Napetostne pore pri celicah v hipotonični raztopini

Prša, Aleks (author), Ziherl, Primož (mentor), Božič, Bojan (comentor)

V zaključni nalogi smo raziskali spreminjanje prostornine celic in pojav napetostnih por celic v različnih hipotoničnih raztopinah vode in gojišča ter v ekvivalentnih hipotoničnih raztopinah saharoze. Pri poskusih smo uporabili epitelne celice kitajskega hrčka [angl. Chinese hamster ovary (CHO) cell], ki so bile gojene v naprednem minimalnem mediju z dodatkom 5% fetalnega telečjega seruma [angl. fetal bovine serum (FBS)] in antibiotikov v CO2 inkubatorju pri 37°C. Vse poskuse smo opravili s konfokalnim mikroskopom, ki omogoča, da naenkrat presvetlimo in zajamemo le določeno rezino celice. Za poskuse spreminjanja prostornine celic smo celicam označili membrano z označevalnim sredstvom CellMask, za opazovanje napetostnih por celic pa smo celice označili s fluorescentnim označevalcem kalcein. Za določanje prostornine celice smo vsebino celotne celice razdelili na skladovnico vodoravnih rezin po osi z, ki so bile med seboj razmaknjene za 0,5 ?m. Spreminjanje prostornine celic smo opazovali v gojišču, v destilirani vodi in v hipotoničnih raztopinah gojišča s 40%, 60%, 80% in 90% deležem vode ter v ekvivalentnih hipotoničnih 23, 65, 125 in 315 mOsm/l raztopinah saharoze. Celice smo posneli pred zamenjavo hipotonične raztopine in po 2, 5, 10, 20, 30, 40, 50 in 60 minutah po dodatku hipotonične raztopine. Prostornino celic smo določili tako, da smo izmerili preseke celic na 7 do 14 enakomerno razmaknjenih rezinah. Celice, pri katerih smo opazovali napetostne pore, smo med poskusom opazovali le v eni izbrani goriščni ravnini. Napetostne pore celic smo opazovali v destilirani vodi in v hipotoničnih raztopinah gojišča s 60%, 80%, 85%, 90% in 95% deležem vode ter v hipotoničnih 23, 65 in 125 mOsm/l raztopinah saharoze. Celice v hipotoničnih raztopinah gojišča z višjimi deleži vode in v destilirani vodi smo slikali vsako drugo sekundo 10 minut po zamenjavi raztopine, celice v hipotoničnih raztopinah z nižjimi deleži vode pa smo slikali vsako trideseto sekundo 1 uro po zamenjavi raztopine. Dobljene rezultate meritev prostornine celic smo pojasnili s teoretičnim modelom, ki je bil razvit na Inštitutu za biofiziko Medicinske fakultete Univerze v Ljubljani. Ugotovili smo, da je nenadno razbarvanje oz. nenaden upad intenzitete celic, označenih s kalceinom, posledica nastanka napetostnih por, kar se na posnetkih jasno vidi.

• Language: Slovenian
• Keywords: celice CHO, celična membrana, prostornina, napetostna pora, hipotonična raztopina
• Work type: Final paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FMF - Faculty of Mathematics and Physics
• Year: 2019
• PID: 20.500.12556/RUL-110751
• COBISS.SI-ID: 3371876
• Publication date in RUL: 19.09.2019

Secondary language

• Language: English
• Title: Tension pores in cells in hypotonic solution

Abstract

The volume changes and occurrence of tension pores in cells in different hypotonic medium of Leibowitz solution and water and equivalent hypotonic solution of sucrose were observed. Chinese hamster ovary (CHO) cells were used in experiments. The cells were grown in minimal essential medium supplemented with 5% fetal bovine serum (FBS) and antibiotics in CO2 incubator at 37°C. All experiments were performed with confocal microscope, which allows us to illuminate and capture only one specific slice of a cell at once. For cell volume changes we labeled cell membrane with the CellMask marker and for leakage experiments for observing tension pores in cells we labeled cells with the calcein marker. To determine the cell volume, the content of the whole cell was divided into stack of horizontal slices along the z-axis spaced bg 0.5 ?m. The volume changes of the cells were observed in Leibowitz solution, in distilled water, hypotonic media of Leibowitz solution with 40%, 60%, 80%, and 90% water content and equivalent hypotonic solution of sucrose with osmolarities 23, 65, 125 and 315 mOsm/l. The cells were imaged before we replace hypotonic solution and after 2, 5, 10, 20, 30, 40, 50 and 60 minutes after hypotonic solution replacement. The cell volume was determined by determination the area of the cell section at 7 to 14 evenly spaced slices. For leakage experiments the cells were imaged only in one selected plane. The cells tension pores were observed in distilled water, hypotonic media of Leibowitz solution with 60%, 80%, 85%, 90% and 95% water content and equivalent hypotonic solution of sucrose with osmolarities 23, 65 and 125 mOsm/l. The cells in distilled water, in hypotonic media with higher water content and in hypotonic solutions of sucrose with lower osmolarities were imaged every 2 seconds 10 minutes after hypotonic solution replacement. The cells in hypotonic solutions with a lower water content and in hypotonic solutions of sucrose with higher osmolarities were imaged every 30 seconds 1 hour after hypotonic solution replacement. The obtained results of cell volume measurements were explained in terms of a theoretical model, developed on Institute of Biophysics, Faculty of Medicine, University of Ljubljana. We found that sudden decrease of calcein intensity in marker cells due to formation of the tension pores, which can be clearly seen in the recordings.

• Keywords: CHO cells, cell membrane, volume, tension pore, hypotonic solution