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Respons på oveckade proteiner: kan plasmid pEGFP-XBPdeldDBD-STOP-tagRFPt användas för detektion av celler vars endoplasmatiska retikulum är stressat?
Linköping University, Department of Medical and Health Sciences.
2016 (Swedish)Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesisAlternative title
Unfolded Protein Response : can plasmid pEGFP-XBPdeldDBD-STOP-tagRFPt be used for detection of cells whose endoplasmatic reticulum is stressed? (English)
Abstract [sv]

Bakgrund: Endoplasmatiskt retikulum upprätthåller proteinhomeostasen genom syntes och degradering av proteiner. Tre signalvägar reglerar processen via ”unfolded protein response” på olika sätt. Inositol-requiring enzyme 1 alpha är en signalväg aktiverad av en störd proteinhomeostas. Stressen uppstår om cellen utsätts för kemiska substanser t.ex. i samband med djurförsök vid framtagning av läkemedel. Den aktiverade signalvägens respons leder bl.a. till splicing av X-box-binding Protein 1 mRNA.

Syfte: Projektets syfte är att minska behovet av djurförsök genom att möjliggöra ökad implementering av 3R-principen. Frågeställningen är om den fluorescerande plasmiden pEGFP-XBP1∆dDBD-STOP-tagRFPt som binder till X-box binding protein 1 mRNA kan användas för detektion av celler vars endoplasmatiska retikulum är stressat.

Metod: Plasmid pEGFP-XBP1∆dDBD-STOP-tagRFPt renades ur DH5-Alpha Escherichia coli med JETstar 2.0 Plasmid Purification Midiprep Kit. Humana epiteliala njurceller 293 transfekterades med plasmiden varpå de doserades med tunicamycin. Kontrollen utgjordes av dimetylsulfoxid. Mikroskopering skedde i fluorescensmikroskopet Zeiss Axiovert 200. Mjukvaran AxioVision Ver. 4.8.1.0. Carl Zeiss Imaging Solutions framställde cellerna på dataskärm.

Resultat: Hypotestestets signifians bestämdes till p < 0,05. Resultatet antyder att antalet ER- stressade celler ökar med ökande inkubationstid (p = 0,001 – 0,966). En mindre antydan finns också mot en ökande koncentration av tunicamycin (p = 0,096 – 0,690).

Slutsats: En rimlig slutsats kan inte dras då antalet utförda analyser är för få. 

Abstract [en]

Background: Endoplasmic reticulum maintains protein homeostasis by protein synthesis and degradation. Three signal paths regulates the process by ”unfolded protein response” in different ways. Inositol-requiring enzyme 1 alpha is one path activated by a disturbed protein homeostasis. Stress starts if the cell is exposed to chemical substances as in connection with scientific animal experiments when pharmaceuticals are developed. The response of the activated signal path leads e.g. to splicing of X-box-binding protein 1 mRNA.

Object: The object of the project is to reduce the need of scientific animal experiments by enabling increased implementation of the 3R principle. The question is if the fluorescent plasmid pEGFP-XBP1∆dDBD-STOP-tagRFPt binding to X-box binding protein 1 mRNA can be used for detection of cells whose endoplasmatic reticulum is stressed.

Method: Plasmid pEGFP-XBP1 ∆ dDBD-STOP-tagRFPt was purified from DH5-Alpha Escherichia coli with JETstar 2.0 Plasmid Purification Midiprep Kit. Human epithelial kidney cells 293 were transfected with the plasmid whereupon they were dosed with tunicamycin. The control was made off dimethylsulfoxide. Microscopy was done in the fluorescence microscope Zeiss Axiovert 200. The software AxioVision Ver. 4.8.1.0. Carl Zeiss Imaging Solutions imaged cells on the computer screen.

Result: The significans of the hypothesis test was set to p < 0,05. The result suggests that the number of ER-stressed cells increase with increasing incubation time (p = 0,001 – 0,966). A smaller hint is also pointing toward increasing concentrations of tunicamycin (p = 0,096 – 0,690).

Conclusion: A reasonable conclusion cannot be made because of too few tests. 

Place, publisher, year, edition, pages
2016. , 32 p.
Keyword [en]
unfolded protein response, plasmid, fluorescence, microscopy, 3R
Keyword [sv]
respons på oveckade proteiner, plasmid, fluorescens, mikroskopi, 3R
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:liu:diva-130045OAI: oai:DiVA.org:liu-130045DiVA: diva2:946746
Subject / course
Biomedical Laboratory Science
Presentation
2016-06-09, Linköping, 14:45 (Swedish)
Supervisors
Examiners
Available from: 2016-08-15 Created: 2016-07-05 Last updated: 2016-08-15Bibliographically approved

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