原 著
Angiotensin IIと物理的圧の接着因子発現に及ぼす協調作用　－培養メサンジウム細胞での検討－

荒井　充
埼玉医科大学腎臓内科学教室〔平成15年10月31日 受付〕

Synergistic Action of Angiotensin II and Transmural Pressure on Expressions of Adhesion Molecules in Cultured Rat Mesangial Cells
Mitsuru Arai (Department of Nephrology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

Objective: Mesangial cell dysfunction is produced in response to various stimuli. The role of both physical factors and humoral mediators in mesangial cell have been investigated, however, there are few studies examining the synergistic action of these factors. In the present study, to investigate the role adhesion molecules in the regulation of cellular function, we assessed the effect of pressure and angiotensin II in the production of endotelin-1 (ET-1) and nitric oxide (NO) from cultured rat mesangial cells (RMC). Furthermore, the effect of antisense of E-selectin in the production of ET-1 and NOx in cultured RMC were examined. Design and Methods: Rat mesangial cells were harvested from Sprague Dawly (SD) rats. Rat mesangial cells were plated onto 75-cm²flasks. A pressure loading apparatus was set up by using compresses He gas. Transmural pressure (0, 50, 100 or 200 mmHg) was applied for 24 hours with angiotensin II (10^-6, 10^-7and 10^-8M). Every 6 hours, cultured media was collected for measurements of ET-1 and NOx. The expression of ecNOS-mRNA was measured by using RT-PCR. The expression of adhesion molecules (integrin α5β1, VCAM1, E-selectin) in the cultured RCM were examined by using immunofluorescens method. The antisense of E-selection was added in the cultured RMC and measured the expression of ET-1 and NOx. Then angiotensin type I receptor blocker (CS866) was added onto cultured media. Results: Either transmural pressure or angiotensin II alone did not induce any significant changes in ET-1 concentration and expression of adhesion molecules. The expression of E-selectin appeared in RCM in accordance with the levels of transmural pressure and the concentrations of angiotensin II. Combined treatment with transmural pressure and angiotensin II produced a marked elevation of ET-1 and reduction of expression of ecNOS-mRNA. Administration of antisense of E-selection in RCM induced the reduction of ET-1 accompanied with the suppression of the expression of E-selection. However there was no significant change in the expression of ecNOS-mRNA. Incubation with CS866 in RMC, adhesion molecule of E-selectin disappeared accompanied with the reduction of ET-1. Conclusions: In conclusion, synergistic action of transmural pressure and renin-angiotensin system may induce the expression of adhesion molecule E-selection accompanied with the increase of ET-1 and reduction of NO in the cultured RMC. These findings suggest that RMC can produce E-selection in response to synergistic action of intraglomerular pressure and angiotensin II and resulted in nephrosclerosis. E-selection plays an important role in the regulation of the production of ET-1 in cultured media. On the contrary, NO production was not related to the expression of E-selection.
Keywords: hypertention, E-selection, adhesion molecule, endothelin-1 (ET-1), Nitric Oxide (NO)
J Saitama Med School 2004;31:1-12
(Received October 31, 2003)

原 著
アリストロキア酸中毒性腎症に対するHepatocyte Growth Factor (HGF)の線維化抑制作用の検討

渡辺　裕輔
埼玉医科大学腎臓内科学教室〔平成15年11月1日 受付〕

Hepatocyte Growth Factor Attenuates Reactive Renal Fibrosis in Aristolochic Acid Nephrotoxicity
Yusuke Watanabe (Department of Nephrology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　Despite the diverse initial causes, chronic renal disease that progress to end-stage renal failure is a remarkably monotonous process that is characterized by the relentless accumulation of extracellular matrix (ECM) leading to widespread interstitial fibrosis. Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocyte, shows mitogenic, morphogenic and anti-apoptotic activities for a wide variety of cells including renal tubular epithelial cells. And HGF has been demonstrated to attenuate acute tubular necrosis and interstitial fibrosis in some of rodent models of kidney disease. But the mechanism of anti-fibrotic effects of HGF has been poorly understood in detail. Then, using HGF transgenic mice, we investigated how HGF could affect chronic toxic nephropathy/interstitial fibrosis caused by a nephrotoxin, aristrochic acid (AA). To find out molecular mechanisms of anti-fibrotic effects of HGF, cultured murine tubular epithelial cells (mProx24) were also employed. Significant tubular degeneration was observed both in the transgenic and the wild-type mice to the same degree after 2 week's treatment with AA. Interstitial fibrosis subsequently developed in the wild-type mice 4 weeks after cessation of AA administration. However, the transgenic mice manifested less fibrotic changes. Decreased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) could partially account for the attenuation of fibrogenesis in the transgenic mouse kidney. HGF at 10 ng/mL and 100 ng/mL could block TIMP-1 gene expression in mProx24 induced by epidermal growth factor (EGF), but a decrease in the number of mProx24 via apoptosis induced by AA was blocked only by HGF at 100 ng/mL.
　In conclusion, circulating transgene-derived HGF (2～10 ng/mL) could not prevent tubular degeneration caused by AA, but facilitate its regeneration without significant fibrogenesis. These findings suggest possible therapeutic efficacy for renal interstitial fibrosis following tubular degeneration even of low-dose HGF.
Keywords: hepatocyte growth factor (HGF), aristolochic acid (AA), renal interstitial fibrosis, tissue inhibitor of metalloproteinase-1 (TIMP-1), epidermal growth factor (EGF), type I collagen
J Saitama Med School 2004;31:13-24
(Received November 1, 2003)

