Name: Door geneesmiddelen geïnduceerde senescentie in levercellen bevordert polarisatie van M2-macrofagen: implicaties voor tyrosinekinaseremmer-geassocieerde hepatotoxiciteit (Video) | JoVE Journal
Uploaded: 2025-10-17T13:10:00.000+00:00
Duration: 9 min 32 s
Description: Ontdek hoe tyrosinekinaseremmers senescentie in levercellen veroorzaken en M2-macrofaagpolarisatie bevorderen via SASP. Deze methode onthult mechanismen van geneesmiddel-geïnduceerde hepatotoxiciteit.

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<table><tbody><tr><td>HepG2 cell line</td><td>NCCS</td><td></td><td>Adherent cell line</td></tr><tr><td>RAW264.7 cell line</td><td>NCCS</td><td></td><td>Adherent cell line</td></tr><tr><td>DMEM (Dulbacco's Modified Eagle Medium)</td><td>Himedia</td><td>AT066</td><td>Hoge glucose, Poeder</td></tr><tr><td>MEM (Modified Eagle Medium</td><td>Gibco</td><td>611100-053</td><td>Poeder</td></tr><tr><td>PBS (Phosphate Buffer Saline)</td><td>Himedia</td><td>TS1101</td><td>Poeder</td></tr><tr><td>Trypsin-EDTA Solution</td><td>Himedia</td><td>TCL007</td><td>Oplossing</td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Himedia</td><td>RM1112</td><td>Vloeistof</td></tr><tr><td>MTT Dye</td><td>Himedia</td><td>TC191</td><td>Poeder</td></tr><tr><td>Lapatinib</td><td>Selleckchem</td><td>S2111</td><td>Poeder</td></tr><tr><td>Neratinib</td><td>Cayman Chemical Company</td><td>18404</td><td>Poeder</td></tr><tr><td>DMSO (Dimethyl Sulfoxide)&nbsp;</td><td>Himedia</td><td>TC185</td><td>Oplossing</td></tr><tr><td>Formaldehyde&nbsp;</td><td>Rankem</td><td>F0080</td><td>Oplossing</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-aldrich</td><td>340855</td><td>Oplossing</td></tr><tr><td>Citric Acid monohydrate, Pure</td><td>Himedia</td><td>GRM229</td><td>Poeder</td></tr><tr><td>Sodium phosphate dibasic anhydrous&nbsp;</td><td>Himedia</td><td>MB024</td><td>Poeder</td></tr><tr><td>Potassium ferrocyanide, K4[Fe(CN)6]</td><td>Fisher Scientific&nbsp;</td><td>P-236-500</td><td></td></tr><tr><td>Potassium ferricyanide, K3[Fe(CN)6]</td><td>Fisher Scientific&nbsp;</td><td>P-232-500</td><td></td></tr><tr><td>NaCl</td><td>SRL</td><td>41721</td><td>Poeder</td></tr><tr><td>MgCl2 hexahydrate</td><td>Himedia</td><td>GRM-1068</td><td>Poeder</td></tr><tr><td>X-Gal</td><td>SRL</td><td>45904</td><td>Poeder</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma-aldrich</td><td>L6529</td><td></td></tr><tr><td>Human Recombinant Interferon-&gamma;</td><td>Himedia</td><td>CF040-20MCG</td><td></td></tr><tr><td>Cell ROX deep red reagent</td><td>Thermo fisher scientific</td><td>C10422</td><td></td></tr><tr><td>Hoechst</td><td>Invitrogen, Thermo Fisher Scientific</td><td>H3570</td><td>Poeder</td></tr><tr><td>MitoTRACKER Green</td><td>Invitrogen, Thermo Fisher Scientific</td><td>1842298</td><td>Poeder</td></tr><tr><td>Anti-ARG1 antibody</td><td>Affinity</td><td>DF6657</td><td>Oplossing, Berekend MW- 35kDa, Waarneembaar MW - 65kDa</td></tr><tr><td>Anti-NOS2 antibody</td><td>Cloudo Clone Corp.</td><td>PAA837Hu01</td><td>Oplossing, Berekend MW- 130kDa, Waarneembaar MW - 65kDa</td></tr><tr><td>Anti-iNOS antibody</td><td>Affinity</td><td></td><td>Oplossing, Berekend MW- 130kDa, Waarneembaar MW - 120kDa</td></tr><tr><td>Anti-GAPDH antibody</td><td>Biobharati LifeScience Pvt.Ltd</td><td>BB-AB0060</td><td>Oplossing</td></tr><tr><td>Anti-Rabbit Antibody</td><td>Cayman Chemical Company</td><td>10004301</td><td>Oplossing</td></tr><tr><td>Nitrocellulose membrane&nbsp;</td><td>Merck Millipore Ltd.&nbsp;</td><td>HATF00010 (Productnummer)</td><td></td></tr><tr><td>femtoLUCENTTMPLUS HRP</td><td>G Bioscience&nbsp;</td><td>786-003</td><td></td></tr><tr><td>60mm Dishes</td><td>NUCK</td><td>150462</td><td></td></tr><tr><td>96well plate,&nbsp;</td><td>NUCK</td><td>165306</td><td></td></tr><tr><td>Cell Culture Incubator&nbsp;</td><td>Thermo Fisher Scientific</td><td>371</td><td></td></tr><tr><td>Microscope&nbsp;</td><td>1. LEICA Inverted Microscope&nbsp;&nbsp; 2. Nikon Confocal Microscope&nbsp;</td><td>1. DMIL LED&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2. Ti2E, A1R</td><td>&nbsp;Beiden zijn modelnummer</td></tr><tr><td>iBright Imaging System</td><td>Invitrogen, Thermo Fisher Scientific</td><td>CL1500 (modelnummer)</td><td></td></tr><tr><td>Plate-reader</td><td>Bio-RAD</td><td>iMark Microplate Reader</td><td></td></tr><tr><td>Centrifuge&nbsp;</td><td>Thermo Fisher Scientific</td><td>Sorvall ST 8R (modelnummer)</td><td></td></tr></tbody></table>

