Name: fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro
Uploaded: 2018-06-20T12:30:00
Description: Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Forfatterne anerkender støtten fra forskningstilskud fra National Natural Science Foundation of China (31371390), programmet for staten High-Tech Development Project (2014AA020702) og programmet for Guangdong videnskab og teknologi (2016B030231001).

Alle dyr arbejde blev udført i overensstemmelse med animal welfare act af institutionelle retningslinjer og godkendt af institutionelle Animal Care og brug udvalget, Sun Yat-sen Universitet.
1. forberedelse af TEM
<ol><li>Udføre mus af cervikal dislokation og fjerne tunger ved hjælp af steril kirurgiske saks og pincet.</li>
	<li>Fordyb tunger i 75% ethanol i 3 min og derefter sætte hver tungen i en 1,5 mL Eppendorf (EP) rør med 1 mL af 10 mM sterile phosphat bufferet løsning (PBS). ...

<ol>
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Oncol. 29, 1488-1494 (2011).</li><li>Zhao, L., Huang, L., Yu, S., Zheng, J., Wang, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+tongue+tissue+as+an+in+vitro.+model+for+studying+tongue+cancer+and+tongue+regeneration.">Decellularized tongue tissue as an in vitro. model for studying tongue cancer and tongue regeneration.</a> Acta Biomaterialia. 58, 122-135 (2017).</li><li>Ng, S. L., Narayanan, K., Gao, S., Wan, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restricted+progenitors+for+the+repopulation+of+decellularized+heart.">Lineage restricted progenitors for the repopulation of decellularized heart.</a> Biomaterials. 32, 7571-7580 (2011).</li><li>Ott, H. 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En veletableret protokol for decellularized ECM fabrikation bør bevare de indfødte ECM sammensætning mens fjernelse af cellulære komponenter i væv næsten helt21. Trods i øjeblikket rapporteret decellularization protokoller, som kræver perfusion gennem Vaskulaturen fjerne materialer ved Konvektiv transport, blev mekanisk agitation vedtaget her, kendt som en traditionel enkel og billig metode22 , 23 , 24<...