原 著
前立腺癌でみられるアンドロゲンレセプター変異体のリガンド結合の特異性

床鍋　繁喜
埼玉医科大学泌尿器科学教室〔平成15年11月13日 受付〕

Ligand Binding Specificity of Androgen Receptor Mutants detected in Prostate Cancer
Shigeki Tokonabe (Department of Urology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　Advanced prostate cancer may often progress as an androgen independent tumor in spite of androgen ablation therapies or medication of antiandrogen drugs. This diverse resistance to the hormonal therapies is thought to be due to the amplified, overexpressed or mutated androgen receptor (AR). Previous studies indicated that the mutated AR loses the specificity to the ligands. However, the functional relationship between mutations of AR and specificity of ligands are poorly understood. In this report, the author shows the dissociation constants (Kd) of mutated ARs to eight ligands including an antiandrogen drug (hydroxyflutamide). Only one amino acid mutation in the ligand-binding pocket, can cause a significant change on the binding affinity. Loss of ligand specificity is observed in the mutated AR such as L701H, H874Y, T877A and T877S, of which mutation is located in 3 or 11 α-helix of the ligand binding domain. Various changes of the affinity are seen among the mutants, which suggest that the mutated ARs could be different mechanisms for tumor growth in prostate cancer cell.
Keywords: androgen receptor, mutation, prostate cancer
J Saitama Med School 2004;31:25-31
(Received Nov 13, 2003)

原 著
Survivinの甲状腺濾胞上皮細胞増殖および分化に対する影響

鈴木　美穂
埼玉医科大学内科学教室（内分泌糖尿病内科部門）〔平成15年11月28日 受付〕

Role of Survivin on the Proliferation and Differentiation of Thyroid Epithelial Cells
Miho Suzuki （Department of Endocrinology and Diabetes, Institute of Internal Medicine, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan）

　Survivin is one of the IAP (inhibitor of apoptosis proteins), which is expressed during embryonic development, but is not expressed in terminal differentiated adult tissue. Overexpression of survivin is observed in a variety of human tumors including thyroid cancers. We examined the role of survivin on the proliferation and differentiation of thyroid epithelial cells. Rat thyroid epithelial cell line FRTL-5 was stably transfected with human sense and antisense survivin genes. FRTL-5 cells transfected with a survivin sense gene (S cells) proliferated without TSH. In the presence of TSH, the proliferation of S cells was significantly faster than that of cells transfected with an empty vector (E cells) or cells transfected with a survivin antisense gene (AS cells). The proliferation of AS cells were retarded as compared with that of E cells. Western blot analysis demonstrated that phosphorylation of Rb protein at serine 795 was enhanced in S cells. Activation of E2F-1 was also enhanced in S cells as demonstrated by gel shift assay. Northern blot analysis showed that expression of Pax-8 mRNA was significantly enhanced in S cells, and was significantly decreased in AS cells. Expression of thyroglobulin (Tg) and TSH receptor (TSHR) mRNA was decreased in AS cells. There was no significant change in TTF-1 and TTF-2 mRNA expression. These observations suggest that survivin enhances the proliferation of thyroid epithelial cells through activation of E2F-1, and also enhance the expression of Pax-8, which then affect the expression of thyroid specific proteins. Survivin appears to play an important role in the proliferation as well as differentiation of thyroid epithelial cells.
Keywords: Survivin, thyroid cancer, cell cycle, differentiation, Rb protein, E2F-1, Pax-8, Tg, NIS, TSHR
J Saitama Med School 2004;31:33-39
(Received November 28, 2003)

原 著
心不全の形成過程におけるEstrogenの心臓リモデリングおよび腎組織にあたえる影響
－Dahl食塩感受性ラット心不全モデルを用いた検討－

福島　理恵
埼玉医科大学腎臓内科学教室〔平成15年12月10日 受付〕

Effects of Estrogen on the Process of Cardiac Remodeling and Nephrosclerosis in Dahl Hypertensive Rats with Heart Failure
Rie Fukushima （Department of Nephrology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan）

Objective: This study was attempted to elucidate the role of estrogen in the process of cardiac remodeling and nephrosclerosis in Dahl-S female rats with myocardial infarction. Methods: 48 Dahl-S rats with myocardial infarction (MI) were divided into four groups as follows; Group 1: MI rats with normal-salt diet (LS, 0.5%NaCl diet), Group 2: MI rats with high-salt diet (HS, 8%NaCl diet), Group 3: MI rats with ovariectomy (OVX, 8%NaCl diet), Group 4: OVX rats with supplementation of estrogen (EST) (8%NaCl diet, 17βestradiol 15 mg/pellet/90 days subcutaneous pellet). Myocardial infarction was made by coronary artery ligation. One week after surgery, ovariectomy was performed. After ovariectomy, high salt diet was fed and water was given ad libitum for 12 weeks. During the study, blood pressure (BP), heart rate, body weight, urine volume and urinary excretions of protein and sodium were measured every 2 weeks. Transthoracic echocardiogram (ejection fraction: EF, inter ventricular septal thickness: IVST) was performed under anesthesia at 12 weeks after surgery. At the end of the study, the heart and the kidney tissues were obtained for light microscopic examination and evaluations of the expression of mRNA of ACE (angiotensin converting enzyme) and ecNOS (nitric oxide synthase). Results: 1. Blood pressure in HS group showed a gradual elevation from 132＋/－8 to 152＋/－10 mmHg (12 weeks) accompanied with the reduction of EF (LS:64＋/－4% vs. HS:46＋/－3%, P<0.01) and increases of IVST. Ovariectomy induced significant reduction in EF (OVX:26＋/－3%, P<0.01 vs. HS) and increases in IVST without any significant changes in systemic BP (OVX:174＋/－12 mmHg). 2. Ovariectomy induced no significant changes in plasma concentration of ACE, PRA (plasma renin activity), angiotensin I and angiotensin II. 3. In the heart obtained from OVX rats, expression of ecNOS-mRNA was decreased and ACE-mRNA was increased. Ovariectomy induced aggravation of cardiac hypertrophy and fibrosis. Treatment with estrogen in OVX rats improved EF (EST:38＋/－4%, P<0.01 vs. OVX: 26＋/－3%) and cardiac hypertrophy, although blood pressure remained at the levels of 168＋/－8 mmHg. 4. In the heart obtained from EST rats, expression of ecNOS-mRNA was significantly increased and ACE-mRNA was significantly suppressed compared to HS. 5. In HS rats, urinary excretion of protein was increased (182.2＋/－40.2 mg/day). Ovariectomy induced the reduction of urinary excretion of protein (125.4＋/－23.4 mg/day) and estrogen supplementation induced a significant increases of urinary excretion of protein (352.2＋/－48.0 mg/day). 6. Light microscopic findings in the kidney showed glomerulosclerosis and interstitial fibrosis in HS rats. Estrogen supplementation induced the aggravation of glomerulosclerosis and interstitial fibrosis accompanied with microangiopathy and thrombosis. Conclusion: These data indicate that estrogen might protect the development of cardiac hypertrophy and consuming heart failure. However, estrogen promotes microangiopathy and thrombosis in the kidney with progression of kidney insufficiency in Dahl-S rats with heart failure.
Keywords: Hypertension, Myocardial Infarction, salt-sensitivity
J Saitama Med School 2004;31:41-54
(Received December 10, 2003)