door geneesmiddelen geïnduceerde senescentie in levercellen bevordert polarisatie van m2-macrofagen: implicaties voor tyrosinekinaseremmer-geassocieerde hepatotoxiciteit

Drug-Induced Senescence in Liver Cells Promotes M2 Macrophage Polarization: Implications for Tyrosine Kinase Inhibitor-Associated Hepatotoxicity

Door geneesmiddelen geïnduceerde senescentie in levercellen bevordert polarisatie van M2-macrofagen: implicaties voor tyrosinekinaseremmer-geassocieerde hepatotoxiciteit

Lapatinib and neratinib are tyrosine kinase inhibitors (TKIs) approved for treating HER2-positive metastatic breast cancer, but their clinical use is ...

drug-induced-senescence-liver-cells-promotes-m2-macrophage

Research

JoVE Journal

Cancer Research

Kankeronderzoek

Cellular veroudering, de onomkeerbare toestand van de celcyclus, kan worden geïnduceerd door verschillende cellulaire stress. Hier beschrijven we protocollen bij cellulaire veroudering en methoden ertoe te brengen markers van veroudering te beoordelen.

Technieken voor het induceren en te kwantificeren Cellulaire senescentie

M2-achtige tumor-geassocieerde macrofagen (TAM) zijn geassocieerd met tumorprogressie en slechte prognose bij kanker. Dit protocol dient als een gedetailleerde gids om THP-1 monocytachtige cellen binnen 14 dagen reproduceerbaar te differentiëren en te polariseren in M2-achtige macrofagen. Dit model is de basis om de ontstekingsremmende effecten van TAM in de tumormicro-omgeving te onderzoeken.

Macrofaagdifferentiatie en polarisatie in een M2-achtig fenotype met behulp van een menselijke monocytachtige THP-1 leukemie cellijn

Hier presenteren we een op flowcytometrie gebaseerde methode voor visualisatie en kwantificering van meerdere senescentie-geassocieerde markers in enkele cellen.

