정상 및 HPV 불멸화 인간 구강상피의 삼차원적 배양 Three Dimensional Culture of Normal & HPV-Immortalized Human Oral Keratinocy
- 언어KOR
- URLhttp://db.koreascholar.com/Article/Detail/292813
pp.313-336
Epithelial-mesenchymal interaction is well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular micro-environment which provide a epithelial-mesenchymal interaction. The aims of this study were to develop and evaluate the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted human normal oral kertinocyte(NHOK) and immortalized human oral keratinocytes(IHOK) by histological and immunohistochemical analysis. The results were as follows;
1. Best condition of three dimensionally reconstituted IHOK & HaCaT cells are 14 days air-exposure cultivation, 3 days of submerged state, and dermal equivalent consisting type I collagen and IGF cells.
2. In comparison to IHOK, there was better preservation of the overall epidermal strucutures in oral cracioma cells (HN30) & HaCaT cells by organotypic cultures. But orgnaotypic co-culture of the normal keratinocyte showed the thinnest epithelial layer formation.
3. PCNA was detected primarily in the basal layer of normal mucosa and NHOK, whereas was shown throught the epithellium except surface layer of IHOK cells, and it's expression was similar to that of CIS of biopsied patient's tissue
4. Involucrin is expressed in the upper layer of oral mucosa and NHOK raft, but staining for involucrin was induced in the IHOK rafts indicating differentiation is incomplete, and the staining pattern in the IHOK raft was not uniform.
5. Normal oral keratinocyte raft showed weak immunostaining for p53, and p53 expression of IHOK raft increased rahter than in NHOK. In organotypic cultures of normal cells and IHOK, p53 expression was restricted to the proliferative part of epithelium. This is consistent with expression pattern in biopsy specimens of the normal and CIS tissue.
6. In artifically reconstructed NHOK, the pattern of keratin staining showed both similarity and differences from that of intact normal mucosa. An obvious difference was increased expression of CK10 & CK19, and decreased expression of CK6 in a reconstructed NHOK rather than in normal mucosa, and similar expression was in CK4 and CK16.
7. CK19 & CK16 were strongly positive in HPV immortlaized keratinocyte rafts rahter than in NHOK, an indicator of premalignant or malignant changes, while CK10 & CK6 were decreased, and organotypic cultures of IHOK express was similar keratin expression as epithelial dysplasia or CIS tissue.
These results suggest that three-dimensional organotypic co-culture of normal oral & immortalized keratinocytes with dermal equivalent consisting type I collagen and fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. Thus this organotypic system can be used for studing mechanism of epitehlial-mesenchymal interaction in particular regulating epidermal diffenentiation and morphogenes
II. 연구재료 및 방법
1. 사람 구강 각 화 세 포 (normal human oralkeratinocyte; NHOK ) 배양
2. 불멸화된 구강각화세포 배양; HPV16의 트란스펙션 및 IHOK(immortalized human oralkeratinocyte) 클론 선택후 세포배양
3. 치 은 섬 유 아 세 포 (human gingivalfibroblasts, GF) 배양
4. 삼차원 배양된 구강 치은 조직의 형태학적 특징분석
5. 면역조직화학 염색
III. 연구결과
1. 대기노출 배양기간에 따른 삼차원적 재건의 조직학적 형태 변화
2. 대 기 노 출 전 배 양 액 에 잠 긴 배 양 기 간(submerged state) 에 따른 조직학적 형태변화
3. 진피대체물로서 섬유모세포 종류에 따른 삼차원적 조직학적 형태 변화
4. 상피세포 종류에 따른 조직학적 형태 변화
5. 정상 및 전암조직과, 악성 구강 상피세포 종류에따른 삼차원적 배양후 증식 및 분화에 따른 면역조직화학적 차이
IV. 고찰
V. 결 론
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