논문 상세보기
pp.151-164
콘택트렌즈 소독제로 쓰이는 H2Û2올 중화용 백긁 판올 이용하여 수회 반복 중화한 용액이 배양 각막상피세포의 Apoptosis룰 유도하는지 여부룹 조사하기 위해 본 연구 를 시행하였다 3% H202(AOSept)를 6시간 동안 9회에 걸쳐 통일 백금 판으로 풍화 한 용액을 배양 각막 상피세포에 배양액의 25% 농도로 1--2 띤 동안 처리하여 처음 중화한 용액과 9번째 중화한 용액음 중심으로 세포독성과 Apoptosis를 조사하였다. 세포똑성은 MTT assay를 이용하였으며 lnverted pþase-contrast microscope로 형 태 를 확얀하고 Toluiclin blue 염 색으로 Apoptotic Cell올 확언하였으며 Hoechst 33342 로 형광 염색하여 Apoptotic Index를 조사하였다. 또한 Annexin V - FITC깨ropiclium Iodide(PDstaíning, DePsipher TM assay. M30 CytoDEATH. TUNEL (TdT-dUTP terminal nick-end labelling) assay를 통해 형태적인 변화될 확인하였다. 생화학적 변 화인 DNA Fragmentation은 Agarose gel electrophoresis룹 사 행하였으며 Fas Antigen 의 발현을 조사하기 위하여‘ Irnmunocytochemistry와 Westem blotting올 시 행하 였다. H20z의 독성올 약화시키기 위해 사용되는 백금 판을 수회 재사용하여 중화한 용액닫 25% 농도로 1-2 일간 처리하였을 때 각막 상피세포의 Apoptosis가 유도되는 것 이 형 광 염 색， DNA ladder. Irnmunocytochemistry. Westem blotting등의 방법 으로 확인되었다. 따라서 중화된 H써2룹 25% 농도로 1-2 일 동안 처리한 결과， 세포 증식 저해와 Apoptosis 가 유도되므로 중화용 백금 판의 이용 횟수활 세한하거나 중화시간 을 증가시킬 필요가 있는 것으로 사료된다. 또한 완전하게 중화된 소독용 H202는 더 이상 소독효파가 없으므로 장시간 동안 렌즈의 소쪽보관 용액으로 사용해서는 안 된 다는 교육이 반드시 펼요할 것으로 판단된다.
To investigate neutralized hydrogen peroxide(H2Ü'z)-induced Apoptosis in cultured human comeal epithelialU-ICE) cell, this study was performed many kind of methods for detecting morphological and biochemical changes and protein expression. A continuous HCE cell line was treated with neutralized HZ02 for 24 hours or 48 hours. Neutralized HzOz solutions were prepared by neutralization with re-using platinum disc for 6 hours. The percentage of dead cells as measured by MTT assay, was high in re-use of old disc manner. Morphologìc changes and guantification of apoptotic cells were determined and counted under fluorescence microscope after neutralized H20z-trea않d HCE cells for 1 or 2 days with Hoechst 33342 staining, Annexin V -FITC!PI staining, M30 CytoDEA TH and DePsipher assay. And Apoptotic changes were also observed by in--situ images, Toluidin Blue staining and TUNEL assay under light microscope. DNA extraction from neutralized H20z-treated HCE cells was electrphoresed by the agarose gel for DNA ladder pattem. The expression of Fas protein was measured by immunocytochemistry and westem blot. A significant decrease of membrane integrity with chromatin condensation was observed with neutralized HzÜ2. Neutralized H202 elicited typical apoptotic morphologic changes(chromatic condensation, nucleus fragmentations, non orangered colored mitochondria) and expressed Fas protein highly. The loss of mitochondrial transmembrane potential appeared in earliest phase; subsequently, the chromatin condensatìon and DNA -fragmentation occurred. DNA extraction from neutralized H202-treated cells showed a typical DNA ladder pattem. A functional Fas-mediated apoptotic pathway was present in cultured HCE cells and could be activated by upregulation of Fas expression with neutralized HzÜz. This study suggests that the re-use of old discs must be prohibited or the soaking time for old discs should be increase for protection of H20Z induced Apoptosis. Once neutralization is complete, the remaining solution is unpreservd. It is necessary for patient to educate the neutralized solution care because viable organisms residing in any lens case biofilm can lead to r앉ontamination
