Name: delivery of exogenous artificially synthesized mirna mimic to the kidney using polyethylenimine nanoparticles in several kidney disease mouse models
Uploaded: 2022-05-10T14:00:00
Description: Watch this Scientific Journal Video about Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models at JoVE.c

Dette arbejde blev delvist støttet af JSPS KAKENHI (tilskud nr. 21K08233). Vi takker Edanz (https://jp.edanz.com/ac) for at redigere udkast til dette manuskript.

Alle dyreforsøgsprotokoller blev godkendt af den dyreetiske komité ved Jichi Medical University og udført i overensstemmelse med retningslinjerne for brug og pleje af forsøgsdyr fra Jichi Medical University Guide for Laboratory Animals. Her demonstrerede vi miRNA-efterligning til nyrerne, hvilket resulterede i dets overekspression ved hjælp af UUO-mus. Denne undersøgelse blev godkendt af den etiske komité ved Jichi Medical University [godkendelse nr. 19-12 for nyrefibrose, 17-024 for akut nyreinfektion (AKI) og 19...

<ol>
	<li>Mohr, A. M., Mott, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+microRNA+biology.">Overview of microRNA biology.</a> Seminars in Liver Disease. 35 (1), 3-11 (2015).</li><li>Bushati, N., Cohen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA+functions.">microRNA functions.</a> Annual Review of Cell and Developmental Biology. 23, 175-205 (2007).</li><li>Simpson, K., Wonnacott, A., Fraser, D. J., Bowen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNAs+in+diabetic+nephropathy:+From+biomarkers+to+therapy.">microRNAs in diabetic nephropathy: From biomarkers to therapy.</a> Current Diabetes Reports. 16 (3), 35 (2016).</li><li>Yheskel, M., Patel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+microRNAs+in+polycystic+kidney+disease.">Therapeutic microRNAs in polycystic kidney disease.</a> Current Opinion in Nephrology and Hypertension. 26 (4), 282-289 (2017).</li><li>Lv, W., 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=Therapeutic+potential+of+microRNAs+for+the+treatment+of+renal+fibrosis+and+CKD.">Therapeutic potential of microRNAs for the treatment of renal fibrosis and CKD.</a> Physiological Genomics. 50 (1), 20-34 (2018).</li><li>Dykxhoorn, D. M., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+silent+revolution:+RNA+interference+as+basic+biology,+research+tool,+and+therapeutic.">The silent revolution: RNA interference as basic biology, research tool, and therapeutic.</a> Annual Review of Medicine. 56, 401-423 (2005).</li><li>Dykxhoorn, D. M., Palliser, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+silent+treatment:+siRNAs+as+small+molecule+drugs.">The silent treatment: siRNAs as small molecule drugs.</a> Gene Therapy. 13 (6), 541-552 (2006).</li><li>Stewart, S. 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=Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells.">Lentivirus-delivered stable gene silencing by RNAi in primary cells.</a> RNA. 9 (4), 493-501 (2003).</li><li>Deng, M., 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=Klotho+gene+delivery+ameliorates+renal+hypertrophy+and+fibrosis+in+streptozotocin-induced+diabetic+rats+by+suppressing+the+Rho-associated+coiled-coil+kinase+signaling+pathway.">Klotho gene delivery ameliorates renal hypertrophy and fibrosis in streptozotocin-induced diabetic rats by suppressing the Rho-associated coiled-coil kinase signaling pathway.</a> Molecular Medicine Reports. 12 (1), 45-54 (2015).</li><li>Zhou, 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=Suppressor+of+cytokine+signaling+(SOCS)+2+attenuates+renal+lesions+in+rats+with+diabetic+nephropathy.">Suppressor of cytokine signaling (SOCS) 2 attenuates renal lesions in rats with diabetic nephropathy.</a> Acta Histochemica. 116 (5), 981-988 (2014).</li><li>Tenenbaum, L., Lehtonen, E., Monahan, P. E. <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+risks+related+to+the+use+of+adeno-associated+virus-based+vectors.">Evaluation of risks related to the use of adeno-associated virus-based vectors.</a> Current Gene Therapy. 3 (6), 545-565 (2003).</li><li>Lukashev, A. N., Zamyatnin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+therapy:+Current+state+and+clinical+perspectives.">Viral vectors for gene therapy: Current state and clinical perspectives.</a> Biochemistry. Biokhimiia. 81 (7), 700-708 (2016).</li><li>Morishita, 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=Delivery+of+microRNA-146a+with+polyethylenimine+nanoparticles+inhibits+renal+fibrosis+in+vivo.">Delivery of microRNA-146a with polyethylenimine nanoparticles inhibits renal fibrosis in vivo.</a> International Journal of Nanomedicine. 