3), 449-460 (2017).</li><li>Hu, Y., 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=Targeting+the+MALAT1/PARP1/LIG3+complex+induces+DNA+damage+and+apoptosis+in+multiple+myeloma.">Targeting the MALAT1/PARP1/LIG3 complex induces DNA damage and apoptosis in multiple myeloma.</a> Leukemia. , (2018).</li><li>Lennox, K. A., Behlke, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+localization+of+long+non-coding+RNAs+affects+silencing+by+RNAi+more+than+by+antisense+oligonucleotides.">Cellular localization of long non-coding RNAs affects silencing by RNAi more than by antisense oligonucleotides.</a> Nucleic Acids Research. 44 (2), 863-877 (2016).</li><li>Kam, N. W., O'Connell, M., Wisdom, J. A., Dai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+nanotubes+as+multifunctional+biological+transporters+and+near-infrared+agents+for+selective+cancer+cell+destruction.">Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (33), 11600-11605 (2005).</li><li>Zeineldin, R., Al-Haik, M., Hudson, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+polyethylene+glycol+integrity+in+specific+receptor+targeting+of+carbon+nanotubes+to+cancer+cells.">Role of polyethylene glycol integrity in specific receptor targeting of carbon nanotubes to cancer cells.</a> Nano Letters. 9 (2), 751-757 (2009).</li><li>Amodio, N., D'Aquila, P., Passarino, G., Tassone, P., Bellizzi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+modifications+in+multiple+myeloma:+recent+advances+on+the+role+of+DNA+and+histone+methylation.">Epigenetic modifications in multiple myeloma: recent advances on the role of DNA and histone methylation.</a> Expert Opinion on Therapeutic Targets. 21 (1), 91-101 (2017).</li><li>Ahmad, N., Haider, S., Jagannathan, S., Anaissie, E., Driscoll, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+theragnostics+for+the+clinical+management+of+multiple+myeloma.">MicroRNA theragnostics for the clinical management of multiple myeloma.</a> Leukemia. 28 (4), 732-738 (2014).</li><li>Amodio, N., 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=Drugging+the+lncRNA+MALAT1+via.+LNA+gapmeR+ASO+inhibits+gene+expression+of+proteasome+subunits+and+triggers+anti-multiple+myeloma+activity.">Drugging the lncRNA MALAT1 via. LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity.</a> Leukemia. , (2018).</li><li>Highleyman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+approves+fomivirsen,+famciclovir,+and+Thalidomide.+Food+and+Drug+Administration.">FDA approves fomivirsen, famciclovir, and Thalidomide. Food and Drug Administration.</a> BETA. 5, (1998).</li><li>Smith, R. J., Hiatt, W. 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Beviser har vist, at lncRNAs at deltage i reguleringen af mange fysiologiske og patofysiologiske procedurer i kræftformer, herunder MM7,8,9; de har potentialet til at være målrettet til behandling af cancer, som kan realiseres af antisense oligonukleotider20,21,22. Den amerikanske Food and Drug Administration (FDA) har godkendt flere...

SWCNT er en ny nanomateriale, der kan levere forskellige typer af lægemidler, såsom proteiner, små molekyler og nukleinsyrer, stabilt og effektivt med ideelle tolerabilitet og minimum toksicitet i vitro1 og in vivo2. En functionalized SWCNT har stor biokompatibilitet og vand opløselighed, kan bruges som en shuttle til mindre molekyler, og kan bære dem for at trænge ind i cellemembranen3,4,5.
lncRNAs er en klynge af RNA (> 200 nt) der er transskriberet fra genomet til mRNA men kan ikke omsættes til proteiner. Stadig flere beviser har vist at lncRNAs deltage i reguleringen af gen expression6 og er involveret i indledningen og progression af de fleste typer af kræft, herunder MM7,8,9. MALAT1 er en nuklear-beriget noncoding udskrift 2 (NEAT2) og en yderst velbevarede lncRNA10. MALAT1 er i første omgang anerkendt i metastatisk ikke-småcellet lungekræft (NSCLC) kræft11, men har været overexpressed i mange tumorer5,12,13; Det er en af de mest højt udtrykt lncRNAs og er korreleret med en dårlig prognose i MM8,14. Udtryk niveauet af MALAT1 er betydeligt højere i fatal kursus extramedullary MM patienter sammenlignet med dem kun diagnosticeret som MM15.
I en tidligere undersøgelse, har vi bekræftet, at anti-MALAT1 oligos håndfast fører til DNA-skader og apoptose i MM16 ved hjælp af gapmer DNA antisense oligonukleotider rettet mod MALAT1 (anti-MALAT1) i MM celler. Gapmer DNA er sammensat af antisense DNA og forbundet af 2'-OMe-RNA'er, som kunne bede MALAT1 spaltning af RNase H aktivitet en gang bundet17. In vivo levering effektiviteten af antisense oligos stadig begrænser dens kliniske anvendelse.
For at teste levering functionalized effekten af SWCNT til anti-MALAT1 gapmer oligos, anti-MALAT1 gapmer DNA er konjugeret til DSPE-PEG2000-Amin SWCNT. SWCNT-anti-MALAT1 derefter indsprøjtes intravenøst i en MM formidlet musen model; en slående hæmning er observeret efter fire behandlinger.

