原 著
動脈硬化抑制に関わるAT₁受容体拮抗薬 とエストロゲンの作用機序: 酸化ストレスに対する相互作用を中心に

津田　昌宏
埼玉医科大学腎臓内科学教室〔平成16年11月23日 受付〕

Mechanism of Inhibitory Action of AT₁Receptor Blocker and Estrogen on Atherosclerosis
Masahiro Tsuda (Department of Nephrology, Saitama Medical School, Moroyama, Iruma-gun, Saitama, 350-0495, Japan)

　In the present study, we investigated a gender difference of atherosclerotic changes induced in apoE-deficient (ApoEKO)mice, focusing on oxidative stress, and the possible interaction between olmesartan, an AT₁(angiotensin type 1 receptor)blocker(ARB), and estrogen. After treatment with high cholesterol diet(HCD) for 6 weeks, apparent atherosclerotic lesion formation including lipid deposition and increase in superoxide production and p47phox expression were observed in ApoEKO mice. These changes were significantly greater in male than in female mice, although plasma cholesterol level was not different. Preceding ovariectomy enhanced atherosclerotic lesion and oxidative stress 6 weeks after HCD. The changes in ovariectomized mice were reversed by 17β-estradiol(80μg/kg/day)replacement. On the other hand, olmesartan(3 mg/kg/day)inhibited both atherosclerosis and oxidative stress observed in ApoEKO mice treated with HCD. The inhibitory effect of olmesartan on atherosclerosis was significantly stronger in female than in male and ovariectomized ApoEKO mice. Neither the smaller dose of estrogen(20μg/kg/day)nor olmesartan(0.5 mg/kg/day)influenced atherosclerosis and oxidative stress. However, co-administration of olmesartan and estrogen at these smaller doses attenuated atherosclerosis as well as oxidative stress. We further investigated interaction between AT₁receptor stimulation and estrogen on NADPH oxidase activity using cultured VSMC, which mainly was expressed AT₁receptor. The NADPH oxidase activity in cultured VSMC was increased by Angiotensin II. 17β-estradiol attenuated NADPH oxidase activity induced by angiotensin II without affecting the expression of AT₁receptor. These results indicate that estrogen enhances the inhibitory effect of AT₁receptor blocker on atherosclerosis. Our results also suggest that estrogen and ARB act synergistically to inhibit oxidative stress, by attenuating NADPH oxidase activity.
Keywords: atherosclerosis, estrogen, AT₁receptor blocker, NADPH oxidase
J Saitama Med School 2005;32:1-11
(Received November 23, 2004)

原 著
腎間質線維芽細胞におけるレニン-アンギオテンシン系の局在と機能

菊田　知宏
埼玉医科大学腎臓内科学教室〔平成16年12月3日 受付〕

Existence and Function of Renin-Angiotensin System in Renal Interstitial Fibroblast
Tomohiro Kikuta (Department of Nephrology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　Tubulointerstitial fibrosis (TIF) is considered a valid marker of progression of diabetic and non-diabetic nephropathy, that correlates negatively with creatinine clearance (CCr), and functional outcome. Since a number of clinical trails have revealed that angiotensin converting enzyme inhibitors (ACEi) slowed the rate of decline of renal function in proteinuric patients, it is suggested that ACEi can directly and/or indirectly affect TIF. Therefore, to test this hypothesis, we performed a prospective study for 3 years focusing on the effects of ACEi on functional outcome of patients with IgA nephropathy (IgAN) in relation to the degree of TIF. In the control group treated with amlodipine, the degree of TIF was negatively correlated with the reduction rate of CCr (dCCr), which was consistent with previous observation. By contrast, in the group treated with ACEi, the dCCr index was attenuated compared with the controls, and there was no correlation between the degree of TIF and the dCCr index. The latter suggested that ACEi had independent effects on renal fibrogenesis. Subsequently, we performed in vitro experiments to test whether angiotensin II(Ang II) and aldosterone (ALD) had direct profibrotic effects on cultured human renal fibroblasts. Human renal fibroblasts expressed Ang II type 1 receptor (AT1R) in vivo and in vitro. Ang II stimulated fibroblast proliferation, and type I collagen production of human renal fibroblasts via AT1R, especially in fibroblasts derived from a fibrosing kidney; this effect was partially mediated by secreted TGF-β. ALD could also promote proliferation of fibrosis-derived fibroblasts. In conclusion, ACEi can efficaciously retard the progression of IgAN with and without TIF equally, which is supposed to be partially due to its direct effects on renal fibrogenesis.
Keywords: angiotensin converting enzyme inhibitor, IgA nephropathy, proteinuria, fibrosis, fibroblast
J Saitama Med School 2005;32:13-21
(Received December 3, 2004)

