Name: how to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases
Uploaded: 2016-09-20T08:00:00
Duration: 13 min 18 s
Description: Watch this Scientific Journal Video about How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases at JoVE.com

We thank Simon Hayward (Vanderbilt University Medical Center) for the BPH-1 cells; and Thomas Cox and Janine Erler (Biotech Research &amp; Innovation Centre, University of Copenhagen) for their assistance with rheological measurements. MR-T was funded by Worldwide Cancer Research, formerly The Association of International Cancer Research (Grant 08-0803 to JS), The British Embassy Montevideo and Agencia Nacional de Investigacion e Innovacion (UK_RH_2015_1_2 to MR-T). MC was supported by Prostate Cancer UK (Grant S14-017 to JS and GS). KW was funded by The China Scholarship Council. MAM was funded by The Saudi Arabian Cultural Bureau.

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J., 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=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li><li>Levental, K. R., 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=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li><li>Carson, W. 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S., Hall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+breast+and+prostate+tissues+under+compression.">Elastic moduli of breast and prostate tissues under compression.</a> Ultrason Imaging. 20 (4), 260-274 (1998).</li><li>Zhang, 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=Quantitative+characterization+of+viscoelastic+properties+of+human+prostate+correlated+with+histology.">Quantitative characterization of viscoelastic properties of human prostate correlated with histology.</a> Ultrasound Med Biol. 34 (7), 1033-1042 (2008).</li><li>Corona, G., 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=Benign+prostatic+hyperplasia:+a+new+metabolic+disease+of+the+aging+male+and+its+correlation+with+sexual+dysfunctions.">Benign prostatic hyperplasia: a new metabolic disease of the aging male and its correlation with sexual dysfunctions.</a> Int J Endocrinol. , (2014).</li></ol>

The authors have nothing to disclose.

