Wydanie 2/2003

Poszukiwanie komórek macierzystych oraz badanie żywotności komórek rąbka rogówki ludzkiej z użyciem mikroskopu konfokalnego

Search for stem cells and cell viability investigations in human corneal limbus using confocal microscope

Piotr Skopiński1,2, Jacek P. Szaflik1, Marek Kujawa2, Ewa  Jankowska2, Cezary Kowalewski3, Małgorzata Brix4, Jerzy Szaflik1,4

1Katedra i Klinika Okulistyki II Wydziału Lekarskiego Akademii Medycznej w Warszawie
Samodzielny Publiczny Kliniczny Szpital Okulistyczny w Warszawie
Kierownik: prof. dr hab. med. Jerzy Szaflik
2Katedra i Zakład Histologii i Embriologii Centrum Biostruktury Akademii Medycznej w Warszawie
Kierownik: prof. dr hab. med. Stanisław Moskalewski
3Klinka Dermatologiczna Akademii Medycznej w Warszawie
Kierownik: prof. dr hab. med. Maria Kostanecka-Błaszczyk
4Warszawski Bank Oczny
Kierownik: prof. dr hab. med. Jerzy Szaflik


Summary: We described two applications of laser confocal microscope in studies concerning human corneal limbus. Firstly, for searching limbal stem cells and secondly, for studies of limbal cell viability in stored for 6 days in Optisol or MK Medium corneoscleral discs. Limbal stem cells are responsible for physiological renewal of corneal and conjunctival epithelium. They are also important for regeneration of the epithelium in case of cornea injury. In the first part of our investigations we performed indirect immunohistochemical reaction with antibody against epiligrin (laminin 5 - basement membrane component) followed by second antibody labelled with indodicarbocyanine (cyanine 5; Cy5) as well as staining with Rhodamine 123. Three populations of cells differing in the intensity of staining with Rhodamine were observed and localised close to basement membrane. Cells with the weakest staining or no staining at all may be stem cells, since it has been demonstrated previously by others that some types of stem cells are able to pomp out xenobiotics (e. g., Hoechst 33342 and Rhodamine 123 stains). In the second part of our studies we investigated limbal cell viability using indirect immunohistochemical reaction with antibody against epiligrin followed by second antibody labelled with FITC and staining with SYTO 62 (to visualise living cells) as well as propidium iodide (to visualise dead cells). Such a triple staining allows for basement membrane localisation and quantitative evaluation of dead cell percentage. All observations were performed under confocal Radiance 2000 Scanning System microscope using argon and diode lasers.

Keywords: confocal microscope, limbal stem cells, corneal epithelium


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