Thesis
各種Epstein-Barr virus 関連Bリンパ芽球様細胞株の比較解析

益田　倫夫
埼玉医科大学小児科学教室
（指導：佐々木　望教授）

医学博士　乙第947号　平成16年6月25日 （埼玉医科大学）現　防衛医科大学校法医学講座

Molecular-Biological Analysis of Various Epstein-Barr Virus Related B Lymphoblastoid Cell Lines. A Comparative Study with Burkitt Lymphoma Cell Lines
Tomoo Masuda (Department of Pediatrics, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan, National Defense Medical College, Department of Forensic Science, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan)

　To clarify the mechanism of malignant transformation associated with Epstein-Barr virus (EBV) infection, we established EBV related B lymphoblastoid cell lines that transformed spontaneously during long term culture of peripheral blood lymphocytes obtained from patients with infectious mononucleosis (spontaneous-BLCLs). The characteristics of spontaneous-BLCLs which were subcultured for more than ten years were compared with those of Burkitt lymphoma cell lines (BL-CLs). In this study, spontaneous-BLCLs and BL-CLs were examined by expression of p53 and sequence ofp53 mRNA, determination of telomere length and telomerase activity, and comprehensive analysis of mRNA by the differential display method. The results showed that, (1) there were mixed cell population with or withoutp53 mRNAmutation in spontaneous-BLCLs, while there were only one type of cells withp53 mRNAmutation in BL-CLs. (2) Immortalization of spontaneous-BLCLs and BL-CLs was not associated with telomere length and telomerase activity. However, there was correlation between telomerase activity and the logarithm of telomere length in spontaneous-BLCLs and BL-CLs (r=-0.85). (3) There were 45 genes related to the oncogenes and the cell cycle regulatory genes which were analyzed by differential display method. In the 45 genes, genes expressed only in BL-Cls wereRas,Raf,RCK/p54etc, and the genes expressed only in spontaneous-BLCLs wereMM-1,XP-G,p58/HHR23Betc, and the genes expressed in both BL-CLs and spontaneous-BLCLs wereHDM-2, activators of RB pathway, etc. Mutant-p53 was only detected in part in immortalized spontaneous-BLCLs, while it was detected in all cell population of BL-CLs with immortalization and malignant transformation. Different oncogenes were expressed between insufficiently tumorigenic spontaneous-BLCLs and sufficiently tumorigenic BL-CLs. With above mentioned finding, we concluded that the expression of mutant-p53 has no role in immortalization of spontaneous-BLCLs, on the other hands, the activation of Rb pathway has major role in immortalization of spontaneous-BLCLs. Suppression ofMM-1andXpgene and/or activation of mutant-p53 and Ras/Raf were probably required in malignant transformation of spontaneous-BLCLs.
Keywords: Epstein-Barr virus, B lymphoblastoid cell lines, p53, telomerase, Rb pathway

医学博士　甲第932号　平成16年3月31日 （埼玉医科大学）

Thesis
Analysis of Galectin-1 Expression in Human Placenta Trophoblasts with Gal3D11, a Novel Monoclonal Anti-Galectin-1 Antibody

Yuriko Yoshino-Ito
Department of Obstetrics and Genecology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan

BACKGROUND: Galectin-1 belongs to the galectin family of lectins, and is expressed in a variety of tissues including placenta, liver, skeletal and smooth muscle. Galectin-1 is thought to be a versatile modulator of cell function, but the biological significance of galectin-1 in human placenta is unclear. The galectin family comprises 14 proteins on the basis of amino acid sequence. METHODS: I raised a novel monoclonal antibody, Gal3D11, against human galectin-1, and characterized the epitope of the antibody using immunoblotting, high performance liquid chromatography, and mass spectrometry. RESULTS: The epitope of Gal3D11 comprises amino acids 29-48 of the galectin-1 protein. In this study, I used Gal3D11 to clarify the expression of galectin-1 at various stages of placental development. I found that galectin-1 was expressed specifically and ubiquitously in placenta trophoblasts, especially in syncytiotrophoblasts, during the first, second, and third trimester. CONCLUSIONS: The widespread expression of galectin-1 in placenta suggests that this protein plays an important role during the development of the placenta. In addition, Gal3D11 would appear to be an indispensable monoclonal antibody with which to further study the function of galectin-1 in different tissues.