10, 3475-3488 (2015).</li><li>Ishii, 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=MicroRNA+expression+profiling+in+diabetic+kidney+disease.">MicroRNA expression profiling in diabetic kidney disease.</a> Translational Research: The Journal of Laboratory and Clinical. 237, 31-52 (2021).</li><li>Aomatsu, 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=MicroRNA+expression+profiling+in+acute+kidney+injury.">MicroRNA expression profiling in acute kidney injury.</a> Translational Research: The Journal of Laboratory and Clinical. (21), 00283-00288 (2021).</li><li>Lungwitz, U., Breunig, M., Blunk, T., Gopferich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+non-viral+gene+delivery+systems.">Polyethylenimine-based non-viral gene delivery systems.</a> European Journal of Pharmceutics and Biopharmaceutics. 60 (2), 247-266 (2005).</li><li>Swami, 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+unique+and+highly+efficient+nonviral+DNA/siRNA+delivery+system+based+on+PEI-bisepoxide+nanoparticles.">A unique and highly efficient nonviral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles.</a> Biochemical and Biophysical Research Communications. 362 (4), 835-841 (2007).</li><li>Kaneko, 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=Detection+of+microRNA+expression+in+the+kidneys+of+immunoglobulin+a+nephropathic+mice.">Detection of microRNA expression in the kidneys of immunoglobulin a nephropathic mice.</a> Journal of Visualized Experiments: JoVE. (161), e61535 (2020).</li><li>Yanai, 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=Quantitative+real-time+PCR+evaluation+of+microRNA+expressions+in+mouse+kidney+with+unilateral+ureteral+obstruction.">Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction.</a> Journal of Visualized Experiments: JoVE. (162), e61383 (2020).</li><li>Aomatsu, 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+quantitative+detection+method+for+microRNAs+in+the+kidney+of+an+ischemic+kidney+injury+mouse+model.">A quantitative detection method for microRNAs in the kidney of an ischemic kidney injury mouse model.</a> Journal of Visualized Experiments: JoVE. (163), e61378 (2020).</li><li>Kushibiki, T., Nagata-Nakajima, N., Sugai, M., Shimizu, A., Tabata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+anti-fibrotic+activity+of+plasmid+DNA+expressing+small+interference+RNA+for+TGF-beta+type+II+receptor+for+a+mouse+model+of+obstructive+nephropathy+by+cationized+gelatin+prepared+from+different+amine+compounds.">Enhanced anti-fibrotic activity of plasmid DNA expressing small interference RNA for TGF-beta type II receptor for a mouse model of obstructive nephropathy by cationized gelatin prepared from different amine compounds.</a> Journal of Controlled Release: Official Journal of the Controlled Release Society. 110 (3), 610-617 (2006).</li><li>Xia, Z., 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=Suppression+of+renal+tubulointerstitial+fibrosis+by+small+interfering+RNA+targeting+heat+shock+protein+47.">Suppression of renal tubulointerstitial fibrosis by small interfering RNA targeting heat shock protein 47.</a> American Journal of Nephrology. 28 (1), 34-46 (2008).</li><li>Tanaka, 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=In+vivo+gene+transfer+of+hepatocyte+growth+factor+to+skeletal+muscle+prevents+changes+in+rat+kidneys+after+5/6+nephrectomy.">In vivo gene transfer of hepatocyte growth factor to skeletal muscle prevents changes in rat kidneys after 5/6 nephrectomy.</a> American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2 (9), 828-836 (2002).</li><li>Hamar, 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=Small+interfering+RNA+targeting+Fas+protects+mice+against+renal+ischemia-reperfusion+injury.">Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (41), 14883-14888 (2004).</li><li>Ma, 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=Xenon+preconditioning+protects+against+renal+ischemic-reperfusion+injury+via+HIF-1alpha+activation.">Xenon preconditioning protects against renal ischemic-reperfusion injury via HIF-1alpha activation.</a> Journal of the American Society of Nephrology: JASN. 20 (4), 713-720 (2009).</li><li>Wei, 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=Short+hairpin+RNA+knockdown+of+connective+tissue+growth+factor+by+ultrasound-targeted+microbubble+destruction+improves+renal+fibrosis.">Short hairpin RNA knockdown of connective tissue growth factor by ultrasound-targeted microbubble destruction improves renal fibrosis.</a> Ultrasound in Medicine and Biology. 42 (12), 2926-2937 (2016).</li></ol>