<table border="0" cellpadding="0" cellspacing="0" style="width:1168px;" width="1167">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:324px;">SWCNTs</td>
			<td style="width:199px;">Millipore-Sigma</td>
			<td style="width:209px;">704113</td>
			<td style="width:436px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DSPE-PEG2000-Amine</td>
			<td>Avanti Polar Lipids</td>
			<td>880128</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">bath sonicator</td>
			<td>VWR</td>
			<td>97043-992</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4 mL centrifugal filter</td>
			<td>Millipore-Sigma</td>
			<td>Z740208-8EA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">UV/VIS spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>accuSkan GO UV/Vis Microplate Spectrophotometer</td>
			<td>extinction coefficient of 0.0465 L/mg/cm at 808 nm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sulfo-LC-SPDP</td>
			<td>ProteoChem</td>
			<td>c1118</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DTT solution</td>
			<td>Millipore-Sigma</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NAP-5 column</td>
			<td>GE Healthcare</td>
			<td>17-0853-01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">in vivo imaging system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NOD.CB17-Prkdcscid/J mice</td>
			<td>Charles River lab</td>
			<td>250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow cytometer</td>
			<td>Becton Dickinso</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td>Lipofectamine2000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RMPI-1640 medium</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MALAT1-QF:</td>
			<td>synthesized by IDT Company</td>
			<td style="width:209px;"></td>
			<td>5&#8217;- GTTCTGATCCCGCTGCTATT - 3&#8217;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MALAT1-QR:</td>
			<td>synthesized by IDT Company</td>
			<td style="width:209px;"></td>
			<td>5&#8217;- TCCTCAACACTCAGCCTTTATC - 3&#8217;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GAPDH-QF:</td>
			<td>synthesized by IDT Company</td>
			<td style="width:209px;"></td>
			<td>5&#8217;- CAAGAGCACAAGAGGAAGAGAG - 3&#8217;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GAPDH-QR:</td>
			<td>synthesized by IDT Company</td>
			<td style="width:209px;"></td>
			<td>5&#8217;- CTACATGGCAACTGTGAGGAG - 3&#8217;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Quantitative PCR using SYBR Green PCR master mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25780</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RevertAid first-stand cDNA synthesis kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K1621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-MALAT1</td>
			<td>synthesized by IDT Company</td>
			<td></td>
			<td>5&#8217;-mC*mG*mA*mA*mA*C*A*T*T 
			*G*G*C*A*C*A*mC*mA*mG*mC*mA-3&#8217;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Viability Assay Kit</td>
			<td>Promega Corporation</td>
			<td>G7570</td>
			<td>CellTiter-GloLuminescent Cell Viability Assay Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">accuSkan GO UV/Vis Microplate Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifugal filter</td>
			<td>Millipore-Sigma</td>
			<td>UFC910008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPSS software</td>
			<td>IBM</td>
			<td>version 24.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Luciferin</td>
			<td>Millipore-Sigma</td>
			<td>L9504</td>
			<td></td>
		</tr>
	</tbody>
</table>

repression of multiple myeloma cell growth in vivo by single-wall carbon nanotube (swcnt)-delivered malat1 antisense oligos

Enkelt-væg kulstof nanorør (SWCNT) er en ny type af nanopartikler, der er blevet brugt til at levere flere slags medicin ind i celler, såsom proteiner, oligonukleotider og små-molekyle designerdrugs. SWCNT har tilpasselig dimensioner, en stor overfladisk område, og kan fleksibelt binde med narkotika gennem forskellige ændringer på dens overflade; Derfor er det et ideelt system til transport af stoffer ind i celler. Længe noncoding RNA'er (lncRNAs) er en klynge af noncoding RNA længere end 200 nt, som ikke kan oversættes til protein, men spiller en vigtig rolle i biologiske og patofysiologiske processer. Metastase-associerede lunge adenocarcinom udskrift 1 (MALAT1) er en yderst velbevarede lncRNA. Det blev påvist, at højere MALAT1 niveauerne er relateret til den dårlig prognose af forskellige kræftformer, herunder myelomatose (MM). Vi har afsløret, at MALAT1 regulerer DNA reparation og celle død i MM; altså, MALAT1 kan betragtes som en terapeutisk destination for MM. Effektiv levering af antisense oligo at hæmme/knockdown MALAT1 in vivo er imidlertid stadig et problem. I denne undersøgelse, vi ændre SWCNT med PIND-2000 og konjugat en anti-MALAT1 oligo til det, teste levering af dette stof in vitro-, injicere det intravenøst i en dissemineret MM musen model og observere en betydelig hæmning af MM progression, som angiver at SWCNT er en ideel levering direkte til anti-MALAT1 gapmer DNA.