原 著
歯槽骨外傷後のデンタルインプラント術後成績

野島淳也，今井謙一郎，都丸泰寿，内藤　実，坂田康彰，安部貴大，嶋村由美子，福島洋介，小林明男，依田哲也
埼玉医科大学口腔外科学〔平成17年1月18日 受付〕

Postoperative Results of a Dental-Implant Technique for Missing Teeth with Alveolar Bone Fracture
NOJIMA Junya, IMAI Kenichiro, TOMARU Yasuhisa, NAITO Minoru, SAKATA Yasuaki, ABE Takahiro, SHIMAMURA Yumiko, FUKUSHIMA Yosuke, KOBAYASHI Akio, YODA Tetsuya (Department of Oral and Maxillofacial Surgery, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan) (Chief: Prof. YODA Tetsuya)

　We evaluated the implant treatment for missing teeth accompanied with an alveolar bone fracture due to trauma. Eight patients were treated with dental implants (AQB system®). In general, for the success of the implant treatment a suitable implant insertion site in the bone is necessary. However, it was difficult to accurately determine the site for the placement of implants since the alveolar bone has a sharp and spiny ridge. To evaluate the postoperative state of implants, imaging diagnosis was used. Dental implantation was established with osseointegration and was fixed in the narrow bone space by cross-sectional CT imaging. Since its osseointegration grew in early period, we recognized the hydroxyapatite (HA)-coated implant system was suitable for narrow bone space for its insertion. However, in some cases, alveolar ridge augmentation becomes necessary when severe bone loss has occurred.
Keywords: alveolar bone fracture, cross-sectional imaging
J Saitama Med School 2005;32:27-30
(Received January 18, 2005)

Original
Anti Tumor Effects of Thalidomide Against Liver Metastasis in a Murine Model of Colon Cancer

Shutaro Ozawa, Hideyuki Tawara, Isamu Koyama
First Department of Surgery, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan

Background: Thalidomide was introduced in the 1950s as a nontoxic sedative, but was removed from the market because of its teratogenicity. Recent studies have demonstrated that the most likely etiology of limb defects produced by fetal exposure to thalidomide is the inhibition of angiogenesis in the developing limb bud. Also many studies have shown that thalidomide inhibits tumor growth in several malignancies by the inhibition of VEGF. Methods: We determined whether the systemic administration of thalidomide inhibits colon cancer liver metastasis in mice. We also evaluated the optimal schedule of this treatment against murine colon cancer liver metastasis. Murine colon cancer CT-26 cells were implanted into the spleens of BALB/c mice. 7 days after tumor implantation, the mice received an intra-peritoneal injection of thalidomide (0, 30 mg/kg) daily or every other day (3 times per week). After the treatments, all mice were sacrificed. The numbers of liver metastases were counted and the expression of VEGF and the micro vessel density were analyzed by immunohistochemistry against liver metastases. Results: 4 out of 10 mice which received a daily administration of thalidomide (30 mg/kg) died. But in this group, we found a significant reduction in the number of liver metastases compared with the control group (0 mg/kg). All mice which received thalidomide every other day survived. In this group, there was also a significant reduction in the number of liver metastases compared with the control. Immunohistochemical analysis revealed lower expressions of VEGF and CD31 in the liver metastases of mice which received thalidomide every other day compared with the control. Conclusion: The systemic administration of thalidomide inhibits liver metastasis of colon cancer in mice by the downregulation of VEGF and angiogenesis. The every other day administration of thalidomide was the optimal schedule in this model.
Keywords: Thalidomide, VEGF, Liver metastasis, tumor vasculature
J Saitama Med School 2005;32:31-36
(Received January 25, 2005)

Abbreviations: FBS, fetal bovine serum; HBSS, Hanks' balanced salt solution; EMEM, Eagle's minimum essential medium; VEGF, vascular endothelial growth factor