Ved hjælp af protokollen præsenteret i dette manuskript kan PEI-NP'er levere miRNA-efterligninger til nyrerne for at inducere overekspression af mål-miRNA'er, hvilket resulterer i behandlingseffekter i in vivo-musemodeller af flere nyresygdomme, herunder nyrefibrose, diabetisk nyresygdom og AKI.
Metoden til at forberede komplekset af PEI-NP'er og miRNA-efterligning er meget enkel. Den positivt ladede overflade af PEI-NP'er fanger miRNA-efterligningen, når de bare blandes 13,14,15,...

miRNA'er, små ikke-kodende RNA'er (21-25 baser), der ikke oversættes til proteiner, hæmmer masser af målbudbringer-RNA'er (mRNA'er) ved at destabilisere dem og hæmme deres oversættelse i forskellige nyresygdomme 1,2. Derfor er genterapi ved hjælp af eksogene kunstigt syntetiserede miRNA-efterligninger eller -hæmmere en potentiel ny mulighed for at hæmme udviklingen af mange nyresygdomme 3,4,5.
På trods af løftet om miRNA-efterligninger eller hæmmere til genterapi er levering til målorganer fortsat en stor hindring for in vivo-eksperimenter for at udvikle deres kliniske potentiale. Fordi kunstigt syntetiserede miRNA-efterligninger eller -hæmmere udsættes for øjeblikkelig nedbrydning af serum-RNase, forkortes deres halveringstid ved systemisk administration in vivo6. Derudover er effektiviteten af miRNA-efterligninger eller hæmmere til at krydse plasmamembranen og transfektcytoplasma generelt meget lavere uden passende vektorer 7,8. Disse beviser tyder på, at udviklingen af miRNA-efterligninger eller inhibitorer leveringssystem til nyrerne er påkrævet for at muliggøre deres anvendelse i kliniske omgivelser og gøre dem til en ny behandlingsmulighed for patienter med forskellige nyresygdomme.
Virale vektorer er blevet brugt som bærere til at levere eksogene miRNA-efterligninger eller hæmmere til nyrerne 9,10. Selvom de er udviklet til biosikkerhed og transfektionseffektivitet, kan virale vektorer stadig forårsage interferonrespons og / eller genetisk ustabilitet11,12. For at overvinde disse bekymringer udviklede vi et miRNA-efterligningsleveringssystem til nyrerne ved hjælp af polyethylenaminnanopartikler (PEI-NP'er), en ikke-viral vektor, i flere musemodeller af nyresygdom13,14,15.
PEI-NP'er er lineære polymerbaserede NP'er, der effektivt kan levere oligonukleotider, herunder miRNA-efterligninger, til nyrerne og anses for at foretrække til fremstilling af ikke-virale vektorer på grund af deres langsigtede sikkerhed og biokompatibilitet13,16,17.
Denne undersøgelse demonstrerer virkningerne af systematisk eksogen miRNA-efterligning med PEI-NP'er via haleveneinjektion i nyrefibrosemodelmus produceret ved ensidig ureterobstruktion (UUO). Derudover demonstrerer vi virkningerne af systematisk eksogen miRNA-efterligning med PEI-NP'er via haleveneinjektion i diabetiske nyresygdomsmodelmus (db / db-mus: C57BLKS / J Iar - + Lepr db / + Leprdb) og akut nyreskade modelmus produceret af renal iskæmi-reperfusionsskade (IRI).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole for staining to nucleus</td>
			<td>Thermo Fisher Scientific</td>
			<td>D-1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer RPE</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>Wash buffer 2</td>
		</tr>
		<tr>
			<td>Buffer RWT</td>
			<td>Qiagen</td>
