Denne undersøgelse blev støttet af National Natural Science Foundation of China (82090013).

Den overordnede protokol kræver 44 dage, herunder 1 dag til fremstilling af luftvejsepitelceller isoleret fra murine luftrør, 15 dage til celleproliferation og 28 dage til CSE-stimulering ved luft-væske-grænsefladen. Alle forsøgsdyr er anbragt i SPF Barrier Animal Room på Animal Experiment Center of Capital Medical University og er blevet gennemgået og godkendt af Animal Experiment and Laboratory Animal Ethics Committee of Capital Medical University (AEEI-2020-100) for at opfylde kravene i ARRIVE-retningslinjer<su...

In vitro-model af murine luftvejsepitelceller og kronisk røgeksponering

<ol>
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KOL er en almindelig kronisk inflammatorisk sygdom i luftvejene. Eksponering for tobaksrøg fører til kronisk luftvejsbetændelse, luftvejsombygning og lungestrukturel ødelæggelse, som er resultatet af interaktionen mellem forskellige strukturelle celler og immunceller10. Som frontlinjen i det medfødte immunsystem i lungen spiller luftvejsepitelceller en meget vigtig rolle under udviklingen af sygdommen11. I dette synspunkt er det et videnskabeligt problem at afklare, h...

Kronisk obstruktiv lungesygdom (KOL) er en heterogen lungesygdom med komplekse karakteristika, mens patienter med KOL gradvist har tendens til at være yngre1. Rygning, en primær risikofaktor for KOL2, har en dybtgående indvirkning på luftvejsepitelceller, der fungerer som den første barriere mod tobaksrøg. På trods af denne kendte sammenhæng er de detaljerede mekanismer, hvorigennem tobaksrøg inducerer ændringer i luftvejsepitelceller, stadig utilstrækkeligt udforsket. En grundig forståelse af disse molekylære ændringer er afgørende for at identificere tidlige diagnostiske markører og terapeutiske mål for KOL.
For at afhjælpe dette hul udviklede vi en ny in vitro-model ved hjælp af murin luftvejsepitelceller. Disse celler blev udsat for langtidsstimulering med cigaretrøgekstrakt (CSE), hvilket gjorde det muligt for os at overvåge dynamiske cellulære ændringer og udforske ændringerne i luftvejsepitelceller under langvarig tobaksstimulering og den underliggende mekanisme. I denne model blev transwell brugt til at tilvejebringe luft-væske-grænsefladen mellem luftvejsepitelceller, og cigaretrøgekstrakt blev stimuleret i det tidlige stadium af epitelcelledifferentiering indtil slutningen af differentieringen efter 28 dage. Tidligere undersøgelser undersøgte kun differentiering og kortvarig stimulering af luftvejsepitelceller (cellelinjedominans). De var begrænset til en enkelt reguleringsvej 3,4,5,6. Protokollen, der præsenteres her, bruger imidlertid primære musens luftvejsepitelceller og optimerer celleekstraktionsprocessen for bedre celleaktivitet end tidligere luftvejsepitelcellekulturmetoder. Denne model fokuserer på ændringer i cellulær differentiering sammen med omfattende transkriptomiske, proteomiske, metabolomiske og epigenomiske analyser. Ved at anvende immunfluorescens og avancerede multi-omics-teknikker sigtede vi mod at belyse luftvejsepitelcellernes cellulære responser på kronisk eksponering for tobaksrøg og derved bidrage til en dybere forståelse af KOL-patogenese. Denne model kan bruges til at udforske ændringerne i luftvejsepitelcelledifferentieringsmønsteret og dets mekanisme forårsaget af langvarig stimulering af forskellige forurenende stoffer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x Penicillin/Streptomycin solution</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm Transwell with 0.4 &#181;m Pore Polyester Membrane Insert, Sterile</td>
			<td>BIOFIL</td>
			<td>TCS016012</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>500x Gentamicin/Amphotericin Solution</td>
			<td>Gibco</td>
			<td>R01510</td>
			<td></td>
		</tr>
		<tr>
			<td>acetylated &#945;-Tubulin</td>
			<td>CST</td>
			<td>#5335</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-&#945;-Tubulin (Lys40) (D20G3)XP Rabbit mAb&#160;</td>
			<td>cellsignal</td>
			<td>#5335</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Component Free Cell Dissociation Kit</td>
			<td>Stemcell</td>
			<td>05426</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-pan Cytokeratin antibody</td>
			<td>abcam</td>
			<td>ab7753</td>
			<td></td>
		</tr>
		<tr>
			<td>Cigarette</td>
			<td>Marlboro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Claudin3</td>
			<td>immunoway</td>
			<td>YT0949</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclase I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclase I from bovine pancreas</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F-12</td>
			<td>Sigma-Aldrich</td>
			<td>N6658</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Solution&#160;</td>
			<td>Stemcell</td>
			<td>07980</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone Stock Solution</td>
			<td>Stemcell</td>
			<td>07925</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin 5AC</td>
			<td>abcam</td>
			<td>ab212636</td>
			<td></td>
		</tr>
		<tr>
			<td>Occludin</td>
			<td>proteintech</td>
			<td>27260-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cytosci</td>
			<td>CBS004S-BR500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI 
			Basal Medium</td>
			<td>Stemcell</td>
			<td>05002&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI 10x Supplement</td>
			<td>Stemcell</td>
			<td>05003&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI Maintenance Supplement</td>
			<td>Stemcell</td>
			<td>05006</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-Ex Plus 50x Supplement</td>
			<td>Stemcell</td>
			<td>05042</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-Ex Plus Basal Medium</td>
			<td>Stemcell</td>
			<td>05041</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat tail collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Stemcell</td>
			<td>07050&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro model for studying differentiation and changes of multi-omics on murine airway epithelial cells stimulated with cigarette smoke extract