原 著
レニン-アンジオテンシン系抑制は非糖尿病性慢性腎疾患患者の血管伸展性を改善する

三村　卓
埼玉医科大学腎臓内科学教室〔平成17年1月27日 受付〕

Blockade of Renin-Angiotensin System with Angiotensin Converting Enzyme Inhibitors and Angiotensin Receptor Blocker Improves Vascular Distensibility in Non-Diabetic Chronic Kidney Diseases
Taku Mimura (Department of Nephrology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　We examined the effects of angiotensin inhibition on pulse wave velocity (PWV) for the patients with non-diabetic kidney diseases. Patients were divided into 4 groups according to their renal function and medication. Patients were followed for a year, and PWV was annually measured. Age, heart rate and systolic blood pressure correlated to PWV (R2=0.58, p<0.0001). Regardless of medications, patients with normal kidney function showed well blood pressure control (130±1/72±1 to 129±2/73±2 mmHg, n=48) and no deterioration of renal function (Scr: 0.85±0.03 to 0.89±0.04 mg/dl), but only patients under angiotensin inhibition manifested a decrease in PWV (1914±84 to 1744±64 cm/sec, n=40, p<0.01). Although blood pressure was maintained below 130/80 mmHg in renal insufficiency patients, renal dysfunction progressed (2.21±0.11 to 2.75±0.18 mg/dl, p<0.01, n=43). With angiotensin inhibition, PWV decreased in this population (1703±64 to 1567±57 cm/sec, n=38, p<0.01). The present findings indicate that age, heart rate and systolic blood pressure constitute important determinants of PWV, and suggest that angiotensin blockade helps keep arterial distensibility in patients with chronic non-diabetic kidney diseases.
Keywords: angiotensin, blood pressure, cardiovascular disease, pulse wave velocity
J Saitama Med School 2005;32:37-43
(Received January 27, 2005)

原 著
インドシアニングリーンを用いた，皮下乳腺全摘出術後の乳頭乳輪の血液灌流の測定

山口　悟，原科　孝雄，Jean Yves Petit^＊
埼玉医科大学総合医療センター形成外科，^＊ヨーロッパがんセンター形成外科（イタリア共和国，ミラノ市）〔平成17年2月15日 受付〕

Evaluation of Skin Perfusion After Nipple-Sparing Mastectomy by Indocyanine Green Dye
Satoru Yamaguchi, Takao Harashina, Jean Yves Petit^＊(Department of Plastic and Reconstructive Surgery, Saitama Medical Center, Kawagoe, Saitama 350-8550, Japan,^＊Department of Plastic and Reconstructive Surgery European Institute of Oncology, Milan, Italy)

　The aim of this study is to investigate the blood supply of the normal nipple-areola complex (NAC) and the spared nipple-areola complex after a nipple-sparing mastectomy by the analysis of the fluorescence resulting from the indocyanine green dye (ICG) injection. At European Institute of Oncology (Milan, Italy), the nipple-sparing mastectomy is a subcutaneous mastectomy which spares a small glandular tissue underneath the areola to preserve the blood supply of the NAC, associated with intraoperative radiotherapy of the remaining glandular tissue. From December 2002 to July 2003 we performed the ICG analyses in 10 monolateral nipple-sparing mastectomy cases (10 healthy breasts and 10 postoperative breasts). In all cases, the resulting fluorescence was registered in three different zones: surrounding mammary skin, areola, and nipple. The slope of the each fluorescence curve was measured. On the healthy breast, the nipple showed a very high perfusion compared with other zones. On the contrary, after the mastectomy the fluorescent pattern was completely altered, being the perfusion of the nipple very low. The perfusion of the surrounding mammary skin was not so changed after surgery. Even more, the perfusion was superior than the healthy breast. In conclusion, these preliminary cases confirm the applicability of the ICG technique to evaluate the perfusion of the healthy and spared nipple-areola complex after surgery. In the future, we can apply this technique to estimate the future-results just after a nipple-sparing mastectomy to avoid NAC necrosis and hypoprojection of the nipple.
Keywords: breast, Surgical Flaps, Dyes, Indocyanine Green, Perfusion
J Saitama Med School 2005;32:45-50
(Received February 15, 2005)

Thesis
大腸癌原発巣におけるThymidylate Synthase, Dihydropyrimidine Dehydrogenase発現の臨床的意義　－フッ化ピリミジン系抗癌剤の治療効果予測との関連－