For at demonstrere hæmning effekten af anti-MALAT1 gapmer DNA i MM, vi væltede udtryk af MALAT1 og brugte det i H929 og MM.1S celler. 48 timer senere, celler blev indsamlet til analyse af knock-down effektivitet og apoptose status i celler transfekteret med anti-MALAT1 gapmer eller kontrol DNA. qRT-PCR resultaterne viste, at anti-MALAT1 gapmer DNA væltede MALAT1 udtrykket i H929 og MM.1S celler effektivt (fig. 2A). Status for apoptose var ...

Watch this Scientific Journal Video about Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos at JoVE.com

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos

Dette manuskript beskriver syntesen af en enkelt-væg kulstof nanorør (SWCNT)-konjugeret MALAT1 antisense gapmer DNA oligonukleotid (SWCNT-anti-MALAT1), som viser den pålidelig levering af SWCNT og den potent terapeutiske effekt af anti-MALAT1 in vitro og in vivo. Metoder anvendes til syntese, ændring, konjugering, og injektion af SWCNT-anti-MALAT1 er beskrevet.

Undertrykkelse af myelomatose cellevækst In Vivo af enkelt-væg kulstof nanorør (SWCNT)-leveres MALAT1 Antisense Oligos

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as ...

Den metode, vi bruger her, kan bidrage til at pakke oligonukleotid med envægsnanorør, som er en ny type nanopartikel til levering af lægemidler. Den største fordel ved denne teknik er mere effektiv levering af oligonukleotid narkotika i celler in vivo. Konsekvenserne af denne teknik omfatter forskning myelomatose, fordi det er indført nanopartikel til at forbedre leveringen af oligonukleotid narkotika.Gennem denne metode kan give indsigt i behandling for myelomatose. Det kan også anvendes på andre sygdomme og konjugeret med andre last, såsom proteiner og små molekyler. Først blandes et milligram enkeltvægget kulstofnanorør, fem milligram DSPE-PEG 2000-amin og fem milliliter steriliseret nukleasefrit vand i et 20-milliliter glasscintillationshætteglas.Sonikere hætteglasset i et vandbad sonikerator på et effektniveau på 90 watt i en time ved stuetemperatur. Efter sonikering centrifuger hætteglasset ved 24.000 gange g i seks timer. Derefter indsamles supernatant opløsningen.Der tilsættes en milliliter af enkeltvægget carbon nanorørsopløsning til en 100-kilodalton molekylvægt cutoff centrifugalfilterenhed efterfulgt af fire milliliter steriliseret nukleasefrit vand. Centrifuge i 10 minutter ved 4,000 gange g ved stuetemperatur for at fjerne den overskydendeamine. Tilføj 0,5 milligram Sulfo-LC-SPDP til 450 mikroliter af den amin-funktionaliserede enkelt-væg carbon nanorør.Derefter tilsættes 50 mikroliter af 10x PBS, og inkuberes i to timer ved stuetemperatur. Dernæst tilsættes reaktionsblandingen til en 100-kilodalton molekylvægt cutoff centrifugalfilter enhed, efterfulgt af fire milliliter nukleasefrit vand. Centrifuge prøven i 10 minutter ved 4,000 gange g ved stuetemperatur for at fjerne den overskydende Sulfo-LC-SPDP.Opsaml sulfo-LC-SPDP-behandlede amin enkeltvægget carbon nanorørsopløsning, der efterlades i filteret, og fortynd den med 500 mikroliter af tidligere forberedt anti-MALAT1-Cy3-opløsning. Derefter inkubere prøven ved fire grader Celsius natten over. For at opnå de bedste sammenligningsresultater arrangerer du tilfældigt 14 mus i forsøgs- eller kontrolgrupper med syv i hver gruppe og med samme forhold mellem mænd og kvinder i hver gruppe.Dernæst rense operation bænken og sterilisere det med 70% ethylalkohol. Brug en varmelampe til at varme en af musene for at hjælpe halevene til at dukke op. Hold musen ordentligt tilbage med en hale injektion fastholdelsesren.Indsprøjtning MM.1S-luc/mCherry celler i 50 mikroliter af PBS gennem hale venen. Tryk på injektionsstedet med en spritserviet i 30 sekunder. Marker derefter den injicerede mus og returnere den til et rent bur.På dag syv, efter MM1S celle injektion, injicere 50 mikroliter af PBS indeholder enkelt-væg carbon nanorør anti-MALAT1 i halen vene af musen. Tryk derefter på injektionsstedet med en spritserviet i 30 sekunder. Overhold den lokale blødning på halen og