			<td>1067933</td>
			<td>Wash buffer 1</td>
		</tr>
		<tr>
			<td>Control-miRNA-mimic (artificially synthesized miRNA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>Not assigned</td>
			<td>5&#8217;-UUCUCCGAACGUGUCACGUTT- 3&#8217; (sense) 
			5&#8217;-ACGUGACACGUUCGGAGAATT-3&#8242; (antisense)</td>
		</tr>
		<tr>
			<td>Cy3-labeled double-strand oligonucleotides</td>
			<td>Takara Bio Inc.</td>
			<td>MIR7900</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein-labeled Lotus tetragonolobus lectin</td>
			<td>Vector Laboratories Inc</td>
			<td>FL-1321</td>
			<td></td>
		</tr>
		<tr>
			<td>In vivo-jetPEI</td>
			<td>Polyplus</td>
			<td>101000021</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96-well reaction plate for qRT-PCR</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316813</td>
			<td>96-well reaction plate</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>Adhesive film for 96-well reaction plate</td>
		</tr>
		<tr>
			<td>miRNA-146a-5p mimic (artificially synthesized miRNA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>Not assigned</td>
			<td>5&#8217;-UGAGAACUGAAUUCCAUGGGU 
			UT-3&#8242; (sense) 5&#8217;-CCCAUGGAAUUCAGUUCUCAUU -3&#8242; (antisense)</td>
		</tr>
		<tr>
			<td>miRNA-146a-5p primer</td>
			<td>Qiagen</td>
			<td>MS00001638</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>miRNA-181b-5p mimic (artificially synthesized miRNA)</td>
			<td>Gene design</td>
			<td>Not assigned</td>
			<td>5&#8217;-AACAUUCAUUGCUGUCGGUGG 
			GUU-3&#8217;</td>
		</tr>
		<tr>
			<td>miRNA-181b-5p primer</td>
			<td>Qiagen</td>
			<td>MS00006083</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>miRNA-5100-mimic (artificially synthesized miRNA)</td>
			<td>Gene design</td>
			<td>Not assigned</td>
			<td>5&#8217;-UCGAAUCCCAGCGGUGCCUCU -3&#8242;</td>
		</tr>
		<tr>
			<td>miRNA-5100-primer</td>
			<td>Qiagen</td>
			<td>MS00042952</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>miRNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>Membrane anchored spin column in a 2.0-mL collection tube</td>
		</tr>
		<tr>
			<td>miScript II RT kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>miScript SYBR Green PCR kit</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>QIA shredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>Biopolymer spin columns in a 2.0-mL collection tube</td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td>Phenol/guanidine-based lysis reagent</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Flex Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>Real-time PCR instrument</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Software version 1.2.1.</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>Real-time PCR instrument software</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Qiagen</td>
			<td>129112</td>
			<td></td>
		</tr>
		<tr>
			<td>RNU6-2 primer</td>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT (Optimal Cutting Temperature Compound)</td>
			<td>Sakura Finetek Japan Co.,Ltd.</td>
			<td>Not assigned</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of exogenous artificially synthesized mirna mimic to the kidney using polyethylenimine nanoparticles in several kidney disease mouse models