原 著
IgA腎症患者の末期腎不全進行における腎疾患関連遺伝子多型の影響

小滝　周平
埼玉医科大学腎臓内科学教室〔平成15年12月9日受付〕

Impacts of Polymorphisms in Genes Encoding Renal Disease-related Molecules on Progression to End-stage Renal Failure in Patients with IgA Nephropathy
Shuhei Kotaki (Department of Nephrology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

Background/Purpose: Polymorphisms found in genes encoding proinflammatory/profibrotic molecules have been demonstrated to be associated with several kinds of renal diseases. We investigated these polymorphisms in IgA nephropathy (IgAN) patients undergoing hemodialysis (HD) and their significance on progression to end-stage renal failure (ESRF). Methods: To evaluate the role of polymorphisms in genes described below, we analyzed the association of these polymorphisms with progression to ESRF in histologically-proven IgAN patients undergoing HD using Kaplan-Meier method. Employed genes are angiotensin-converting enzyme, angiotensinogen, monocyte chemoattractant protein-1, chemokine receptor (CCR5), transforming growth factor-β1, E-selectin (SELE), and L-selectin (SELL). Results: The duration between renal biopsy and ESRF (induction to HD) was significantly shorter in IgAN patients with T1402 allele at the SELE gene, G(-642) allele or T712 allele at the SELL gene than those without them. Any other polymorphisms were not significantly associated with progression to ESRF in this study. Conclusion: This work provides evidence that the T1402C polymorphism of the SELE gene, A(-642)G and C712T polymorphisms of the SELL gene are associated with rapid progression to ESRF in IgAN patients.
Keywords: IgA nephropathy, end-stage renal failure, polymorphism, E-/L-selectin
J Saitama Med School 2004;31:55-66
(Received December 9, 2003)

原 著
高齢者大動脈弁狭窄症例における大動脈弁石灰化度と頸動脈動脈硬化病変との関連に関する検討

長崎　治能
埼玉医科大学循環器内科学教室〔平成15年11月28日 受付〕

Correlation Between Calcific Aortic Valvular Changes and Atherosclerosis in Carotid Arteries in Elderly Patients with Aortic Stenosis
Harutaka Nagasaki (Division of Cardiology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　To examine the association among the sclerotic and calcified changes in the aortic valve, atherosclerotic changes in carotid arteries and cardiovascular risk factors in the elderly patients (>60 years old) with aortic stenosis (AS)<1.5 cm2(aortic valve area), 49 consecutive patients (average 75.5 years old, 23 male and 26 female) were prospectively studied. The degree of calcification of the aortic valve was classified into four groups as follows, group I; no calcification, group II; mildly calcified (small isolated spots), group III; moderately calcified (multiple larger spots), and group IV; heavily calcified (extensive thickening and calcification of all cusp). We quantitatively evaluated the carotid atherosclerosis by using carotid ultrasonography. The upper limit of normal for the IMCT (intima-media complex thickening) was defined as 1.0 mm, and lesions with an IMCT≧1.1 mm were defined as atheromatous plaques. The traditional cardiovascular risk factors (smoking, hypertension, total cholesterol and HDL-cholesterol, diabetes) and high-sensitivity CRP and fibrin were simultaneously assessed. All patients had the calcified aortic valve. The patients in group III (n=22) and IV (n=18) were 81% of patients studied. Twenty-three (66%) of 35 patients who underwent carotid ultrasonography had atheromatous lesions. There was no significant correlation between calcific aortic valve changes and stenotic severity in this series. Although the increased LDL-cholesterol level, decreased HDL-cholesterol level and increased HbA1c% were significantly associated with severity of atherosclerotic changes in carotid arteries, they were not correlated with the severity of calcific aortic valvular changes. In conclusion, severity of calcific valvular changes in the elderly patients with aortic stenosis were not associated with cardiovascular risk factors, but carotid atherosclerosis was significantly correlated with them. These results suggest the difference in pathogenesis and development between calcific changes in aortic stenosis and carotid atherosclerosis in the elderly populations.
Keywords: calcific aortic valvular changes, atherosclerosis in carotid arteries, aortic stenosis, cardiovascular risk factors
J Saitama Med School 2004;31:67-72
(Received November 28, 2003)

