NR. 2/2003
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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 |
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| 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. |
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| Key words: |
confocal microscope, limbal stem cells, corneal epithelium |
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