西　直人
埼玉医科大学外科学 （消化器・一般外科部門　消化器・一般外科（II））
（指導：平山　廉三 教授）

医学博士　乙第959号　平成16年12月24日 （埼玉医科大学）

Gene Expression of Thymidylate Synthase and Dihydropyrimidine Dehydrogenase in Primary Colorectal Cancer
Naoto Nishi (Department of Digestive and General Surgery, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

BACKGROUND: Thymidylate synthase (TS) is a target enzyme for 5-fluorouracil (5-FU), and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme for degradation of 5-FU. Recently, determination of intratumoral TS and DPD is of clinical interest because elevated TS and DPD levels can influence the tumor response to 5-FU based chemotherapy through increased inactivation of the agent in tumor cells. In the present study, TS and DPD mRNA levels were evaluated in 12 cases of primary colorectal cancer with liver metastasis that were surgically resected. Moreover, TS and DPD mRNA levels of primary colorectal cancer with metastatic tumor were analysed in terms of the response of UFT/LV based chemotherapy. PATIENTS and METHODS: (1) TS and DPD mRNA levels were evaluated in 12 surgical cases of primary colorectal cancer with liver metastasis. No one had received 5-FU based chemotherapy prior to the study. (2) TS and DPD mRNA levels of the metastatic colorectal cancer patient who received the UFT/LV based chemotherapy were investigated in 37 cases. RESULT: (1) Measurement of the TS and DPD mRNA level in both primary and metastatic lesions were possible in all 12 cases. The TS mRNA level in hepatic metastatic foci was significantly lower than that in primary lesions (median TS/GAPDH ratio 0.89 and 1.09 respectively, p=0.0047, Wilcoxon signed-ranks test). The DPD mRNA level in hepatic metastatic foci was significantly higher than that in primary lesions (median DPD/GAPDH ratio 0.87 and 0.48 respectively, p=0.0047). Both TS and DPD mRNA had linear relationship between primary colorectal cancer and metastatic liver tumor. (2) The response rate of UFT/LV based chemotherapy with low-TS mRNA (TS≦1.0) was significantly higher than that with high-TS mRNA (TS>1.0) (P=0.038). Similarly, the response rate with low-DPD mRNA (DPD≦0.5) was statistically higher than that with high-DPD mRNA (DPD>0.5) (P<0.0001). The patients with low expression of TS mRNA had significantly longer survival than patients with high value of TS mRNA (P=0.0069). The patients with low expression of DPD mRNA had also significantly longer survival than patients with a high value of DPD mRNA (P<0.0001). CONCLUSION: The results of this study showed that TS and DPD gene expression in primary colorectal cancer is associated with metastatic tumor. It will become possible to predict the efficacy of the UFT/LV based chemotherapy for liver metastasis by analyzing TS and DPD levels of colorectal cancer.
Keywords: Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) 5-fluorouracil (5-FU), colorectal cancer

Thesis
5-Fluorouracilの抗腫瘍効果における活性酸素の役割

河相　開流
埼玉医科大学外科学（消化器・一般外科部門　消化器・一般外科（II））
（指導：平山　廉三教授）

医学博士　甲第927号　平成16年3月31日 （埼玉医科大学）

Role of Reactive Oxygen Species as an Anticancer Effect Induced by 5-Fluorouracil
－ Experimental Study Using a Human Gastric Cancer Cell Line (MKN45) －
Kairyu Kaai (Department of General and gastroenterological Surgery, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　It is well known that in a variety of anticancer agent, such as Mitomycin C, Adriamycin, CDDP, reactive oxgen species (ROS) play important roles in cytotoxic effects to tumor cells. However, as to 5-fluorouracil (5-FU) it has not been clear whether ROS can be induced by 5-FU or not and its related to anticancer effect. Therefore, in order to clarify the role of ROS in cytotoxic process induced by 5-FU, we measured ROS and 8-oxoguanidine (8-OHdG) of 5-FU-treated MKN-45 (human poorly differentiated adenocarcinomatous cells of stomach) and HUVEC (Human umbilical vein endothelial cells) cell lines by flow cytometry. In addition, the relation between ROS and apoptosis was investigated by flow cytometry. Trend of various enzymes related to active oxygen and promoting / inhibiting factors of apoptosis also measured by Western blotting method. Levels of ROS and 8-OHdG(specific maker of DNA damage caused by ROS) increased in MKN-45 cells exposed to CDDP or 5-FU, ROS were increased, whereas 8-OHdG showed no change in HUVEC cells. Level of SOD and p22 proteins increased, whereas catalase showed no change in MKN-45 cells exposed to 5-FU. Although DNA degradation induced by ROS via p53 and NFκB enhancement was found in MKN-45 cells exposed to 5-FU. There are no effect of caspase-dependent apoptotic factors. In summery, our results may indicate that anticancer effect of 5-FU is mediated mainly through the caspase-independent apoptosis via the ROS induction.
Keywords: 5-FU, reactive oxygen species, 8OHdG, apoptosis