Original
Development of a Totally Implantable Artificial Heart
Saitama Medical SchoolType TotalArtificialHeart: STAH

Hiroyuki Noda, Shunei Kyo, Ryozo Omoto¹⁾
Department of Cardiovascular Surgery, Saitama Medical School Moroyama, Iruma-gun, Saitama 350-0495, Japan,¹⁾Saitama Medical School Hospital

　A prototype of Saitama Medical School type Total Artificial Heart (STAH) was newly developed. STAH designing is to be installed totally inside the mediastinal cavity replacing the natural heart. Because of the sophisticated shape of the blood pump based on an anatomical view, as well as separation of the blood pump from the actuator, the whole system is resulted in good feasibility for anatomical fitting. The principle of drive mechanism is that two pusher plates eject the blood in the left and the right chambers of the blood pump alternately, by driving a simple cam with an electromechanical actuator. This prototype has proven to eject 4.6 L/min on a mock circulatory system. Although further efforts will be necessary for a practical application, the STAH pump may have a possibility to be one of the most promising devices as a total artificial heart.
Keywords: total artificial heart, electromechanical pump, cam system
J Saitama Med School 2004;31:73-81
(Received September 12, 2003)

原 著
マウス胎仔腎臓由来細胞による培養下でのrenal tubulogenesisにおける形態学的検討

永野　忠相
埼玉医科大学総合医療センター第4内科〔平成16年1月9日 受付〕

The organoid culture method using completely isolated cells from fetal organ is a useful tool for studying organogenesis. Using this method, we investigated the renal development, especially renal tubulogenesis, by a combination of electronmicroscopy, lectin-histochemistry, and reverse transcription linked polymerase chain reaction (RT-PCR). Completely isolated cells from fetal mouse kidneys were seeded onto a membrane filter. After 7 days in culture, a dome-shaped cellular mass consisted of tubule-like structures was formed. Some short microvilli and basement membrane were observed. After 14 days in culture, microvilli became longer, and the number of microvilli was increased. The tubule-like structure showed a positive staining of lectins[Dolicos biflorus agglutinin (DBA) and Wheat germ agglutinin (WGA)]. Expression of aquaporin-2 (AQP-2) could be detected after 1 day in culture. When the culture periods were prolonged, the expression of AQP-2 was not detectable. Based on the findings of morphological features and lectin-histochemical staining patterns, we concluded that the tubule-like structure was recognized as a primitive collecting duct. Organoid culture method presented here is a useful tool for studying the development of the kidney.
Keywords: kidney, culture, tubulogenesis, lectin, collecting duct, aquaporin
J Saitama Med School 2004;31:89-94
(Received January 9, 2004)

原 著
マウスおよびヒト胎盤におけるEPC-1/PEDF遺伝子の発現

臼井　真由美
埼玉医科大学総合医療センター産婦人科〔平成16年1月19日 受付〕

Expression of Early Population Doubling Level cDNA-1(EPC-1)/Pigment Epithelium-derived Factor (PEDF) in Mice Placentae and Human Placentae
Mayumi Usui (Department of Obstetrics and Gynecology, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama 350-8550, Japan)

　Whereas there are many hypotheses on the causes of senescence of the body, it has not yet been elucidated whether the placenta, which is to be delivered from the mother after pregnancy for ten months, is also subjected to senescent changes. Namely, is the senescence already started in the placenta of late pregnancy, as it is a matured placenta?
　In this paper, a gene, early population doubling level cDNA-1/pigment epithelium-derived factor(EPC-1/PEDF), was used as a marker to examine senescent changes. This gene has already been used as an ageing marker for tissues although its expression level is fluctuated according to cell cycle, ageing, cell differentiation, etc. We noticed that, when the expression of EPC-1/PEDF mRNA was detected by RT-PCR, its level was decreasing in the placenta of pregnant mice as pregnancy progressed. Next we performed real-time quantitative PCR to detect EPC-1/PEDF mRNA in 19 cases of human placentas at 27-40 weeks of gestation, obtained upon elective cesarean deliveries done prior to the onset of labor. Its expression was decreased as pregnancy progressed. The 8 cases of vaginal delivery and the 5 cases of emergency cesarean deliveries after onset of labor gave lower values than the cases subjected to elective cesarean delivery. In cases of 8 dizygotic twins pregnancy at 32-38 weeks of gestation including two cases of discordant twins, the expression of EPC-1/PEDF mRNA was detected by real-time quantitative PCR. The placentae from the smaller neonates gave extremely lower values than the normal placentae from the lager ones.
　These findings indicated that the placenta is an organ subjected to senescent changes. The onset of labor may be related with ageing of the placenta. In addition, it was suggested that the placentas with intrauterine growth retardation could show placental dysfunction accompanying senescent changes in the uterus.
Keywords: EPC-1, PEDF, Placenta, Pregnancy, senescent, ageing, onset of labor, Twin, IUGR
J Saitama Med School 2004;31:95-101
(Received January 19, 2004)