En protokol til generering af 3D glandulær acini fra MEC&#39;er i rene RBM geler 6 blev ændret i en tidligere undersøgelse ved tilsætning af 4 mg / ml type I collagen til rbM matrix. Tilsætningen af ​​kollagen resulterede i elasticitetsmodulet af rbM gel stigende fra 175 ± 37 til 1589 ± 380 Pa. Dette 9,1-gange stigning i stivheden modulerede væksten, overlevelse, migration og differentiering af MEC&#39;er 21. Protokollen blev ændret igen ved at indbefatte et behandlingstrin med D - (-) - ribose at fremme ikke-enzymatisk tværbinding af type I kollagen, der var blevet tilsat til ringmekanismen gel. Det resulterende 15-gange stigning i stivhed fandtes at samarbejde med oncogen transformation af MEC&#39;er at fremme deres invasive adfærd 22. Den eksperimentelle tilgang tilføjer type I collagen til RBM geler letter direkte interaktion af MEC&#39;er med kollagen fibre, som kun forekommer i humant væv efter fysisk barriere mellem stromaog epithel tilvejebragt af BM undergår proteolytisk nedbrydning. Ved at generere 3D glandulær acini fra PEC i rene RBM geler forbehandlet med GLA, den nuværende protokol åbner vejen at undersøge, hvordan BM stivhed per se kan udløse deres invasive adfærd (figur 3). Niveauerne af BM stivhed induceret i denne protokol har fysiologiske relevans. Inkubering med 50 mM GLA i 6 timer og 14 timer steg henholdsvis de elastiske moduli af det rene rbM gel til 175 ± 90 og 322 ± 160 i forhold til 122 ± 55 Pascal i RBM geler behandlet med PBS (tabel 1). Dette 1,7-3,2 gange stigning i rbM stivhed rekapitulerer den 2,5- til 3,4 gange stigning i stivhed observeret i maligne forhold til normal prostata eller BPH væv 23-26. Som beskrevet i en nylig publikation 13 de morfologiske forandringer som følge af ophobning af AGE og rbM stivhed i PEC acini kan kvantificeres med en statistisk signifikant skift fra arounded til polygonal form, nedsat luminale / total acinære området, og fremspringende celler migrerer fra acina i AGE-rige rbM (figur 3). Immunoblotting kan også anvendes til at vurdere markører for EMT (f.eks tab af E-cadherin 13) og den kontraktile adfærd (fx phosphoryleret myosin let kæde-2, pMLC2 13) i PEC&#39;er dyrket i normalt versus stiv rbM (figur 3). Kan påføres yderligere evaluering ved hjælp immunfluorescensfarvning og konfokal mikroskopi at visualisere BM (f.eks laminin, kollagen IV og AGE ophobning 13), cellulære apikale-til-basal polaritet (f.eks apikal lokalisering af EEA1: tidlig endosomal antigen 1, og GM130: 130 kDa cis-Golgi markør 13) og cellulære mønstre af adhæsionsmolekyler (fx E-cadherin lokalisering til celle-celle vejkryds 13) (figur 3). <img alt = &quot;Figur 3&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig3.jpg&quot; /&gt; Figur 3:. Oversigt over de forskellige protokoller præsenteres her Diagrammet viser hvordan man kan forberede og stivne den rekonstituerede basalmembran (rbM) med glycolaldehyd (Maillard reaktion), hvordan til frø celler på den stive rbM, hvordan man analyserer den stive rbM ( omfanget af Maillard reaktion) og procedurer, der kan bruges til at analysere de cellulære og molekylære forandringer som følge af AGE-rige rbM. AGE, avancerede glykering slutprodukter; BM, basalmembran; DAPI, 4 &#39;, 6-diamidino-2-phenylindol; EEA1, tidlige endosomale antigen 1; GAPDH, glyceraldehyd-3-phosphat dehydrogenase; GLA, glycolaldehyd; CEE, glycin-ethylester; GM130, 130 kDa cis-Golgi markør; p-MLC2 (Thr18 / Ser19), myosin let kæde-2 phosphoryleret i sites threonin 18 og serin 19; rbM, rekonstitueret basalmembran; SDS-PAGE, natriumdodecylsulfat polyacrylamidgelelektroforese. For RWPE1 acini Scale bar = 10 um; for PC3 tumorcellesfæroider Scale bar = 100 um. Dette tal har været ændret siden henvisning 13. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig3large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a> Fejlfindingstrin vil være nødvendigt, hvis D - (-) - ribose vælges som tværbindingsmidlet for rbM. Under protokol udvikling blev det konstateret, at behandling med 1 M D - (-) - ribose i 72 timer, som tidligere beskrevet for rbM / kollagengeler 22, resulterede i dehydrering og krympning af rettighedsbaserede geler. Evalueringen af ​​lavere koncentrationer af D - (-) - ribose og kortere behandlingstider kan bidrage til at overvinde denne begrænsning. En potentiel begrænsning i fremtidige anvendelser af protokollen kunne opstå, hvis der ønskes højere niveauer af rbM stivhed. Hvis der anvendes længere inkubationstider og højere koncentrationer af GLA at inducere højere niveauer af rbM gel stivhed vil det være nødvendigt at<html> vurdere, om disse behandlingsmuligheder betingelser har en indvirkning på celle overlevelse og spredning, som tidligere beskrevet 13. Det skal også bemærkes, at inkubation af RWPE-1-celler med serum inducerer en fænotypisk EMT-lignende overgang og udsættelse for serum eller serum-holdige materialer bør undgås. For eksempel, hvis eksperimenter involverer transfektion af lille interfererende RNA (siRNA) oligonukleotider, proceduren bør optimeres ved anvendelse RWPE-1 celler dyrket i KSFM, uden at skifte cellerne til lavt serum transfektion medier. Denne ulempe kan være til skade for gen lyddæmpning opnås ved brug af forbigående siRNA tilgange i modellen. For nogle protein-targets ville det rådes til at ansætte inducerbare shRNA vektorer for justerbar gen lyddæmpning og det ønskede fald i protein niveauer. Tilpasninger som omfatter enzymatisk tværbinding ved stromal celle eller tumorcelle associeret lysyloxidase (LOX) 17 kunne også inkorporeres i fremtidige modeller. jove_content &quot;&gt; Denne protokol vil lette den fremtidige undersøgelse af pro-invasive mekanismer udløst af AGE-afhængige BM stivhed i Pecs (RWPE-1, BPH-1) og evaluering af anti-metastatiske mål i invasive PTC&#39;er (PC3). Da BPH anses for at være en stofskiftesygdom 27, denne protokol baner også vejen mod vores bedre forståelse af sammenhængen mellem metaboliske sygdomme og øget prostatakræft risiko. i betragtning af, at BM stivhed fremkaldt af sin eksponering mod AGEs kan være en udløsende faktor for invasiv i andre kræft typer, vil det være af interesse at anvende protokollen til at oprette lignende modeller, der omfatter normale epitelceller og tumorceller fra andre organer (f.eks bryst, tyktarm, æggestokke, bugspytkirtel). Kritiske trin i protokollen sammen med deres tider, er opsummeret i figur 4. Under den indledende skridt er det vigtigt at opretholde stamopløsningen af rbM ved 4 ° C, mens det smelter for at forhindre sin polymerization. Pipettespidser bør ikke placeres i rM stamopløsningen indtil de er kølet til 4 ° C. For det næste trin er det også vigtigt at sikre de kammerskiver har ækvilibreret til 4 ° C, før de overtrækkes med rbM opløsning. det vil så snart temperaturen af ​​rbM opløsningen forøges over 4 ° C undergår irreversibel polymerisation til dannelse af en gel. Det vigtigt, at rbM ikke forstyrres under polymerisationen fase for at sikre, at den danner en jævn overflade egnet til celledyrkning og mikroskopisk analyse. Varigheden af ​​inkubationen med GLA med eller uden inhibitorer af Maillard-reaktionen (natriumcyanoborhydrid og amingoguanidine) bestemmer, hvor stiv RBM gelen bliver. Det anbefales at bruge en 6 timers inkubation med GLA hvis halvstift betingelser er påkrævet, og 14 timers inkubation, hvis stive betingelser er påkrævet (tabel 1). Alternative inkuberingstider eller koncentrationer af GLA kan anvendes, hvis forskellige niveaueraf stivhed er ønsket. I dette tilfælde rheologisk analyse af RBM geler skal indgå som et væsentligt skridt. Efter trinnet til standsning af Maillard-reaktionen ved inkubering med GEE og de efterfølgende vasketrin med PBS, kan ringmekanismen geler anvendes straks eller opbevares ved 4 ° C i op til 48 timer før deres anvendelse til cellekultur. Når cellekulturer er sat op er det vigtigt at ændre dyrkningsmediet (herunder eventuelle behandlinger) hver anden dag. Det anbefales at bevare 3D cellekulturer til 3-12 dage efter de parametre, der undersøges. For 3D PEC acini anbefales det at analysere de kulturer efter 6 dage, og 3D PTC sphæroider analyse anbefales efter 3 dages dyrkning i første instans. <img alt="Figur 4" src="/files/ftp_upload/54230/54230fig4.jpg" /> Figur 4:. Enkel Oversigt over protokollen med kritiske stadier og Timings angivet flow diagram viser hvordan man kan forberede og stivne den rekonstituerede basalmembran (rbM) med glycolaldehyd (Maillard reaktion) med kritiske trin og tider, der er angivet. Punkter, hvor protokollen kan stoppes, og RBM geler gemt, er også angivet. rbM, rekonstitueret basalmembran; GLA, glycolaldehyd; CEE, glycin-ethylester; ON, natten over; PBS, phosphatpufret saltopløsning; RT, stuetemperatur. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig4large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a>