Thesis
ビタミンD 結合タンパク(DBP)遺伝子多型と閉経後女性における骨量・骨代謝マーカーとの関連

清水　省志
埼玉医科大学産婦人科学教室
（指導：畑　俊夫教授）

医学博士　甲第931号　平成16年3月31日 （埼玉医科大学）

　骨は形成・吸収（リモデリング）を繰り返しながら，その構造，骨量，強度および体液のカルシウム濃度を維持している．加齢，閉経等により，リモデリングが破綻して骨量が低下し，骨の微細構造が変化して脆弱性が亢進した疾患が骨粗鬆症であり，高齢化社会が進むなか老後のQOLを左右する大きな因子と考えられる．
　加齢における骨粗鬆症との相関を示す遺伝子マーカーを検索するために，骨代謝に関連すると想定される遺伝性因子（Luteinizing Hormone-β subunit：LH-β，α2-macroglobulin：A2M，Tumor Necrosis Factor-α：TNF-α，Vitamin D-Binding Protein：DBP）に着目し，その遺伝子多型と骨量，骨代謝マーカーについて解析し検討を行った．
　健康なボランティアの閉経後女性236名（年齢41-91歳，閉経後1年以上，平均年齢±SD;64.3±9.2歳）の血液一般生化学検査，骨形成マーカーとしてアルカリフォスファターゼ，オステオカルシン，骨吸収マーカーとしてピリジノリン，デオキシピリジノリンそして脊椎骨密度データを用いた．
　LH-β遺伝子はエクソン2（Trp8Argおよび Ile15Thr置換），A2M遺伝子はVal1000Ile置換，TNF-α遺伝子はプロモーター領域の－308G/A，－857C/Tの2カ所の置換，DBP遺伝子はエクソン11（Glu416Asp およびLys420Thr置換）について検討したところ，LH-β遺伝子，A2M遺伝子，TNF-α遺伝子では多型と骨密度，骨代謝マーカーとの有意な相関は示さなかった．一方，DBP遺伝子多型では骨代謝マーカーであるアルカリフォスファターゼ，オステオカルシンおよびデオキシピリジノリンについて有意な相関が示された．DBPはビタミンDと結合し運搬する役割があり，骨代謝に影響をおよぼしている可能性が考えられるが，DBP遺伝子多型と骨密度との相関は有意ではなかった．
　本研究により，DBP遺伝子多型と種々の骨代謝マーカーとの相関を示し， 骨代謝回転状態や将来における骨粗鬆症発症の遺伝子マーカーの候補となりうることが示唆された．
Keywords: DBP，LH-β，A2M，TNF-α，遺伝子多型，骨粗鬆症，骨吸収，骨形成