原 著
心筋細胞興奮収縮連関におよぼす第3のキナーゼ系Rhoキナーゼの役割について

茆原　るり
埼玉医科大学第二内科学教室〔平成16年1月13日 受付〕

The Role of Rho Kinase : Third Kinase System in the Regulation of Excitation-Contraction Coupling of Cardiac Muscle
Ruri Chihara (Second Department of Internal Medicine, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　It has been known that various neurohumoral factors play important roles in the regulation of contraction and relaxation in cardiac muscle. Earlier reports suggested that angiotensin II play important roles in the pathogensis of chronic heart failure. However, the roles of endothelin 1 have not been fully clarified. Thus, we investigated the roles of endothelin in excitation-contraction coupling and relaxation in isolated single ventricular myocytes. We focused on the third kinase system: Rho dependent protein kinase (ROCK) in addition to the protein kinase A and C. We isolated single ventricular myocytes from Wister rats and measured intracellular calcium transients and cell contraction simultaneously. When we pretreated cells with PKC inhibitor, exposure to endothelin-1 still produced positive inotropic and lusitrophic effects. These effects were not observed when cells were pretreated with PKC inhibitor and ROCK inhibitor or myosin light chain kinase inihibitor. Effects of myosin light chain phosphatase inhibitor simulated those of endothelin-1. Inhibition of myosin light chain phosphatase accelerated the phosphorylation status of cardiac myosin light chain resulting in positive inotropic action similar to endothelin-1. Our results suggested that endothelin might activate myosin light chain not only via PKC but also via ROCK pathway.
　Our working hypothesis is that endothelin 1 may produce positive inotropic effects via ROCK pathway. Thus we try to reveal the role of ROCK pathway in the regulation of contraction and relaxation in cardiac muscle.
Keywords: Endothelin, Cardiac muscle, Myosin light chain, Rho kinase, Protein kinase C, Excitation-contraction coupling
J Saitama Med School 2004;31:103-113
(Received January 13, 2004)

Case Report
Isolated Adrenocorticotropic Hormone (ACTH) Deficiency and Thyroid-Stimulating Hormone (TSH)-Thyroid Hormone Derangement: Report of Three Cases

Shigemitsu Yasuda, Seiki Wada, Miho Suzuki, Akinobu Minagawa, Shinji Kitahama, Makoto Iitaka, Shigehiro Katayama
Department of Endocrinology and Diabetes, Institute of Internal Medicine, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan

Abstract: We present three cases of isolated adrenocorticotropin (ACTH) deficiency accompanied by derangement of the thyroid-stimulating hormone (TSH)-thyroidal axis. Thyroid hormone and TSH levels were evaluated before and after cortisol replacement. Although markedly elevated levels of TSH were noted in one case, this patient also showed typical features of Hashimoto's thyroiditis. In the other two cases, basal TSH levels were increased, and replacement of cortisol reversed the values. We have previously reported that interference of thyroid hormone synthesis and/or secretion by glucocorticoid deficiency is a major cause of TSH-thyroidal axis derangement. However, it has been shown that a considerable number of reported cases exhibit severe hypothyroidism due to Hashimoto's thyroiditis, as was shown in case 2. It has been recognized that, whether the origin is the pituitary or the adrenal gland, polyglandular failure is a complex of autoimmune endocrinopathy. Alternatively, it is assumed that depletion of the physiological concentration of cortisol may worsen hypothyroidism due to Hashimoto's thyroiditis, possibly through modification of T cell function. Since transient abnormalities of the TSH-thyroidal axis and growth hormone could occur in glucocorticoid-deficient patients along with derangement of other pituitary hormones, hormonal evaluation should be carried out after a sufficiently long interval following cortisol replacement.
Keywords: adrenocorticotropic hormone deficiency, hypothyroidism, hypocortisolism, Hashimoto's thyroiditis
J Saitama Med School 2004;31:115-120
(Received February 23, 2004)

Thesis
Port site metastasisモデルの作成と成立過程の組織学的検討

山田　博文
埼玉医科大学総合医療センター外科
（指導：橋本　大定教授）

医学博士　乙第872号　平成15年4月25日 （埼玉医科大学）

Establishment of a Model of Port Site Metastasis in Rabbit: Histological Examination of the Growth
Hirofumi Yamada(Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama 350-8550, Japan)

　The port site metastasis (PSM) is a peculiar phenomenon following laparoscopic tumor surgery. Although the pathomechanisms involved in PSM should be resolved promptly in order to safely expand the indication of laparoscopic tumor surgery, there have been no useful animal models mimicking the PSM that is observed in the clinical setting. Conventionally, small animals such as mice and rats were used as animal experiment model for PSM. This is too far different to represent a model for PSM after laparoscopic surgery in man. The purpose of this study was to establish a model of PSM in rabbits and examine the growth characteristics of this model. In experiment-1, the relationship between the number of tumor cells intraperitoneally inoculated and the frequency of the PSM was examined. Male Japanese rabbits (n＝5, per group) received intraperitoneal inoculation of one of the three different counts of VX₂cancer cells (Group A; 5×10³cells, Group B; 5×10⁴cells, or Group C; 5×10⁵cells). Three days after the tumor inoculation, rabbits underwent carbon dioxide pneumoperitoneum at 8 mmHg for 30 minutes with 9 ports inserted into the abdomen. The frequency of port site metastasis detected on day 17 was greater as the number of inoculated VX₂cells suspension increased. The frequency of port site metastasis in group C was 35.6％. Therefore, this model was used in the next experiment. In experiment-2, the same conditions as in the group C rabbits were used to generate port site metastases in 21 rabbits. The rabbits were sacrificed to examine the growth characteristics of PSM on days 3, 5, 7, 9, 13, 17, or 23. The first histological evidence of PSM formation was found on day 13. On day 17, 76 percent of the port sites developed tumors, and 59％ of them invaded into the muscle layer from subperitoneal layer. On day 23, 100％ of the port sites developed tumors, and 96％ of them were found to invade the muscle layer. These results suggest that the initial site of growth of the PSM is subperitoneal tissue and the metastasis grows and invades the muscle layer. Our model is useful because (1) the rabbits, the largest animals with transplantable tumor cell line, are the closest animal model for PSM in man, (2) it is a relatively stable source of PSM that is similar to the form seen in clinical setting, and (3) the developing process of PSM which begins from embedment in the subperitoneal tissue, leading to invasion of muscular layer. In the future, wide range of applications can be expected from this experimental model.
Keywords: port-site metastasis, VX₂, pneumoperitoneum, histological examination