The basement membrane (BM) is a sheet of specialized extracellular matrix (ECM) that maintains stable tissue borders by separating layers of epithelial cells from the stroma1. Covalent crosslinking between adjacent triple helices of collagen IV in the BM stabilizes their lateral association by establishing an irregular network of super-twisted helices2. These collagen IV lattices act as a scaffold for its interaction with laminin and other BM components1. The structural arrangement of the BM provides it with the mechanical strength and rigidity necessary for the normal development of glandular epithelia3. 
During aging and disease the BM progressively thickens and stiffens3,4. For example, a 3-fold increase in the elastic modulus (E) of the ocular BM occurs between the ages of 50 and 80 in the normal population, and this stiffening is further exacerbated in metabolic disorders like diabetes5. The structural and biomechanical changes in the BM that result in its increased stiffness occur when its ECM components, collagen IV and laminin, become non-enzymatically crosslinked following their exposure to advanced glycation endproducts (AGEs).
The purpose of the method described here was to establish a model for the investigation of how BM stiffness, due to AGE exposure, promotes prostate epithelial cell (PEC) and prostate tumour cell (PTC) invasiveness in the context of the switch to metastatic prostate cancer (PCa). To do this a previous method used for generating 3D glandular acini from mammary epithelial cells (MECs) in reconstituted rBM gels6 was adapted to include an additional step where the rBM gels are pre-treated with glycolaldehyde (GLA). Several techniques for assessing GLA induced crosslinking and stiffening of pre-treated rBM gels are described, including photometric analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), confocal microscopy and rheometric analysis. The prostate cell types selected for culture on the pre-stiffened rBM include: RWPE-1, PECs derived from a normal prostate gland7; BPH-1, PECs derived from a prostate gland affected by BPH8; and PC3, metastatic PTCs derived from a secondary tumor located in the vertebral bone of a prostate cancer (PCa) patient9. 
In addition to advancing the study of prostate gland pathology, the protocol for stiffening of rBM gels by their treatment with GLA can be adapted to investigate how BM stiffness contributes to other age-related pathologies and metabolic disorders. For example, the model can be directly applied to investigate how metastatic cancer is induced by BM stiffness in organs such as the breast, colon, ovary and pancreas by the incorporation of appropriate cell types. Furthermore, the protocol can be adapted to investigate how stiff BM promotes biomechanical mechanisms of disease progression in diabetes-related microvascular disease, retinopathy, nephropathy and neuropathy.

