간 독성 과정 중 투여 된 DL-α-Tocopherol의 손상 회복 효과와 Thymosinβ4, c-Myc, Erb-B2, TGF-β1 유전자 발현 및 조직학적 형태 변화

다국어 초록 (Multilingual Abstract)

In general, DL-a-Tocopherol was known to be highly resistant to damaged cells and tissues. Thymosin-β4 gene was involved in immune response, angiogenesis, and metastasis. c-Myc and Erb-B2 genes were oncogene. TGF-β1 was known as gene of tissue fibrosis.
This study was conducted to investigate the antioxidant activity of alpha-Tocopherol and the degree of expression of TGF-β1, c-Myc, Erb-B2 and Thymosin-β4 in damaged liver tissues. Study was made liver tissue fibrosis model, alcoholic liver damage model, and acute hepatotoxicity model as test methods by oral administration to 10-week-old BALB/c using carbon tetrachloride and alcohol, and studied serological changes of related enzyme and electron microscopic specimen preparation, histological changes through special staining and expression change of TGF-β1, and c-Myc, Erb-B2, Thymosin-β4 through real time PCR, and analyzed antioxidant effect through administration of DL-a-Tocopherol.
Various serological and histological results showing the degree of liver damage after alpha - Tocopherol administration and the degree of gene expression according to hepatic tissue damage were analyzed and the following conclusions could be obtained.
First, the liver tissues of damaged was increased serological enzymes of ALT, AST and ALP. Second, the case of damaged liver tissue was observed fibrosis due to accumulation of glue fibers in cytoplasm by the damage of cell organelles, and observed through histological staining that central vein or surrounding tissue of hepatic triad were highly damaged. Third, the damaged liver tissues was observed that expression of TGF-β1, c-Myc and Thymosin-β4 genes was increased, and Erb-B2 gene was not clearly expressed. Fourth, antioxidant effect of damaged liver tissue by Dl-α-Tocopherol administration was observed that there was antioxidant effect which suppresses tissue damage through serological tests and histological staining. Fifth, antioxidant effect of liver fibrosis tissue by Dl-α-Tocopherol was poor in expression of TGF-β1, c-Myc and Thymosin-β4 genes. Sixth, antioxidant effect of Alcoholic liver tissue by Dl-α-Tocopherol was observed that the expression of TGF-β1, c-Myc and Thymosin-β4 genes were suppressed.
Therefore, based on the antioxidant effect of alpha Tocopherol on liver tissue in the prevention of liver-related diseases and the diagnosis of diseases in existing clinical trials, in addition to serological and histologic examination, by examining gene expression status by TGF-β1, c-Myc, and Thymosin-β4 molecular diagnostic techniques, it is considered to be used as a supplementary data for liver disease diagnosis

국문 초록 (Abstract)

일반적으로 DL-a-Tocopherol은 손상된 세포나 조직의 손상 회복 기능이 강한 것으로 알려져 있으며 Thymosin-β4 유전자는 면역반응이나 혈관 생성, 암전이 등에 관여 하며 c-Myc, Erb-B2 유전자는 종양 관련 유전자이고 TGF- β1는 조직 섬유화 관련 유전자로 알려져 있다.
따라서 본 연구는 손상된 간 조직에서 DL-a-Tocopherol의 항산화 작용과 조직 손상에 따른 TGF-β1, 및 c-Myc, Erb-B2, Thymosin-β4 발현 정도를 알아 보고자 실시하였다. 검사 방법으로는 사염화탄소 및 알코올을 이용하여 10주령 된 BALB/c에 경구 투여함으로써 간조직의 섬유화 모델과 알코올성 간 손상 모델, 그리고 급성 간독성모델을 만들고 혈청학적 관련 효소 변화와 전자현미경적 표본제작, 특수염색을 통한 조직학적 변화, 그리고 Realtime PCR을 통한 TGF-β1, 및 c-Myc, Erb-B2, Thymosin-β4 발현 변화를 연구하였으며 DL-a-Tocopherol의 투여를 통한 항산화 효과 여부를 분석하였다.
DL-α-Tocopherol 투여 후 간 손상 영향 정도를 보여주는 각종 혈청학적, 조직학적 결과와 간 조직 손상에 따른 유전자 발현 정도를 분석하여 본 바 다음과 같은 결론을 얻을 수 있었다.
첫째. 손상 받은 간 조직은 ALT, AST, ALP의 혈청학적 효소가 증가됨을 알 수 있었다. 둘째. 간 조직이 손상 받을 경우 세포 소기관의 손상으로 인하여 세포질에 아교섬유 축적에 따른 섬유화를 볼 수 있었으며 중심정맥이나 간세둥이 주위 조직에 손상이 많음을 조직학적 염색을 통하여 알수 있었다. 셋째. 손상 받은 간 조직에서는 TGF-β1, c-Myc 및 Thymosin-β4 유전자의 발현 증가를 알 수 있었으며 Erb-B2 유전자는 뚜렷한 발현을 알 수 없었다. 넷째. DL-α-Tocopherol 투여에 따른 손상 받은 간조직의 항산화 효과는 혈청학적 검사와 조직학적 염색을 통하여 조직 손상을 억제하는 회복 효과가 있다는 것을 알 수 있었다. 다섯째. 섬유화가 진행된 간 조직에서 TGF-β1 유전자를 제외한 c-Myc 및 Thymosin-β4 유전자의 발현에 대한 DL-α-Tocopherol 항산화 효과는 미비한 결과를 알 수 있었다. 여섯째. 알코올에 의한 간 손상 조직에서 TGF-β1, c-Myc 및 Thymosin-β4유전자의 발현에 대한 DL-α-Tocopherol 투여는 발현을 억제하는 회복 효과가 있다는 것을 알 수 있었다.
따라서 기존 임상에서 간 기능 관련 질병 예방과 질환 판정 시 DL-α-Tocopherol에 의한 간 조직 항산화 효과를 참고로 하여 혈청학적, 조직학적 검사 외에 TGF-β1, c-Myc 및 Thymosin-β4의 분자 진단 기법에 의한 유전자 발현 상태를 검사함으로써 간 질환 판정의 보조 자료로 활용 될 수 있을 것으로 사료 된다.