Thesis
眼窩領域MALTリンパ腫の臨床病理学的検討とBCL10蛋白およびAPI2-MALT1キメラ遺伝子発現について

安達　章子
埼玉医科大学総合医療センター病理部
（指導：糸山　進次教授）

医学博士　乙第899号　平成15年10月24日 （埼玉医科大学）

　眼窩領域悪性リンパ腫のうち，50-70％はMALTリンパ腫（extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue）であり，低悪性度B細胞リンパ腫として特異的な臨床病理像を有する．近年MALTリンパ腫の染色体転座t（1;14）（p22;q32）から単離されたBCL10，およびt（11;18）（q21;q21）から単離されたAPI2-MALT1キメラ遺伝子発現が，その腫瘍発生に関与していることが示唆されている．本研究では眼窩領域リンパ増殖性疾患62症例を集積，臨床病理学的解析を行い，眼窩MALTリンパ腫におけるBCL10蛋白およびAPI2-MALT1キメラ遺伝子の発現について検討した．62症例中58症例は悪性リンパ腫，うち50症例はMALTリンパ腫であった．MALTリンパ腫は他のリンパ腫に比べて局在性の傾向があり，再発は16症例に認められたが，予後は概ね良好であった．免疫組織化学染色により，MALTリンパ腫のうち29症例（58％）にBCL10蛋白の核及び胞体への発現が認められ，BCL10核発現が眼窩領域MALTリンパ腫の腫瘍発育に関与していることが示唆された．BCL10核発現と臨床因子（年齢・性・発生部位・臨床病期・再発・再発までの期間）との間に関連性は認められなかった．また初発および再発MALTリンパ腫67検体のパラフィン切片からRNAを抽出し，multiplex RT-PCR法によるAPI2-MALT1キメラ遺伝子の検出を試みたが，発現は認められなかった．すなわち眼窩領域MALTリンパ腫においてはBCL10核発現とAPI2-MALT1キメラ遺伝子発現の関連性は見られず，両者の関連性が報告されている胃や肺MALTリンパ腫とは異なった結果であった．眼窩領域MALTリンパ腫における高頻度のBCL10核発現はAPI2-MALT1キメラ遺伝子以外の，未知の遺伝子変異の関与が示唆された．

Thesis
交流3,000V 電位負荷及び杜仲葉併用による肉芽形成とコラーゲンの合成促進効果の研究

古賀　義久
埼玉医科大学麻酔学教室
（指導：松本　勲教授）

医学博士　乙第918号　平成16年3月19日 （埼玉医科大学）

Studies on the Stimulative Effect of 3,000 Volts Alternating Current on Granuloma Formation and Collagen Synthesis
Yoshioka Koga (Department of Anesthesiology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　Rats raised on a 6% protein diet (low-protein diet) for three weeks have a reduced ability to synthesize collagen, and because the levels of this diet-induced reduced collagen synthesis are comparable to those for normally aged rats, low-protein diet-fed rats can be used as a model for aged rats. In the present study, 6-week-old male Wistar rats were raised on a low-protein diet for three weeks, and the effects of applying 3,000 V of alternating current and supplementing the low-protein diet with Eucommia ulmoides leaves on collagen synthesis were determined. 1. By applying 3,000 V of alternating current to the rats on the low-protein diet, the weight of granulation tissue and levels of hydroxyproline increased in a time-dependent manner when compared with the control group. After six weeks of applying 3,000 V of alternating current, the weight of granulation tissue and levels of hydroxyproline increased significantly by 187 and 171%, respectively (p<0.05). 2. By applying 3,000 V of alternating current to rats on the low-protein diet supplemented with Eucommia ulmoides leaves (0.75%), the weight of granulation tissue and levels of hydroxyproline increased significantly within four weeks by 136 and 163%, respectively (p<0.05 and 0.001, respectively). The results of the present study suggest that adding Eucommia ulmoides leaves facilitates collagen synthesis and formation of granulation tissue in aged rats. The fact that levels of hydroxyproline in granulation tissue increased significantly suggested that reduced collagen synthesis in aged rats was activated. The formation of granulation tissue is indicative of the early stages of wound healing, and this formation facilitates wound healing.
Keywords: Aged rat, Eucommia ulmoides leaves, 3,000 V of alternating current, Collagen synthesis, Granuloma formation