Kronisk obstruktiv lungesygdom (KOL) tilskrives i høj grad eksponering for tobaksrøg. At undersøge, hvordan luftvejsepitelceller funktionelt tilpasser sig tobaksrøg, er afgørende for at forstå patogenesen af KOL. Denne undersøgelse var at opstille en in vitro-model ved hjælp af primære murin luftvejsepitelceller for at efterligne den virkelige virkning af tobaksrøg. I modsætning til etablerede cellelinjer bevarer primære celler mere in vivo-lignende egenskaber, herunder vækstmønstre, aldring og differentiering. Disse celler udviser en følsom inflammatorisk respons og effektiv differentiering og repræsenterer således nøje fysiologiske tilstande. I denne model blev primære murine luftvejsepitelceller dyrket i 28 dage under en luft-væske-grænseflade med en optimal koncentration af cigaretrøgekstrakt (CSE), hvilket førte til omdannelsen af et monolag af udifferentierede celler til et pseudostratificeret søjleepitel, hvilket indikerer CSE-akklimatisering. Omfattende multi-omics-analyser blev derefter anvendt til at belyse de mekanismer, hvormed CSE påvirker differentieringen af basale luftvejsceller. Denne indsigt giver en dybere forståelse af de cellulære processer, der understøtter KOL-progression som reaktion på eksponering for tobaksrøg.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition with complex characteristics, while patients with COPD gradually tend to be younger1. Smoking, a primary risk factor for COPD2, has a profound impact on airway epithelial cells, which serve as the initial barrier against tobacco smoke. Despite this known association, the detailed mechanisms through which tobacco smoke induces changes in airway epithelial cells remain inadequately explored. A thorough understanding of these molecular alterations is essential for identifying early diagnostic markers and therapeutic targets for COPD.
To address this gap, we developed a novel in vitro model using murine airway epithelial cells. These cells were subjected to long-term stimulation with cigarette smoke extract (CSE), enabling us to monitor dynamic cellular changes and explore the changes in airway epithelial cells under long-term tobacco stimulation and the underlying mechanism. In this model, transwell was used to provide the air-liquid interface of airway epithelial cells, and cigarette smoke extract was stimulated in the early stage of epithelial cell differentiation until the end of differentiation at 28 days. Previous studies investigated only the differentiation and short-term stimulation of airway epithelial cells (cell line dominance). They were limited to a single regulatory pathway3,4,5,6. However, the protocol presented here uses primary mouse airway epithelial cells and optimizes the cell extraction process for better cell activity than previous airway epithelial cell culture methods. This model focuses on alterations in cellular differentiation alongside comprehensive transcriptomic, proteomic, metabolomic, and epigenomic analyses. Employing immunofluorescence and advanced multi-omics techniques, we aimed to elucidate the cellular responses of airway epithelial cells to chronic tobacco smoke exposure, thereby contributing to a deeper understanding of COPD pathogenesis. This model can be used to explore the changes in airway epithelial cell differentiation pattern and its mechanism caused by long-term stimulation of various pollutants.

Forfatter Spotlight: Udvikling og karakterisering af en in vitro-model til at studere kronisk cigaretrøgeksponering og dens indvirkning på luftvejsepitelceller i KOL-forskning

In vitro-model til undersøgelse af differentiering og ændringer af multi-omics på murine luftvejsepitelceller stimuleret med cigaretrøgekstrakt

Differentiering Mus luftvejsepitelceller differentierede med succes efter dyrkning ved en luft-væske-grænseflade med et differentieringsmedium i 28 dage. Tilstedeværelsen af cilierede celler og bægerceller blev påvist ved immunfluorescensanalyse af ciliamarkøracetyleret α-Tubulin (grøn; Figur 3A) og bægercellemarkøren Mucin5AC, henholdsvis6 (rød; Figur 3B).
Bestemmelse af CSE-koncentration med he...

Her beskriver vi en in vitro-model til isolering og differentiering af murine luftvejsepitelceller med fokus på deres akklimatisering til kronisk cigaretrøgekstrakt (CSE). Modellen kan bruges til omfattende karakterisering af multi-omics-påvirkningen af CSE, hvilket muligvis giver indsigt i de cellulære reaktioner under kronisk røgeksponering.

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

In vitro-model til undersøgelse af differentiering og ændringer af multi-omics på murine luftvejsepitelceller stimuleret med cigaretrøgekstrakt

Abstract