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			<td height="21" style="height:21px;width:628px;">I - Material for monolayer culture</td>
			<td style="width:265px;"></td>
			<td style="width:139px;"></td>
			<td style="width:401px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BPH-1</td>
			<td>CaP Cell Line Database</td>
			<td>PCaCL-132</td>
			<td>Contact: simon.hayward@mcmail.vanderbilt.edu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Complete keratinocyte serum-free media&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>17005-075</td>
			<td>Do not warm at 37 &#186;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum&#160;</td>
			<td>First Link UK Ltd</td>
			<td>02-00-850</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC3&#160;</td>
			<td>American Type Culture Collection</td>
			<td>CRL-1435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (Dulbecco A) tablets</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R5886</td>
			<td>warm in 37 &#186;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RWPE-1&#160;</td>
			<td>American Type Culture Collection</td>
			<td>CRL-11609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">II - Material for 3D culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminoguanidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>396494</td>
			<td>Irritating to eyes, respiratory system and skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chamber slides, 8-well&#160;</td>
			<td>Thermo Scientific Nunc Lab-Tek</td>
			<td>TKT-210-816M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Matrix reconstituted basement membrane (rBM) reduced growth factor extract&#160;</td>
			<td>AMS Biotechnology</td>
			<td>3445-005-01</td>
			<td>Store Basement membrane (BM) at -80 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyanogen bromide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C91492</td>
			<td>Toxic by contact skin and inhalation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>695076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine ethyl ester hydrochloride (GEE)</td>
			<td>Sigma-Aldrich</td>
			<td>50060</td>
			<td>Irritating to eyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycolaldehyde dimer (GLA)</td>
			<td>Sigma-Aldrich</td>
			<td>G6805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium cyanoborohydride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>71435</td>
			<td>Highly flammable; Toxic by contact skin and inhalation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filter 0.22 microns</td>
			<td>Appleton Woods</td>
			<td>BC680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">III - Material to quantify Maillard reaction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>ThemoFisher Scientific</td>
			<td>D3571</td>
			<td>light sensitive and store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cloning cylinders</td>
			<td>Sigma-Aldrich</td>
			<td>C1059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td>
			<td>ThemoFisher Scientific</td>
			<td>A-11001</td>
			<td>light sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate</td>
			<td>ThemoFisher Scientific</td>
			<td>A-11034</td>
			<td>light sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum</td>
			<td>Abcam</td>
			<td>ab7481</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting media&#160;</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-pentosidine clone PEN-12 mAb</td>
			<td>TransGenic Inc</td>
			<td>KH012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-human collagen IV polyclonal antibody&#160;&#160;</td>
			<td>Acris Antibodies</td>
			<td>R1041</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-laminin A/C pAb</td>
			<td>Santa Cruz Biotechnology Inc</td>
			<td>sc-7292</td>
			<td>Store at -20 &#186;C in aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol tert-octylphenyl ether (Triton-X100)</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol sorbitan monolaurate (Tween-20)</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis cassette Slide-A-Lyzer</td>
			<td>ThemoFisher Scientific</td>
			<td>66333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IV - Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ARG2 controlled strain rotational rheometer</td>
			<td>T.A. Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axiovert S100 (20x magnification) microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 controlled humidified incubation chamber for Zeiss Axio S100 microscope</td>
			<td>Solent Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Axiovert 200M (40x, 63x magnification) microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus LH50A microscope fitted with a digital camera using phase-contrast&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PHERAstar Plus plate reader spectrophotometer</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">V - Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image J 1.47v</td>
			<td>National Institute of Health, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MetaXpress</td>
			<td>Molecular Divices</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