Thesis
大腸癌における可溶性Interleukin-2 receptor（IL-2R）値の変動

坂田　秀人
埼玉医科大学第二外科学教室
（指導：平山　康三教授）

医学博士　乙第782号　平成13年7月27日 （埼玉医科大学）

背　景：癌細胞等によって放出されるメディエーターに刺激されて，T細胞が活性化するとIL-2の分泌が始まり，またIL-2受容体α鎖がTリンパ球細胞膜上に出現する．このIL-2が，オートクラインあるいはパラクラインによってIL-2受容体と結合するとT細胞の増殖・分化が始まる．結合後，すぐにIL-2受容体のα鎖の一部が解離し，血中に放出される．これが可溶性interleukin-2 receptor（IL-2R）である．以上の機構より，血中の可溶性IL-2R値が宿主の腫瘍免疫能の多寡を反映して変動することが考えられ，腫瘍マーカーとして利用できるものと推測される．今回，大腸癌患者の血中の可溶性IL-2R値の測定と，癌局所および所属リンパ節におけるIL-2R陽性リンパ球（以下，活性型Tリンパ球とする）の出現状況を探り検討した．
方　法：大腸癌155症例から採取した血清を用いて，ELISA法による可溶性IL-2R 値の測定を行なった．同時に各種の腫瘍マーカーを測定して比較を行なった．さらに抗ヒトインターロイキン-2マウスモノクローナル抗体を用い，ABC法による癌局所ならびに所属リンパ節の活性型Tリンパ球の局在について免疫組織化学的検索を行なった．
結　果：大腸癌患者の血中可溶性IL-2R値は健常コントロール群に比べ有意に高値を示した．IL-2R値は漿膜浸潤症例，およびリンパ節転移をもつ症例では有意に高値を示したが，他の臨床病理学的因子による上昇は認めなかった．リンパ節転移陽性症例についての感度と特異度は，CEAは40.2％と66.0％，IL-2Rは43.2％と77.4％であった．CEAとIL-2Rのコンビネーションアッセイでは63.3％と95.8％に上昇しIL-2R値がリンパ節転移の予測に有用であることが示唆された．ほとんどの癌組織とすべての転移性リンパ節において活性型Tリンパ球の浸潤を認めた．他方，非癌部の大腸組織や転移陰性の所属リンパ節では活性型Tリンパ球の浸潤をみることはなかった．これらの結果から，大腸癌組織や転移リンパ節においてTリンパ球が活性化され，それらが増殖・分化する過程でIL-2Rのα鎖（可溶性IL-2R）が血中に放出されるため，血中の可溶性IL-2Rが上昇する機序が推定された．
結　論：大腸癌患者の血中の可溶性IL-2R値測定により，リンパ節転移症例が判別できる可能性が示唆された．
キーワード： 結腸癌，直腸癌，IL-2R ， CD25蛋白

Thesis
血清チミジンキナーゼおよび可溶性インターロイキン-2受容体の測定値は悪性リンパ腫再発の早期予測に有用な腫瘍マーカーである

若尾　大輔
埼玉医科大学内科学（血液内科部門）
（指導：別所　正美教授）

医学博士　乙第903号　平成15年12月19日 （埼玉医科大学

Serum Thymidine Kinase and Soluble Interleukin-2 Receptor Levels Predict Subclinical Recurrence of Malignant Lymphoma
Daisuke Wakao (First Department of Internal Medicine, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan )

　Before and after anti-neoplastic chemotherapy, serum thymidine kinase (TK) and soluble interleukin-2 receptor (sIL-2R) levels were serially determined in 28 patients with malignant lymphoma (ML). In 15 patients achieving and maintaining complete remission (CR) for more than 2 years, serum TK and sIL-2R levels were unchanged or decreased gradually. In contrast, logarithmic linear increases in TK and sIL-2R levels were observed in 13 relapsed patients. The increments of the serum markers preceded more than 10 months before the relapses. A significant positive correlation between the slope of the line for TK and that for sIL-2R was noted. The doubling time for TK estimated from the slope also showed a positive correlation with that for sIL-2R. Taken together, serum TK and sIL-2R were shown to be quite sensitive and interrelated serum markers for the recurrence of ML. Slopes of logarithmic linear increase, which are proper and specific for the individual patients, are inversely correlated with the doubling time and reflect proliferation of ML. We compared serum TK/sIL-2R and international prognostic index (IPI) for the detection of relapse of ML. IPI is widely used for individual evaluation of prognosis of ML. We conclude that serum TK and sIL-2R are better predictor of relapse than LDH and IPI.
Keywords: thymidine kinase, soluble interleukin-2 receptor, recurrence, logarithmic linear increase, proliferation of lymphoma cells

Thesis
大腸癌におけるorotate phosphoribosyltransferase発現に関する検討

高橋　威洋
埼玉医科大学消化器一般外科(II)部門
（指導：平山　廉三教授）

医学博士　甲第935号　平成16年4月23日 （埼玉医科大学）

Expression of orotate phosphoribosyltransferase in colorectal cancer
Takehiro Takahashi (Department of digestive and general surgery, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

Backgroud: Activation of 5-fluorouracil (5-FU) into nucleotides requires phosphorylation by orotate phosphoribosyltransferase (OPRT). In this study, we investigated the correlation between enzymatic activity and gene expression of OPRT in colorectal cancer tissues, and the association between OPRT gene expression and anti-tumor effect.
Materials and Methods: Enzymatic activity and gene expression of OPRT were measured by radioassay method and a real-time reverse transcriptional-polymerase chain reaction method, respectively, in 20 primary colorectal cancer tissues. In 37 patients treated with tegafur-uracil and leucovorin for metastatic colorectal cancer, OPRT gene expressions were analyzed by the same method.
Results: There was a positive correlation between enzymatic activity and gene expression of OPRT (r＝0.788, P＜0.0001). Responding tumors had statistically higher OPRT gene expression than nonresponding tumours (P＝0.0008), and patients with a high OPRT mRNA expression suvived longer than those with a low OPRT mRNA expression (P＝0.0019).
Conclusion: The expression of OPRT gene might be useful as predictive parameter for the efficacy of fluoropyrimidine-based chemotherapy.
Keywords: orotate phosphoribosyltransferase, colorectal cancer, 5-fluorouracil

原 著
エストロゲン応答遺伝子COX7RP（cytochrome c oxidase subunit 7a related polypeptide)の子宮内膜癌における発現とその調節

林　るつ子
埼玉医科大学総合医療センター産婦人科〔平成16年7月14日 受付〕

Expression and Regulation of an Estrogen-Responsive Gene, Cytochrome C Oxidase Subunit 7a Related Polypeptide (COX7RP) in Endometrial Cancer
Rutsuko Hobo-Hayashi (Department of Obstetrics and Gynecology, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama 350-8550, Japan)

