파라핀 절편의 변성된 단백질을 분석하기 위한 ImmunoMemBlot 방법의 적용 -법랑모세포종과 선양치성종양의 비교- Application of ImmunoMemBlot Method for the Analysis of Denatured Protein Extracts from Paraffin Sections -Ameloblastoma Versus Adenomatoid Odontogenic Tumor-
- 언어KOR
- URLhttp://db.koreascholar.com/Article/Detail/292832
pp.269-278
In order to perform the protein analysis using the paraffin sections previous fixed with formalin, we applied the ImmunoMemBlot (IMB) method1) to detect the epitopes of target proteins with specific antibodies. In this study the protein extracts were obtained from the paraffin sections of each representative case of ameloblastoma, adenomatoid odontogenic tumor (AOT), and normal gingiva, and more a protein extract from fresh tissue of ameloblastoma was also compared to evaluate the IMB results used with 24 different antibodies. First of all, in the comparison between the paraffin section extract and fresh tissue extract of ameloblastoma, the latter consistently showed more positive IMB reaction than the former. Meanwhile, the paraffin section extract of ameloblastoma was more comparable with that of normal gingival, disclosing that most of proliferating genes, oncogenes, and apoptosis related genes, i.e., PCNA, CDK4, c-erbB2, CEA, p53, Bax, Bad, FLIP, FAS, Bcl-2, p21, N-ras, MMP-2, MMP-9, caspase-3, -8, -9, were highly expressed in ameloblastoma, but EGFR, HGF, and VEGF were similarly expressed both in the ameloblastoma and in normal human gingiva. On the other hand, the comparison between ameloblastoma and AOT both in the immunohistochemistry and IMB using their paraffin section extracts clearly demonstrated that the ameloblastoma showed more expression of proliferating genes and oncogenes while the AOT showed more expression of apoptosis related genes, i.e., Bax, Bad, FLIP, and caspase-9. Taken together, these data suggest that the IMB can be used for the primary screening of quantitative protein analysis using the paraffin section extract, and that the IMB results could be evaluated in conjunction with the immunohistochemical observation.
II. 실험재료 및 방법
1. 단백질 추출
2. 단백질 정제
3. MemBlot 장치의 도트 브랏 (dot blot) 형성
4. 면역화학적 염색
5. 3중 항체 간접법
6. 영상처리 및 결과 분석
III. 결과
1. 법랑모세포종의 신선조직 추출 단백질과 파라핀절편 추출 단백질의 비교
2. 법랑모세포종과 정상 치은조직의 파라핀 조직추출 단백질의 유전물질 발현의 비교
3. 법랑모세포종과 선양치성종양의 파라핀 절편에서얻은 단백질의 유전물질 발현의 비교
VI. 총괄 및 고안
V. 참고문헌