Thesis
組織型別にみた胃癌におけるThymidylate Synthase, Dihydropyrimidine Dehydorogenase発現の検討

須藤　謙一
埼玉医科大学外科学（消化器・一般外科部門　消化器・一般外科（II））
（指導：平山　廉三 教授）

医学博士　甲第966号　平成17年2月18日 （埼玉医科大学）

Gene Expression of Thymidylate Synthase and Dihydropyrimidine Dehydrogenase in Gastric Cancer in Tems of Histolgical Type
Kenichi Suto (Department of Digestive and General Surgery, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　S-1 is a new oral fluorinated pyrimidine, in which tegafur (FT) has been combined with two substances as modulator; gimeracil (CDHP), and oteracil potassium (Oxo), in a molar ratio of 1:0.4:1 (FT:CDHP:Oxo). 5-FU is catabolized to dihydrofluorouracil by the first and rate-limiting enzyme, dihydropyrimidine dehydorogenase (DPD).The main mode of action of 5-FU is thought to be through its active metabolite suppresses thymidylate synthase (TS), that subsequently inhibit DNA syntheses. The predictive values of TS and DPD gene expressions in primary gastric cancer in terms of differentiated and undifferentiated type cases were also evaluated and the 2 gene expressions were retrospectively evaluated in patients with gastric cancer treated by a regimen containing S-1. The study population consisted of 78 patients with advanced gastric cancer who underwent surgical treatment and without S-1 chemotherapy 27 patients treated by S-1-alone for metastatic gastric cancer. Laser-captured micro dissection of malignant tissue was performed in formalin-fixed, paraffin-embedded specimens. After extraction RNA, TS and DPD gene expressions were measured by the real-time reverse transcriptional PCR method. TS gene expression was higher in differentiated type cases than undifferentiated type cases (p<0.001).However, DPD gene expression of undifferentiated type cases was statistically higher than that of differentiated type cases (p<0.05). There was no statistical difference in DPD gene expression in term of response in 27 cases treated with S-1. TS mRNA of responding tumors was statistically lower than that of nonresponding ones when treated with S-1 (p<0.005), and patients with low TS gene expression survived longer than those with high TS gene expression, with statistical significance (p<0.0001). TS and DPD gene expressions in primary gastric cancer differ according to degree of histological differentiation. In addition S-1 treatment effect for metastatic gastric cancer might be determined by depending on the amount of TS expressions, regardless of DPD gene expressions.

Thesis
穿孔性腹膜炎におけるビリルビン酸化生成物(Biopyrrins)ならびにヘムオキシゲナーゼの動向　─ 実験的ならびに臨床的検討 ─

菊地　政貴
埼玉医科大学外科学（消化器・一般外科部門　消化器・一般外科（II））
（指導　平山　廉三　教授）

医学博士　乙第971号　平成17年2月18日 （埼玉医科大学）

　Heme oxygenase(HO)-1, the rate-limiting enzyme in heme degradation, is induced by oxidative stress and its major final product, bilirubin, is a potent physiological antioxidant. Bilirubin oxidative metabolites are generated from bilirubin as a result of its scavenging action against reactive oxygen species (ROS). Thus, the production of Biopyrrins, oxidative metabolites of bilirubin, appears to reflect the degree of oxidative stress, and the level of urine Biopyrrins may be useful for evaluation of oxidative stress. The purpose of this study was to clarify the target organs for oxidative stress by acute abdomen, based on the change of urine Biopyrrins levels and HO-1. The levels of urine Biopyrrins and expression of HO-1 were determined from mice cecal ligation and puncture (CLP) model , the lipopolysaccharide injected intraperitoneally (LPS I.P.) and intravenously(LPS I.V.) models. The expression of HO-1 protein was shown at liver in CLP model, at spleen in LPS I.V. model, and at both of liver and spleen in LPS I.P. model. The level of urine Biopyrrins changed in accordance with the expression of HO-1 in all models. The level of urine Biopyrrins in 71 cases of acute abdomen was determined, and compared with the criteria of SIRS (systemic inflammatory response syndrome). In the perforated peritonitis cases, high level of urine Biopyrrins were shown despite of that these cases did not satisfy the criteria of SIRS. These results suggest that the level of urine Biopyrrins is a possible marker for continuous monitoring of clinical severity of perforated peritonitis cases.
Keywords: Biopyrrins, Bilirubin, Heme oxygenase, 活性酸素, 腹膜炎