　Endometrial cancer is one of the most common gynecologic malignancies. Recent epidemiological studies show that the incidence rate of endometrial cancer is increasing in Japan. Although the exact cause of endometrial cancer is still obscured, estrogen appears to play an important role in endometrial cancer because prolonged exposure to unopposed estrogen has been reported as a risk factor for it. Indeed, Ishikawa cells derived from human endometrial cancer that express estrogen receptor α and β(ERα and ERβ) show estrogen dependent proliferation. ERs are ligand dependent transcription factors that regulate target gene transcription. Therefore, estrogen actions such as cell growth in endometrial cancer are exhibited by estrogen-responsive genes. However, it has not been fully elucidated how the estrogen-responsive genes mediate the progression of endometrial cancer.
　COX7RP has been isolated as an estrogen responsive-gene with an ER-binding human genomic fragment, EB1, obtained by the genomic-binding site cloning. COX7RP gene contains a perfect palindromic estrogen-responsive element (ERE) in the first intron. The expression of COX7RP mRNA is up-regulated by estrogen in human breast cancer MCF7 cells. Based on the homology of the amino acids, COX7RP is considered as a member of COX7a family which is a nuclear encoded subunit of cytochrome c oxidase (COX).
　The present study aims to determine whether COX7RP as a novel estrogen-responsive gene associates with endometrial cancer. The expression of COX7RP mRNA is demonstrated to respond to estrogen and repressed by ICI 182, 780, a pure antagonist for ER in human endometrial cancer Ishikawa cells. The ERE-containing EB1 fragment activates transcription in response to estrogen. In addition, the correlations between COX7RP and ER mRNA expression levels are found in clinical samples of endometiral cancer. Western blot analysis detected COX7RP protein in a mitochondrial fraction of cell extracts by a rabbit polyclonal antibody raised against a C-terminal polypeptide of human COX7RP. Moreover, immunohistochemistry with this anti-COX7RP polyclonal antibody and a ERαantibody reveals that the COX7RP protein is localized in cytoplasm of endometrial cancer cells. In most of COX7RP positive cells, we also observed nuclear staining of ERα.
　Taken together, these results suggest that COX7RP is involved in the estrogen actions in endometrial cancer as a primary estrogen-responsive gene.
Keywords: Estrogen, endometrial cancer, COX7RP, estrogen receptor
J Saitama Med School 2004;31:199-206
(Received July 14, 2004)

Thesis
肺癌における組織内Thymidylate synthase（TS）およびDihydropyrimidine dehydrogenase（DPD）活性と予後に関する検討

赤石　亨
埼玉医科大学呼吸器外科
（指導：金子　公一助教授 ）

医学博士　甲第920号　平成16年3月19日 （埼玉医科大学）

Tissue Activities of Thymidylate Synthase (TS) and Dihydroprimidine Dehydrogenase (DPD) and Their Prognostic Relevance in Primary Lung Cancer

　Tissue expressions of thymidylate synthase (TS), a target enzyme for 5-flurouacil (5-FU)-based anticancer agents, and dihydroprimidine dehydrogenase (DPD), a degradation enzyme for 5-Fu-based anticancer agents, have been suggested to be potentially associated with not only antitumor efficacy in anticancer drugs but also tumor characteristics and prognosis. Tissue activities of TS and DPD were measured, and their relationships with tumor characteristics such as histological type, degree of cell differentiation, etc., and prognosis were examined. Further, the relationship between the levels of TS and DPD expressions and those of TS and DPD activities was investigated by immunohistological staining utilizing anti-TS antibody, anti-DPD antibody, cyclin A and ki-67. In a total of 55 patients with surgically resected primary lung cancer, enzymatic activities in cancerous tissues of their resected lung tumors as well as normal lung tissues were measured. TS activity in the tumor lung tissue and normal lung tissue was 0.031 pmol/mg protein (hereafter the unit omitted) and 0.022, respectively, and DPD activity in the tumor lung tissue and normal lung tissue was 302.6 031 pmol/min/mg protein (hereafter the unit omitted)and 90.1, respectively, exhibiting a trend toward an elevated level of both activities in the tumor lung tissues. TS activity showed no difference between the two types(squamous cell carcinoma and adrenocarcinoma) but DPD activity was high in the cases of adrenocarcinoma. TS activity was high at earlier disease stages I and II, while DPD activity was lower at stages I and II, compared to stages III and IV. TS and DPD activities in recurrent cases were 0.027 and 271.1, respectively, where in non-recurrent cases they were 0.037 and 290.1, respectively, thus showing a slightly elevated level of TS activity in the non-recurrent cases. On immunohistological staining of cancerous tissues, in both TS and DPD staining, the level of their activities tended to be high in cases stained positively, and a correlation was observed between enzymatic activity and expression of enzyme. In cases shown positive for TS staining, many cases were positive for cyclin A as well as Ki-67 and a similar pattern was observed in cases shown positive for DPD staining. However, in the DPD staining, marked tissue inhomogeneity was observed and, even in cases stained positively, there were some such cases as the negative part being mixed in a portion of the tissue, which even led us to consider this as the cause of fluctuations in the level of activity. The levels of TS and DPD activities in the lung cancer tissue were seen in part correlated with tumor characteristics and prognosis and, further, their usefulness was also suggested as markers for not only prognostic evaluation but also pre- and post-operative adjuvant chemotherapies.
Keywords: Thymidylate synthase, Dihydroprimidine dehydrogenase, 5-flurouacil, lung cancer, cyclinA, Ki-67