목차 (Table of Contents)

• I. 서 론 ···································································1
• 1. 간 기능····································································1
• 2. CCl4의 간 섬유화 기전 ···················································2
• 3. 알코올의 간 손상 기전 ··················································4
• 4. Thymosin-β4, c-Myc, Erb-B2, TGF-β1 유전자 ·······················5
• 5. DL-α-Tocopherol ·······················································7
• 6. 연구 목적·····································································8
• II. 재료 및 방법 ·······························································9
• 1. 실험동물 ································································9
• 1-1 간섬유화 모델 ·························································9
• 1-2 알코올성 간 손상 조직 모델 ·········································10
• 1-3 급성 간독성 모델······················································11
• 2. 혈액학적검사···························································12
• 3. 조직학적 평가··························································13
• 3-1 전자현미경적 조직관찰 ···············································13
• 3-2 Hematoxylin & Eosin 염색···········································15
• 3-3 마손 삼색 (Masson's Trichrom)염색································17
• 3-4 세망 섬유 (Gomori Reticulum)염색··································19
• 4. 유전자 검사····························································21
• 5. 통계분석································································23
• III. 결 과
• 1. 간 섬유화 과정 중 투여된 DL-α-Tocopherol의 손상 회복 효과와
• Thymosinβ4, c-Myc, Erb-B2, TGF-β1 유전자 발현 및 조직학적 형태
• 변화············································································24
• 1-1. 전자현미경적 조직관찰·············································25
• 1-2. 혈액 검사 측정····· ················································31
• 1-3. 조직학적 변화···· ···················································33
• 1-3-1. Hematoxylin & Eosin 염색···········································35
• 1-3-2. 마손 삼색 (Masson's Trichrom)염색······························37
• 1-3-3. 세망 섬유 (Gomori Reticulum)염색································39
• 1-4. 유전자 분석······ ·· ·· ·· · ·· ··················· ·· ·· ·· · ··············41
• 2. 알코올성 간 손상 과정 중 투여된 DL-α-Tocopherol의 손상 회복 효과와
• Thymosinβ4, c-Myc, Erb-B2, TGF-β1 유전자 발현 및 조직학적 형태
• 변화············································································54
• 2-1. 전자현미경적 조직관찰··· ······ ······································55
• 2-2. 혈액검사 측정···· ······················································58
• 2-3. 조직학적 변화···· ······················································60
• 2-3-1. Hematoxylin & Eosin 염색·······································62
• 2-3-2. 마손 삼색 (Masson's Trichrom)염색······························64
• 2-3-3. 세망 섬유 (Gomori Reticulum)염색·······························66
• 2-4. 유전자 분석···· ······ ·················· ·· · ·· ·· ·· · ··················68
• 3. 급성 간독성에서의 Thymosinβ4, c-Myc, Erb-B2, TGF-β1 유전자
• 발현 및 조직학적 형태 변화················································81
• 3-1. 전자현미경적 조직관찰·················································82
• 3-2. 혈액검사 측정··· ······················································84
• 3-3. 조직학적 변화···· ·····················································86
• 3-3-1. Hematoxylin & Eosin 염색········································86
• 3-3-2. 마손 삼색 (Masson Trichrome)염색······························88
• 3-3-3. 세망 섬유 (Gomori Reticulum)염색······························90
• 3-4. 유전자 분석·· ·· ·· ·· · ·· ······················ ·· ·· · ···· ··············92
• Ⅳ. 고 찰······································································105
• Ⅴ. 요 약······································································111
• Ⅵ. 참고문헌································································113