Thesis
マウス線維芽細胞3T3-L1細胞が脂肪細胞に分化する際の細胞内情報伝達経路の検討

富山　浩二
埼玉医科大学内科学内分泌・糖尿病内科部門
（指導　片山　茂裕　教授）

医学博士　乙第973号　平成17年3月25日 （埼玉医科大学）

　マウス線維芽細胞3T3-L1細胞は，isobutyl-methylxanthine（IBMX）, dexamethasone（DEX）, insulinによって効率良く脂肪細胞に分化する．Insulin受容体はチロシンリン酸化型の受容体の一つで，その生理作用の多くはphosphatidylinositol 3-kinase（PI3-kinase）によって介される．そこで特異的PI3-kinase阻害薬であるwortmanninを用い，その脂肪細胞分化に及ぼす影響を見た．Wortmanninはinsulinで3T3-L1細胞を刺激した際のPI3-kinase活性を濃度依存的に抑制し，また3T3-L1細胞の脂肪細胞への分化も同様に濃度依存的に抑制した．次に分化過程をDay 0-2の初期相とDay 2-4の後期相に分け，どちらか一方のみに薬剤を添加した場合の分化に及ぼす影響を見た．Wortmanninは分化の初期相，あるいは後期相のみに用いた場合も，3T3-L1細胞の分化を約50％に抑制した．同様の実験を3-Hydroxy-3-methylglutaryl coenzyme A（HMG-CoA）還元酵素阻害剤simvastatinで行ったが，wortmannin同様後期相のみに用いても分化は抑制された．これらの結果から，3T3-L1細胞の脂肪細胞への分化においてPI3-kinaseが重要な役割を演じていること，分化の過程における細胞内情報伝達経路は，その持続的な活性化のみでなく，初期相，あるいは後期相それぞれが単独でも重要であることが示唆された．

Thesis
ヒトVα24インバリアントNKT細胞サブセットによる樹状細胞を介した免疫制御機構の解析

鈴木　元晴
埼玉医科大学産婦人科学教室
（指導　石原　理　教授）

医学博士　甲第976号　平成17年3月25日 （埼玉医科大学）

　ヒトインバリアントNKT（iNKT）細胞は，非古典的MHC class I分子であるCD1d分子に拘束される従来のTリンパ球とは異なるTリンパ球サブセットである．iNKT細胞の活性化により，自然免疫応答を担当するNK細胞や樹状細胞（Dendritic cells；DCs），あるいは獲得免疫応答を担当するB細胞やT細胞など，多くの細胞の迅速な活性化，およびこれらの連鎖反応が誘導される．また，iNKT細胞はTh1，Th2タイプの免疫応答を制御するだけでなく，種々の免疫応答において重要なエフェクター機能を有している．これまでiNKT細胞は，Th2応答を促進し，多くの臓器特異的自己免疫疾患発症の防止に寄与しているとの報告がある．しかし一方でiNKT細胞はTh2免疫応答を抑制し，IL-12によって誘導されるTh1免疫応答を促進し，腫瘍拒絶や感染防御などに関与するとの報告もある．これらの矛盾した現象は，異なる機能を持つiNKT細胞サブセットの存在に起因することが示唆される．実際，iNKT細胞には，CD4^＋，CD8^＋，CD4^－CD8^－（double negative：DN）サブセットが存在し，それぞれが産生するサイトカインが異なることが知られている．
　我々は，DCを介したCD4^＋T細胞の分化におけるヒトiNKTサブセットの役割をin vitroで評価する実験系を構築した．このシステムは，異なるiNKT細胞サブセットと共培養されたmonocyte由来DC （Mo-DCs）とアロのnaive CD4^＋T細胞を共培養することでhelper分化を評価するものである．これによって，TCRリガンドによって活性化されたCD4＋，およびDN iNKT細胞サブセットは，DCに対してIL-12p70の産生を促し，完全な成熟を誘導することが明らかとなった．しかし，CD4^＋iNKTと共培養されて成熟したMo-DCは，naive CD4^＋T細胞をTh1に分化誘導した（IFN-γの高産生性とIL-4の低産生性）．一方，DN iNKTと共培養されて成熟したMo-DCは，naive CD4^＋T細胞をTh2に分化誘導した（IL-4の高産生性とIFN-γの低産生性）．さらに，CD4^＋iNKTによって成熟したDCとは対照的に，DN iNKTによって成熟したDCは，stimulator/responder比1：30でMLR抑制活性を示した．これらの効果は，iNKT細胞を蛋白合成阻害剤であるemetineで処理することにより消失した．
　以上より，ヒトiNKT細胞サブセットのバランスは，T細胞応答の方向性を決定するDCの分化に重要な要素であることが明らかとなった．このメカニズムは，ヒトにおける自己免疫疾患の発症に重要な役割を演じているかもしれない．また，自己免疫疾患治療におけるDN iNKT細胞の有用性を示唆するものである．

Analysis of Dendritic Cell-Mediated Immune Regulation by Human Vα24 Invariant NKT Cell Subsets
Motoharu Suzuki (Department of Obstetrics and Gynecology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)