Tungen har forskellige vigtige funktioner, såsom deglutition, artikulation og smagning. Således har værdiforringelse af tungen funktion stor indvirkning på patienternes livskvalitet1. Den mest almindelige malignitet i mundhulen er tungen pladecellekræft (TSCC), som normalt forekommer i mennesker, der drikker alkohol eller ryge tobak2.
I de seneste år er der sket små fremskridt i grundforskning på TSCC. Manglen på effektive in vitro- forskning modeller er fortsat at være en af de største problemer. Således, den ekstracellulære matrix (ECM) viser sig for at være en mulig løsning. Eftersom ECM er et komplekst netværk ramme sammensat af velorganiseret matrix komponenter, være stillads materialer med en ECM-lignende struktur og sammensætning kompetente til kræftforskning. Decellularized ECM kan perfekt niche celler fra samme oprindelse i vitro, som viser sig for at være den mest markante fordel af ECM.
ECM kan opbevares med cellulære komponenter fjernes fra væv gennem decellularization ved hjælp af vaske-og rengøringsmidler og enzymer. Forskellige ECM komponenter, herunder kollagen, fibronektin, og laminin i decellularized matrix giver en indfødt-væv-lignende mikromiljø for dyrkede celler, fremmer overlevelse, spredning og differentiering af celler3. Derudover kan immunogenicitet til transplantation blive reduceret til et minimalt niveau med manglen på cellulære komponenter i ECM.
Hidtil, har fabrikation metoder for decellularized ECM været prøvet i forskellige væv og organer, såsom hjerte4,5,6,7, leveren8,9,10 ,11, lunge12,13,14,15,16,17, og nyre18,19 , 20. imidlertid ingen relevant forskning konstateret på lignende arbejde i tungen til bedste af vores viden.
I denne undersøgelse, blev decellularized tunge ekstracellulære matrix (TEM) fremstillet både effektivt og billigt af en række fysiske, kemiske og enzymatisk behandling. Derefter blev TEM brugt til at sammenfatte TSCC i vitro, viser en passende simulation for TSCC adfærd og udvikling. TEM har god biokompatibilitet samt evnen til at guide cellerne til væv-specifik niche, som angiver, at TEM kan have stort potentiale i TSCC forskning3. Den protokol, der er vist her giver et valg for forskere studerer på enten patogenese eller kliniske behandlinger af TSCC.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57-BL/6J mice</td>
			<td style="width:292px;">Sun Yat-sen University Laboratory Animal Center</td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">HB15-GR-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A100395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dibasic Sodium Phosphate</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">BE14-GR-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic&#160;</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL EP tube</td>
			<td style="width:292px;">Axygen</td>
			<td style="width:119px;">MCT-150-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-low temperature freezer&#160;</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3.5 cm cell culture dish</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">153066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 cm cell culture dish</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">V900502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">449164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium sulphate</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A601988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">158968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">PHR1487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical suture</td>
			<td style="width:292px;">Shanghai Jinhuan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 mL wide-mouth bottle</td>
			<td style="width:292px;">SHUNIU</td>
			<td style="width:119px;">1407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stirrer</td>
			<td style="width:292px;">AS ONE</td>
			<td style="width:119px;">1-4602-32</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">CO2 incubator</td>
			<td style="width:292px;">SHEL LAB</td>
			<td style="width:119px;">SCO5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL syringe</td>
			<td style="width:292px;">Hunan Pingan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL centrifuge tube</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">227270</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cal27 cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Tongue squamous cell carcinoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">U2OS cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes free acid</td>
			<td style="width:292px;">BBI</td>
			<td style="width:119px;">A600264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:292px;">Hyclone</td>
			<td style="width:119px;">SH30084.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4 &#176;C fridge</td>
			<td style="width:292px;">Haier</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water purifier</td>
			<td style="width:292px;">ELGA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
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fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

For at konstruere en effektiv og realistisk model for tunge planocellulært karcinom (TSCC) in vitro-blev metoder skabt til at producere decellularized tungen ekstracellulære matrix (TEM) der giver funktionelle stilladser for TSCC byggeri. TEM giver en in vitro- niche for cellevækst, differentiering og celle migration. Mikrostrukturer indfødte ekstracellulære matrix (ECM) og biokemiske kompositioner bevaret i matrixen decellularized indeholder væv-specifikke nicher for forankring celler. Fabrikation af TEM kan realiseres af deoxyribonuclease (DNase) fordøjelsen ledsaget med en alvorlig af organiske eller uorganiske forbehandling. Denne protokol er nem at betjene og sikrer høj effektivitet for decellularization. TEM viste positiv cytocompatibility for TSCC celler under statisk eller omrørt dyrkningsbetingelser, som giver mulighed for opførelse af TSCC model. En self-made bioreaktor blev også brugt til den vedvarende stirred betingelse for cellekultur. Rekonstrueret TSCC bruge TEM viste egenskaber der minder om kliniske TSCC histopatologi, tyder på potentialet i TSCC forskning.

Denne protokol for forberedelse af TEM viser sig for at være effektive og hensigtsmæssige. TEM viste perfekt decellularization sammenlignet med modersmål væv. Effekten af decellularization blev bekræftet af hæmatoxylin-eosin (han) farvning (figur 1A-B). HAN farvning resultaterne viste fuldstændig forsvundet nukleart farvning i TEM (figur 1B). DNA indhold kvantificering fra tidligere arbejd...

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Fabrikation af tungen ekstracellulære Matrix og rekonstituering af tungen planocellulært karcinom In Vitro

En metode er vist her til forberedelse af tungen ekstracellulære matrix (TEM) med effektiv decellularization. TEM kan bruges som funktionelle stilladser til genopbygningen af et tungen planocellulært karcinom (TSCC) model statisk eller omrørt kultur betingelser.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Research

JoVE Journal

Cancer Research

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

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