Thesis
mRev-Erbβプロモーター/エンハンサー領域の同定とその概日リズム性発現機構の解析

徐　海源
埼玉医科大学　生理学

医学博士　甲第1051号　平成19年3月23日 （埼玉医科大学）

略語：BMAL1 (Brain and Muscle Arnt-like 1)
BMAL2 (Brain and Muscle Arnt-like 2)
CLOCK (Circadian Locomotor Output Cycles Kaput)
NPAS2 (Neuronal PAS Domain Protein 2)
PER (Period)
CRY (Cryptochrome)
ROR (Retinoic acid receptor-related Orphan Receptor)
E4BP4 (Adenovirus E4 promoter binding protein 4)
DBP (D-site of Albumin Promoter Binding Protein)
RORE (ROR/Rev-Erb Response Element)

要旨：時計遺伝子Bmal1は暗期にピークのある概日リズム性の発現がある．このリズム性発現はBmal1遺伝子のプロモーター/エンハンサー領域にある2箇所のROREにROR/REV-ERBファミリー（RORα，RORβ，RORγ，REV-ERBα, REV-ERBβ）に属する転写因子が作用し形成されていると考えられている．この中でREV-ERBβは恒常的抑制作用を有するオーファン核内受容体として知られているが，その機能や発現制御機構に関する報告は少なく，特にプロモーター/エンハンサーに関する詳細な研究はなされていないことから，マウスRev-Erbβプロモーター/エンハンサーをクローニングしその解析を試みた．
　マウスゲノムデーターベースではRev-Erbβ転写開始点の下流にギャップが存在することから，このギャップを挟むようにPCRプライマーを設計し，プロモーター領域から第1エクソンおよびイントロン含む約2.1 kbの領域（mRev-Erbβ-F-L）をクローニングした．さらにNIH3T3の培養細胞系でルシフェラーゼアッセイとリアルタイムモニタリングを用いて，時計遺伝子による転写制御機構とリズム性発現に関する解析を行った．
　mRev-Erbβ遺伝子の第1エクソンを含む上流約1.3 kbの領域 (mRev-Erbβ-promoter/enhancer（1.3 K))には典型的な時計遺伝子結合配列はなかったが，第1イントロン領域(mRev-Erbβ-promoter/enhancer (del-673))には3箇所のE-boxとD-boxが存在した．mRev-Erbβ－F-Lは，Bmal1/CLOCK，BMAL2/NPAS2およびDBPによって転写が促進された．リアルタイムモニタリング法ではmRev-Erbβ-promoter/enhancer(1.3 K)にはリズム性の発現がなく，mRev-Erbβ-F-LおよびmRev-Erbβ-promoter/enhancer(del-673) にはリズム性発現が見られ，しかもmRev-Erbβ-F-L の振幅が大きかったことから，第1エクソンを含む上流域はプロモーターとして，第1イントロンはリズム性発現を制御するエンハンサーとして働いていることが明らかとなった．

Transcription and regulation of the mouse Rev-Erbβgene
Haiyuan Xu (Department of Physiology, Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

Keywords: Circadian rhythm, REV-ERBβ, BMAL1/CLOCK, clock genes, transcriptional regulation

Thesis
膵管内乳頭粘液性腫瘍（IPMN）の病理組織学的・免疫組織化学的研究―とくにその組織亜型分類について―

内藤　善久
埼玉医科大学　国際医療センター　病理診断科　坂戸中央病院外科

医学博士　乙第1065号　平成19年10月26日 （埼玉医科大学）

Histopathological and Immunohistochemical Study of Intraductal Papillar y Mucinous　Neoplasm of the Pancreas with Special Reference to Its Histologic Subtypes
Yoshihisa Naitoh (Department of Pathology, Saitama Medical University International Medical Center,Hidaka, Satiama 350 -1298, and Department of Surgery, Sakado-chuuou Hospital, Sakado, Saitama 350-0233, Japan)