microRNA'er (miRNA'er), små ikke-kodende RNA'er (21-25 baser), der ikke oversættes til proteiner, hæmmer masser af target messenger RNA'er (mRNA'er) ved at destabilisere og hæmme deres oversættelse i forskellige nyresygdomme. Derfor er veksling af miRNA-ekspression ved eksogene kunstigt syntetiserede miRNA-efterligninger en potentielt nyttig behandlingsmulighed til at hæmme udviklingen af mange nyresygdomme. Men fordi serum-RNAase straks nedbryder systematisk administrerede eksogene miRNA-efterligninger in vivo, er levering af miRNA til nyrerne fortsat en udfordring. Derfor er vektorer, der kan beskytte eksogene miRNA-efterligninger mod nedbrydning af RNAase og levere dem signifikant til nyrerne, nødvendige. Mange undersøgelser har brugt virale vektorer til at levere eksogene miRNA-efterligninger eller hæmmere til nyrerne. Imidlertid kan virale vektorer forårsage interferonrespons og / eller genetisk ustabilitet. Derfor er udviklingen af virale vektorer også en hindring for den kliniske anvendelse af eksogene miRNA-efterligninger eller hæmmere. For at overvinde disse bekymringer vedrørende virale vektorer udviklede vi en ikke-viral vektormetode til at levere miRNA-efterligninger til nyrerne ved hjælp af haleveneinjektion af polyethylenaminnanopartikler (PEI-NP'er), hvilket førte til signifikant overekspression af mål-miRNA'er i flere musemodeller af nyresygdom.

Mål-miRNA'erne for nyrefibrose, diabetisk nefropati og AKI beskrevet nedenfor blev valgt baseret på mikroarray, qRT-PCR og / eller databaseforskning til genterapiapplikationer. For yderligere oplysninger henvises til de tidligere publikationer13,14,15.
Levering og virkninger af miRNA-146a-5p-efterligning ved hjælp af PEI-NP'er i nyrefibrosemus13</str...

Watch this Scientific Journal Video about Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models at JoVE.c

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

Her leverer vi eksogene kunstigt syntetiserede miRNA-efterligninger til nyrerne via haleveneinjektion af en ikke-viral vektor og polyethylenaminnanopartikler i flere nyresygdomsmusemodeller. Dette førte til signifikant overekspression af mål-miRNA i nyrerne, hvilket resulterede i hæmmet progression af nyresygdom i flere musemodeller.

Levering af eksogen kunstigt syntetiseret miRNA-efterligning til nyrerne ved hjælp af polyethylenaminnanopartikler i flere nyresygdomsmusemodeller