how to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

Her beskriver vi en protokol, som kan anvendes til at studere den biofysiske mikromiljø relateret til øget tykkelse og stivhed af basalmembranen (BM) under aldersrelaterede patologier og metaboliske lidelser (fx cancer, diabetes, mikrovaskulær sygdomstilstande, retinopati, nefropati og neuropati) . Udgangspunktet for modellen er ikke-enzymatisk krydsbinding af rekonstitueret BM (rbM) matrix ved behandling med glycolaldehyd (GLA) for at fremme avanceret glykering slutprodukt (AGE) generation via Maillard reaktion. Eksempler på laboratoriemetoder, der kan anvendes til at bekræfte AGE generation, ikke-enzymatisk tværbinding og øget stivhed i GLA behandlet rbM er skitseret. Disse omfatter fremstilling af nativt rbM (behandlet med phosphatbufret saltvand, PBS) og stiv rbM (behandlet med GLA) til bestemmelse af: dens alder indhold ved fotometrisk analyse og immunofluorescens-mikroskopi, dens ikke-enzymatisk tværbinding ved natriumdodecylsulfat polyacrylamidgelelektroforese (SDS PAGE) samt konfokal mikroskopi, og dets øgede stivhed ved hjælp rheometri. Den her beskrevne fremgangsmåde kan anvendes til at forøge stivheden (elasticitetsmoduler, E) på rbM op til 3,2 gange, i overensstemmelse med målinger foretaget i rask versus sygdomsramt humant prostatavæv. For at genskabe den biofysiske mikromiljø forbundet med den aldrende og syge prostata tre prostata celletyper blev introduceret på indfødte rbM og stiv rbM: RWPE-1, prostata epitelceller (PEC) afledt af en normal prostata; BPH-1, PEC&#39;er afledt af en prostata ramt af benign prostatahyperplasi (BPH); og PC3, metastatiske celler afledt af en sekundær knogle tumor stammer fra prostatacancer. Flere parametre kan måles, herunder størrelse, form og invasive egenskaber af 3D glandulær acini dannet af RWPE-1 og BPH-1 på nativ versus stiv rbM og gennemsnitlig celle længde, vandrende hastighed og persistens af celle bevægelse af 3D Sphereoids dannet af PC3 celler under de samme betingelser. Celle signalveje og den subcellulære lokalisering af proteiner kan også vurderes.