　【Background and Aims】Intraductal papillary mucinous neoplasm (IPMN) is one of major cystic neoplasms of the pancreas, uniquely developing in the ductal system. IPMNs have been classified into some subtypes according to their histology and mucin phenotypes. However, the pathologic features of IPMNs in terms of their histologic subtypes have not been fully investigated. The aims of this study were: 1) to reevaluate the histologic subtypes and mucin profiles of IPMNs, 2) to clarify the pathologic features of IPMNs with regard to their histologic subtypes, and 3) to investigate differential expression of some tumor-related factors among the histologic subtypes of IPMNs. 【Design】91 cases of surgically resected IPMNs of the pancreas were subjected to examination. Formalin-fixed and paraffin-embedded sections of each lesion were cut for hematoxylin and eosin staining (HE) and immunohistochemistry. Pathologic features of each lesion were evaluated on HE slides. Immunohistochemistry for representative sections of each lesion were performed for mucins (MUC1, MUC2, MUC5AC, and MUC6), c-erbB-2, COX-2, and cyclin D1, using ENVISION system, and the results were evaluated semiquantitatively. 【Results】 The IPMNs were classified into the following histologic subtypes: (1) 50 cases of Gastric type (showing some resemblance to gastric foveolar epithelium), (2) 30 cases of Intestinal type (showing a colorectal villous tumor-like appearance), (3) 4 cases of Gastric＋Intestinal type (coexistence of Gastric type area and Intestinal type area), and (4) 7 cases of Oncocytic type (arborizing nodular growth of oncocystic epithelium). The mucin phenotype of Gastric type was mostly MUC5AC＋/MUC2－, whereas that of Intestinal type was MUC5AC＋/MUC2＋. Furthermore, histopathologic features were different in many respects between Gastric type and Intestinal type. However, an obvious difference was not determined with respect to the expression of c-erbB-2, COX-2, or cyclin D1. Oncocytic type showed a distinctive mucin phenotype, i.e. MUC1＋ and MUC6＋ in addition to MUC5AC＋. IPMN-derived invasive carcinoma was observed in 7 out of 30 Intestinal type IPMNs (23％) and one out of 50 Gastric type IPMNs (2％). The histologic type of IPMN-derived invasive carcinoma included mucinous carcinoma and conventional type invasive ductal carcinoma. In Intestinal type IPMN-derived invasive carcinoma, MUC2 expression was preserved in mucinous carcinoma in contrast to the conventional type invasive ductal carcinoma. 【Conclusions】IPMNs of the pancreas are classified into several histologic subtypes. The major subtypes are Intestinal type and Gastric type, which show distinctive pathologic features, possibly reflecting different biological behaviors. With regard to IPMN-derived invasive carcinoma, mucinous carcinoma seems to be unique as Intestinal type IPMN-derived invasive carcinoma. Oncocytic type represents a distinctive feature, whereas it is possible that it is related to Gastric type. Histologic subtypes should be considered in the diagnosis and clinical management of IPMNs of the pancreas.
Keywords: pancreas, intraductal papillary mucinous neoplasm (IPMN), histologic type, mucin, immunohistochemistry

Thesis
肝細胞癌に対するラジオ波焼灼療法―段階的焼灼法および必要最小限展開法の開発―

今村　雅俊
埼玉医科大学　内科学消化器・肝臓内科部門
（指導：持田　智 教授）

医学博士　乙第1062号　平成19年9月21日 （埼玉医科大学）

Radiofrequency Ablation for Hepatocellular Carcinoma　- Invention of “the stepwise deployment ablation method” and “the array deployment with minimum requirement diameter method”-
Masatoshi Imamura (Division of Gastroenterology & Hepatology, Internal Medicine, Saitama Medical University,Moroyama, Iruma-gun, Saitama 350 - 0495, Japan )

　The applicant utilizes the expandable arrays technology for this RFA therapy, and invented the two methods of RFA therapies. The first one is “the stepwise deployment ablation method”, which coagulates the center of the tumor first by half deployment, then coagulates entire tumor by full deployment. The other one is “the array deployment with minimum requirement diameter method”, which coagulate the minimum tumor lesion by deploying the arrays with minimally required.
To prove the efficacy of those methods, the applicant conducted the basic research of the process of coagulation in dead cow liver. It was found that the stepwise deployment method enables to create necrotic lesion shorter time than the ablation by the manufacturers recommended ablation algorithm. On the other hand, this method is also clarified that all practical HCC tumor treatment cases are surely able to achieve “roll-off”, the measurement sign of the complete necrosis. In addition, it was clarified that the array deployment with minimum requirement diameter method is minimum invasive ablation procedure, because it does not coagulate the lesions of “non-tumor part” around the ablation target tumor when the tumor size is less than 1.5cm in diameter.
Moreover, these methods enabled to be applied in the tumors such as large-scale HCC or the tumors located right under the diaphragm, which tumors were not in the RFA treatment criteria for the reason of the safety.
Therefore, the RFA treatment therapy by utilizing “the stepwise deployment ablation method” and “the array deployment with minimum requirement diameter method” can be considered as safe and effective therapy for liver malignant tumors.
Keywords: RFA, HCC, The stepwise deployment ablation method, The array deployment with minimum requirement diameter method

Thesis
Blue native 電気泳動法を用いたミトコンドリア呼吸鎖異常症の診断

本多　正和
埼玉医科大学　小児科部門

医学博士　甲第1041号　平成19年3月23日 （埼玉医科大学）

RBlue native polyacrylamide gel electrophoresis is a powerful tool for screening of mitochondrial respiratory chain disorders.
Masakazu Honda (Department of Pediatrics, School of Medicine, Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　Abstract:Mitochondrial respiratory chain disorders are not only the most common but the most clinically diverse group of inborn errors of metabolism. It is often difficult to establish an accurate diagnosis and then to determine the molecular basis of respiratory chain disorders. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a form of native electrophoresis for resolving membrane protein complexes. We used BN-PAGE combined either with in gel enzyme staining or with immunoblotting to analyze 20 patients with possible, probable or definite mitochondrial respiratory chain disorders as defined by the criteria of Bernier et al. A potential complex I abnormality was detected in 11 out of 20 patients, but no abnormalities were found either in the amounts or activities of other complexes. Complex I abnormalities were categorized into three groups based on amount and size of assembled complex I. Combined with conventional enzyme assay, 4 out of 11 patients having complex I abnormality in BN-PAGE analyses were diagnosed as definite complex I deficiency. Tissue specificity in three sisters with complex I deficiency was also identified. We conclude that BN-PAGE is a powerful tool both in the correct diagnosis and as a useful guide to future molecular analysis for respiratory chain disorders.
Keywords: Blue native polyacrylamide gel electrophoresis; Mitochondrial respiratory chain disorders; In gel enzyme staining; Immunoblotting; Tissue specificity