Gelijktijdige beeldvorming en flowcytometrie-gebaseerde detectie van meerdere fluorescerende senescentiemarkers in therapie-geïnduceerde senescente kankercellen

Dit protocol biedt gereflecteerd licht confocale microscopie voor objectieve, enkelceldetectie van senescence-geassocieerde bèta-galactosidase (SA-β-gal) activiteit in de stromale vasculaire fractie die is geïsoleerd uit menselijk vetweefsel. De methode is compatibel met immunocytochemie, bevat pH-controles voor kwantitatieve beoordeling en biedt bovendien een gevoeliger en minder subjectief alternatief voor conventionele SA-β-gal kleuring.

Senescentiedetectie met behulp van gereflecteerd licht in de vetachtige stromale vaatfractie

Deze methode bestudeert hoe senescente huidfibroblasten gezonde fibroblasten beïnvloeden. Senescentie wordt geïnduceerd door UVB-blootstelling, en het geconditioneerde medium wordt toegepast op normale fibroblasten. Het helpt bij het evalueren van bioactieve verbindingen die gericht zijn op veranderde cel-tot-cel communicatie bij huidveroudering.

Nieuwe combinatie van antioxidanten om gezonde intercellulaire communicatie te herstellen in een in vitro model met behulp van een geconditioneerd medium uit senescente cellen

Het begrijpen van het kwaadaardige gedrag van kanker vereist het creëren van nauwkeurige modellen van hoe tumorcellen interageren met componenten van de tumormicro-omgeving, zoals macrofagen. Hier beschrijven we twee methoden om de interactie van glioblastoomcellen met tumorgeassocieerde macrofagen en microglia te bestuderen, waarbij het effect op glioblastoominvasie wordt beoordeeld.

Coculture Testen om te studeren macrofagen en microglia Stimulatie van glioblastoma Invasion

Hier presenteren we een protocol voor antilichaam arrays om te identificeren van de wijzigingen in de signaalroutes in verschillende cellulaire modellen. Deze veranderingen, veroorzaakt door drugs/hypoxie/ultra-violet licht/straling, of overexpressie/Downregulatie/uitsparingen, zijn belangrijk voor verschillende modellen van de ziekte en kunnen aangeven of een therapie effect sorteren zullen of mechanismen van drugs kan identificeren weerstand.

Beoordeling van weerstand tegen Tyrosine Kinase remmers door een ondervraging van signaaltransductie trajecten door antilichaam Arrays

Dit artikel is een nuttige in vitro assay om het vermogen van geconditioneerd medium van tumorcellen te evalueren om macrofagen aan te trekken.

In vitro assay om tumor-macrofaag interactie te bestuderen

Deze studie beschrijft een methode om exosoom-verrijkte extracellulaire blaasjes te isoleren die immuunstimulerende granulocyten macrofaagkolonie-stimulerende factoren dragen uit embryonale stamcellen.

Isolatie van exosoom-verrijkte extracellulaire blaasjes met granulocyt-macrofaag koloniestimulerende factor uit embryonale stamcellen

Macrofaag fagocytische activiteit tegen kankercellen, in het bijzonder 4T1 muis borstklier carcinoom cellen, werd afgebeeld in deze studie. Het cocultuurmodel van de levende cellen werd vastgesteld en waargenomen met behulp van een combinatie van fluorescerende en differentiële interferentie contrast microscopie. Deze beoordeling is gemaakt met behulp van imaging software om multipoint time-lapse-video te ontwikkelen.

Time-lapse 2D-beeldvorming van fagocytische activiteit in M1 macrophage-4T1 muis borstkanker cellen in co-culturen

Ontdek hoe tyrosinekinaseremmers senescentie in levercellen veroorzaken en M2-macrofaagpolarisatie bevorderen via SASP. Deze methode onthult mechanismen van geneesmiddel-geïnduceerde hepatotoxiciteit.

Door geneesmiddelen geïnduceerde senescentie in levercellen bevordert polarisatie van M2-macrofagen: implicaties voor tyrosinekinaseremmer-geassocieerde hepatotoxiciteit (Video) | JoVE Journal

Door geneesmiddelen geïnduceerde senescentie in levercellen bevordert polarisatie van M2-macrofagen: implicaties voor tyrosinekinaseremmer-geassocieerde hepatotoxiciteit

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