Opredelitev imunogenosti gojenega limbalnega tkiva.

Mikec, Špela (author), Kopitar, Andreja Nataša (mentor)

Limbalne epitelijske matične celice (LEMC) se nahajajo v roženičnem limbusu in omogočajo obnovo roženičnega epitelija. Okvara in pomanjkanje teh celic vodi v boleznsko stanje. Eden od načinov zdravljenja pomanjkanja LEMC je s presaditvijo vzgojenega limbalnega tkiva v kulturi. Določali smo imunogenost gojenega roženičnega tkiva iz keratolimbalnih eksplantov kadavrskih roženic do tretje pasaže na plastiki in intaktni amnijski membrani (AM). S pretočno citometrijo smo določali prisotnost dveh podtipov dendritičnih celic (DC), ki smo jih ločili po izražanju naslednjih označevalcev, za plazmacitoidne DC (pDC): CD123+, CD303+, CD11c-, in za mielodine DC (mDC): CD11c+, CD123-. Merili smo tudi izražanje molekule MHC II, kostimulatornih molekul CD80 in CD86 ter aktivacijske molekule CD83. Merili smo še izražanje molekul CK12 in ABCB5. Prve so značilne za populacijo zrelih epitelijskih roženičnih celic, druge pa za pa populacijo LEMC. Za vse parametre smo naredili modele napovedi povprečnih deležev po pasažah. Pri vseh parametrih se je izkazalo, da razlika med rastjo celic na plastiki in na AM za gojenje ni statistično značilna (p>0,05). Z višanjem pasaž smo opazili trend znižanja deleža pDC in mDC, vendar pa vrednosti niso bile statistično značilne. Trdimo lahko, da je padec števila zrelih epitelijskih roženičnih celic iz prve v drugo (p=0,007) in iz druge v tretjo pasažo (p=0,01) statistično značilen. Prav tako je statistično značilen padec LEMC iz prve v drugo pasažo (p=0,02). Glede na rezultate menimo, da je na obeh podlagah smiselno gojiti celice do vključno prve pasaže, tako dobimo tkivo, ki ima zadostno količino epitelijskih roženičnih celic, LEMC in zmanjšan delež mDC in pDC.

• Language: Slovenian
• Keywords: roženica, limbalne epitelijske matične celice, pomanjkanje limbalnih epitelijskih matičnih celic, gojeno limbalno tkivo, eksplantna tehnika, amnijska membrana
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [Š. Mikec]
• Year: 2018
• PID: 20.500.12556/RUL-103043
• UDC: 602.9:591.81:611.841(043.2)
• COBISS.SI-ID: 9047417
• Publication date in RUL: 13.09.2018

MIKEC, Špela, 2018, Opredelitev imunogenosti gojenega limbalnega tkiva. [online]. Master’s thesis. Š. Mikec. [Accessed 14 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=103043

Secondary language

• Language: English
• Title: Determining the immunogenicity of cultured limbal tissue

Abstract

Limbal epithelial stem cells (LESCs) are found in the corneal limbus and are important for corneal epithelium renewal. Lack of these cells leads to a limbal stem cell deficiency (LSCD). We determined the immunogenicity of cultivated tissue from keratolimbal explants of cadaveric corneas up to the third passage grown on plastics and on intact amniotic membrane (AM). Flow cytometry was used to determine the presence of two subtypes of dendritic cells (DC), which were determined by measuring the expression of the following markers: for plasmacitoid DC (pDC): CD123+, CD303+, CD11c-, and for myeloid DC (mDC): CD11c+, CD123-. We also measured the expression of MHC II molecule, the CD80 and CD86 costumulatory molecules, and CD83 activation molecule. Populaton of mature epithelial cells was determined by measuring the expression of CK12, and the population of LESC by the expression of ABCB5. Models of forecasts of average percentage by passages were made. The difference between plastics and AM was not statistically significant (p>0.05) for any measured parameter. Increasing passages reduced the percentage of pDC and mDC, but the values were not statistically significant. The average percentage of mature epithelial corneal cells fell from first to second (p=0.007) and from second to third passage (p=0.01). The average percentage of LEMC from the first to the second passage (p=0.02) also dropped. According to our results, it is best to cultivate the cells up to and including the first passage for both substrates, thus obtaining a tissue that has a sufficient amount of epithelial corneal cells, LEMC, and a reduced proportion of mDC and pDC.

• Keywords: cornea, limbal epithelial stem cells, limbal stem cell deficiency, cultured limbal tissue, explant technique, amniotic membrane