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

Denne metode kan hjælpe med at besvare nøglespørgsmål i terapi af mikroRNA til nyrerne, hvilket fortsat er en udfordring. Den største fordel ved denne teknik er, at den har gjort det muligt at behandle mikroRNA-efterligninger til nyrerne uden at forårsage interferonrespons eller genetisk ustabilitet. Begynd med at opløse 10 mikroliter PEI-NP'er i 90 mikroliter 5% glucoseopløsning i et 1,5 milliliter mikrocentrifugerør.Derefter hvirvel røret forsigtigt og drej ned. Bland 50 mikroliter kunstigt syntetiserede miRNA'er opløst i 100 mikromolært nukleasefrit vand med 50 mikromolære 10% glucoseopløsning i 1,5 ml mikrocentrifugerør. Vortex røret forsigtigt og drej ned.Denne proces giver miRNA opløst i 5% glucoseopløsning. Bland derefter 100 mikroliter PEI-NP'er i 5% glucoseopløsning og 100 mikroliter miRNA-efterligning i 5% glucoseopløsning. Vortex røret forsigtigt og drej ned.Inkuber blandingen i 15 minutter ved stuetemperatur for at fremstille et stabilt kompleks af PEI-NP'er miRNA efterligning. Opløs to mikroliter PEI-NP'er i 98 mikroliter 10% glucoseopløsning i et 1,5 ml mikrocentrifugerør. Vortex røret forsigtigt og drej ned.Derefter tilsættes 100 mikroliter Cy3-mærket miRNA-efterligning til 100 mikroliter PEI-NP'er i 10% glucoseopløsning og hvirvel røret forsigtigt. Inkuber blandingen i 15 minutter ved stuetemperatur for at fremstille et stabilt kompleks af PEI-NPs-Cy3-mærket-miRNA-efterligning. Placer UUO-musen med hovedet først i et 50 ml centrifugerør uden bedøvelse, og placer derefter halen gennem et forberedt hul i hætten.Fyld en milliliter sprøjte med en 30-gauge nål med 200 mikroliter PEI-NPs-Cy3-mærket-miRNA-efterligningskompleks og injicer komplekset via musens halevene. Lav derefter et snit i hud, muskler og ribben med kirurgisk saks og tang for at udsætte hjertet. Efter at have lavet et snit i højre atrium, injiceres PBS i venstre ventrikel, indtil nyrefarven ændres til lysegul.Dette indikerer, at hele musekroppen er perfuseret med PBS. Efter opsamling af nyrerne skal du fjerne nyrekapslerne og vaske dem to gange med PBS. Integrer nyrevævsprøverne i OCT-forbindelse og frys dem i flydende nitrogen.Brug en kryostat til at forberede sektioner og montere sektionerne på silanbelagte glasskinner. Fix dem med 4% paraformaldehyd og vask forsigtigt diasene to gange med PBS. Endelig visualiseres fluorescensfarvningen ved fluorescensmikroskopi ved 100x og 400x forstørrelse og passende billedbehandlingssoftware.Det repræsentative billede viser strukturen af PEI-NPs-miRNA-efterligningskomplekset. Levering og virkninger af miRNA-146a-5p-efterligning ved anvendelse af PEI-NP'er i renal fibrose er afbildet i disse billeder. Fluorescerende mikroskopianalyse viste, at haleveneinjektion af PEI-NP'er kunne levere miRNA-efterligningen til det tubulointerstitielle rum.I nyrefibrosemus produceret af UUO omfattede dette renale tubulære celler, som ikke har en regelmæssig form i nyrerne og glomerulus. qRT-PCR-analyse viste, at injektion af PEI-NPs-miRNA-146a-5p-mimic inducerede signifikant overekspression af miRNA-146a-5p i nyrerne. Den histologiske analyse med Sirius røde farvning er vist her.Fluorescerende mikroskopianalyse viste, at haleveneinjektion af PEI-NP'er kunne levere miRNA-efterligning til glomerulus og tubulointerstitiel plads i nyrerne hos diabetiske nyremodelmus in vivo. Derudover viste qRT-PCR-analysen, at injektion af PEI-NPs-miRNA-181b-5p-mimic inducerede signifikant overekspression af miRNA-181b-5p i nyrerne. Histologisk analyse med periodisk syre-Schiff-farvning viste, at injektion af PEI-NPs-miRNA-181b-5p-mimic en gang om ugen fra 10 til 20 uger hæmmede progressionen af diabetisk nyresygdom hos mus.Levering og virkninger af PEI-NPs-miRNA-5100-efterligning i akutte nyreskademodelmus produceret af iskæmi-reperfusionsskade er vist her. Dataene fra fluorescerende mikroskopianalyse qRT-PCR-analyse til evaluering af overekspressionseffekterne af miRNA-5100 efter injektion af PEI-NPs-miRNA-5100-efterligning og den histologiske analyse af hæmatoxylin-eosinfarvet nyre er afbildet i disse billeder. PEI-NP'er injiceret via halevenen to dage før iskæmi-reperfusionsskaden, eller IRI, procedure forhindrer akut nyreskadeprogression induceret af IRI.Når du udfører denne teknik, skal du forberede fem mikrometer tykke sektioner, der indeholder så meget nyrevæv som muligt. Du kan også forberede syv mikrometer tykke sektioner.

Research

JoVE Journal

Medicine

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Spectral karyotyping til Studieinformationen kromosomfejl hos mennesker og mus med polycystisk nyresygdom

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Vurdering af vaskulære funktion hos patienter med kronisk nyresygdom

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

En mulig Zebrafisk Model af polycystisk nyresygdom: Knockdown af<em&gt; Wnt5a</em&gt; Årsager cyster i zebrafisk Nyrer

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Mus nyretransplantation: modeller af allotransplantatafstødning

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Anvendelse af Ultra-high Field MRI i Small gnavermodeller af polycystisk nyresygdom for<em&gt; In vivo</em&gt; Fænotypebestemmelse and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Murine Kidney Transplant Technique

Murine nyretransplantation Teknik

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Normotermisk<em&gt; Ex vivo</em&gt; Nyre Perfusion for Bevarelse af Nyre grafts før Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Intravascular Delivery of Biologics to the Rat Kidney

Intravaskulær Levering af Biologics til rottenyre

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

Vurdering af nyrefunktion i musemodeller af glomerulær sygdom

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...