musens generelle opførsel i et minut, og vend den derefter tilbage til et rent bur.Overhold alle injicerede mus igen, før du returnerer buret til stativet for at sikre, at injektionerne tåles godt. Hver mus vejes før dissektion. Indsamle perifert blod fra hjertet for en komplet blodtælling assay udført af en blodcelle counter maskine.Dernæst indsamle væv i knoglemarven, milt, lymfeknude, nyre, og væv med synlig metastase. Ekstrakt RNA og protein fra knoglemarvsprøverne. Fix alle resterende væv i formalin.Efter 24 timers fiksering nedsænkes knogleprøverne i en 0,5-molar EDTA-opløsning i fem dage til afkalkning. Derefter nedsænkes alle prøver i 75% ethylalkohol til langtidsopbevaring. QRT PCR resultater viste, at anti-MALAT1 gapmeR DNA bankede ned MALAT1 udtryk og induceret apoptose betydeligt i H929 og MM.1S celler.Single-wall carbon nanotube anti-MALAT1-Cy3 blev leveret i kernen af MM-celler, og betydeligt undertrykt den endogene MALAT1 niveau i både H929 og MM.1S celler. Levedygtigheden af BMEC-1 celle har ingen signifikant forskel efter behandling af enkelt-væg carbon nanorør anti-MALAT1-Cy3 eller kontrol ved forskellige doser og tidspunkter. Dette indikerer, at toksiciteten af en-væg carbon nanorør anti-MALAT1-Cy3 har en begrænset og acceptabel toksicitet for normale celler ved høj dosis.En menneskelig MM-formidlet mus model blev etableret for at vurdere behandlingseffekter af enkelt-væg carbon nanorør anti-MALAT1 in vivo. Luciferon-signalerne fra mus i enkeltvægget carbon nanotube anti-MALAT1 behandlingsgruppe var bemærkelsesværdigt lavere sammenlignet med kontrollen efter 21 dages behandling. Fra disse resultater, Det blev konkluderet, at enkelt-væg carbon nanorør anti-MALAT1 behandling via intravenøs injektion kunne levere anti-MALAT1 oligos til tumorceller effektivt.Der blev ikke observeret væsentlige bivirkninger ved behandlingen, hvilket indikerer, at enevægsinnanorør anti-MALAT1-injektion er en sikker behandling for mus. Mens du forsøger denne procedure, Er det vigtigt at huske, at cellen molekyle sammensætning vil påvirke levering, effektivitet, og terapeutisk effekt af oligonukleotid narkotika. Efter denne procedure, andre metoder som bøjning af proteiner eller små molekyler kan udføres for at besvare yderligere spørgsmål.Ligesom ved hjælp af en-væg kulstof nanorør til at levere andre lægemidler.

Research

JoVE Journal

Cancer Research

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

En protokol til Rapid Post-mortem Cell Culture af Diffus Intrinsic Pontine gliom (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR vurdering af pH, pO2, Redox Status, og koncentrationen af fosfat og glutathion i Tumor mikromiljø

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multimodal en bioluminescerende og Positron-emissions-tomografi/edb tomografi billeddannelse af myelomatose knoglemarv Xenografts i NOG mus

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Neutrofile ekstracellulære fælder genereret af Low Density neutrofiler fremstillet af Peritoneal Lavage væske mægle tumorcellevækst og vedhæftet fil

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Evaluering af biomarkører i gliom af Immunhistokemi på Paraffin-Embedded 3D gliom Neurosphere kulturer

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

In vivo hæmning af MicroRNA at mindske tumor vækst i mus

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

En musemodel til at undersøge rollen som kræftassocierede fibroblaster i tumorvækst

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

En Ex Vivo Brain Slice Model til at studere og målrette brystkræft hjernemetastatisk tumorvækst

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Definition af genfunktioner i tumorigenese ved Ex vivo Ablation af floxede alleler i maligne perifere nerveskede tumorceller

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In vivo Immunogenicitetsscreening af tumorafledte ekstracellulære vesikler ved flowcytometri af milt-T-celler

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...