3D Prostata acini dyrket på Stiff rbM Efter 6 dage i kultur, PEC&#39;er afledt fra normale prostatavæv (RWPE-1) (figur 1A) og BPH tissue (BPH-1) (figur 1B) formular acini på native (PBS-behandlede) rbM der er organiseret i ensartede sfæroider af epitel celler. Disse acini har også karakter af højt organiserede PEC&#39;er med apikal-til-basal polaritet og en synlig luminale rum 13,20. Den acini dannet af PEC&#39;er afledt fra normale prostatavæv (RWPE-1) (figur 1A) og BPH tissue (BPH-1) (figur 1B) på stivnede (AGE-rige) rbM (behandlet med GLA) har et afbrudt arkitektur (skiftende fra kugleformet til polygonal i form og celler fremspringende / migrering fra acini i AGE-rige rbM) (figur 1A). Disse<html> acini er også karakteriseret ved meget uorganiserede PEC, der har mistet deres apikale-til-basal polaritet med en lille eller ikke-eksisterende luminale rum 13. <img alt="figur 1" src="/files/ftp_upload/54230/54230fig1.jpg" /> Figur 1:. Prostata epitelceller dyrkes som 3D Glandulær acini på Native og Stiv Rekonstitueret Basement Membrane (rbM) (A) lysfelt billeder af RWPE-1 celler dyrket i 12 timer op til 6 dage på RBM geler behandlet med PBS (native) eller 50 mM glycolaldehyd for 14 timer (AGE-rige, stiv); Scale bar = 50 um. (B) BPH-1-celler, dyrket som beskrevet i panel A; Scale barer = 50 um; data er repræsentative for 3 uafhængige forsøg. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a> <html> Tabel 1 &quot;src =&quot; / files / ftp_upload / 54230 / 54230table1.jpg &quot;/&gt; Tabel 1:. Karakteristik af prostata epithelial RWPE-1 acini dyrket på Native, Semi-Stiff og Stiff Rekonstitueret Basement Membrane (rbM) RWPE-1 acini blev dyrket på rbM forbehandlet med PBS for 14 timer (native), glycolaldehyd (GLA ) i 6 timer (semi-stiv) eller GLA for 14 timer (stiv). For acinar form, den procentvise (%) ± standardafvigelse (SD) af runde blev semi-polygonale og polygonale acini beregnet ud fra 5 uafhængige forsøg (50 acini kvantificeret pr betingelse). Relativ acinar størrelse blev beregnet (native rbM = 100%) fra 3 uafhængige forsøg. For invasivitet, blev% ± SD acini med en eller flere udragende celler beregnet ud fra 3 uafhængige eksperimenter. Fold ændring beregnes ved at dividere den gennemsnitlige værdi opnået under semi-stive eller stive forhold ved den tilsvarende værdi for native betingelser. P-værdier beregnet ved anvendelse af Students t-test (α = 0,05). Indholdsproduktion &quot;fo: holde-together.within-side =&quot; 1 &quot;&gt; ALDER afhængig øget rbM stivhed fremmer PC3 prostata tumor celle migration PC3 celler dyrket på indfødte rbM migrere ved at opretholde kontinuerlig celle-celle kontakt, mens PC3 celler dyrket på AGE-rige (stiv) rbM flytte uafhængigt af hinanden (figur 2A). Efter 72 timer i kultur PC3 celler danner foci (sphæroider) på native (PBS behandlede) rbM, mens PC3 celler på stiv (AGE-rige) rbM ikke fra sfæroider og migrere uafhængigt (figur 2B). PC3 celler på stiv (AGE-rige) rbM er mere langstrakt end PC3 celler dyrket på indfødte rbM (Figur 2C). PC3 celler på stiv rbM migrere hurtigere end PC3 celler dyrket på indfødte rbM (figur 2D). PC3 celler på stiv rbM vise et fald i vedholdenhed i forhold til PC3 celler dyrket på indfødte rbM (Figur 2E). <img alt = &quot;Figur 2&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig2.jpg&quot; /&gt; Figur 2:. Prostata Tumor Cell Migration på Native og Stiv Rekonstitueret Basement Membrane (rbM) (A) lysfelt billeder af PC3 celler dyrket på RBM geler behandlet med PBS (native) eller 50 mM glycolaldehyd for 14 timer (AGE-rige, stiv) . Celler blev afbildet ved hjælp af et lysfelt mikroskop (10X objektiv), og et opkøb på 1 billede pr time for 12 timer efterfulgt af celle sporing at generere baner. Viste billeder svarer til de tidspunkter efter 0, 3, 6, 9 og 12 timer. Baner enkeltceller er vist for 12 hr tidspunkt. Scale bar = 100 um. (B) PC3-celler dyrket på nativt eller stiv rbM i 72 timer, og afbildes som beskrevet i panel (A). Scale bar = 100 um. Detalje viser valgte område ved 2X forstørrelse. (C) gennemsnit ± SD celle længde (um); signifikant forskel mellem nativt rbM og stiv rbM (p = 1,2 x 10 -23). ( <strong&gt; D) Middel ± SD hastighed (um / time) beregnet ud fra celle baner; signifikant forskel mellem nativt rbM og stiv rbM (p = 0,004). (E) gennemsnit ± SD persistens af cellebevægelse (forhold D / T, hvor D = afstand fra start til afslutning celle bane, T = samlet længde på celle bane); signifikant forskel mellem nativt rbM og stiv rbM (p = 0,0007). For paneler CE&gt; 10 celler blev analyseret, data er repræsentativt for 3 uafhængige forsøg. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig2large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a>

Watch this Scientific Journal Video about How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases at JoVE.com

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Her forklarer vi en protokol til modellering af biofysiske mikromiljø hvor krydsbinding og øget stivhed af basalmembranen (BM) induceret af avancerede glykering slutprodukter (alder) har patologisk relevans.

Sådan Undersøgelse Basement Membrane stivhed som en biofysisk Trigger i prostatakræft og andre aldersrelaterede patologier eller metaboliske sygdomme

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

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