原 著
JAM-Bノックアウトマウスにおける白血球浸潤

坂口　武久
埼玉医科大学ゲノム医学研究センター発生・分化・再生部門〔平成17年11月11日 受付〕

Leukocyte Transmigration in JAM-B Disrupted Mice
Takehisa Sakaguchi (Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical School 1397-1, Yamane, Hidaka, Saitama 350-1241, Japan)

　Junctional Adhesion Molecule (JAM)-B is known as one of the members of the immunogloblin super family and is involved in formation of tight junction of many types of cells including epithelial and endothelial cells. It has recently been demonstrated that, in response to inflammation, JAM-B present on the surface of endothelial cell forms heterophilic interactions with JAM-C on the leukocyte during transmigration from blood vessels into affected tissues. The formation of JAM-B－JAM-C complex strongly suggests that this heterophilic interaction plays an important role in the transmigration of the leukocytes. To address this question, I generated mice lacking JAM-B. Subsequently, I examined leukocyte transmigration in JAM-B－/－ mice by Contact Hyper Sensitivity (CHS) model using oxazolone as sensitizing agent and thioglycollate-induced peritonitis model. From these analyses, I found that levels of leukocyte transmigration in JAM-B－/－ mice were similar to those of wild-type mice. Thus, our results established that the gene is dispensable for this event.
Keywords: JAM-B, Transmigration, Gene Targeting, Leukocyte, Endothelial Cell
J Saitama Med School 2006;33:1-10
(Received November 11, 2005)

Thesis
Tumor Necrosis Factor-αのマウス肝細胞アポトーシス抑制作用におけるInhibitor of Apoptosis Proteins の関与

菅原　通子
埼玉医科大学内科学消化器・肝臓内科部門

共同研究者：名越　澄子（埼玉医科大学内科学消化器・肝臓内科部門），善本　隆之（東京医科大学内科学難病治療研究センター），持田　智（埼玉医科大学内科学消化器・肝臓内科部門），藤原　研司（独立行政法人労働者健康福祉機構横浜労災病院）

医学博士　甲第978号　平成17年3月25日 （埼玉医科大学）

Inhibitory Action of Tumor Necrosis Factor-α on Hepatocyte Apoptosis may Depend on Inhibitor of Apoptosis Proteins in Mice
Kayoko Sugawara (Gastroenterology & Hepatology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

【Background and Aims】Tumor Necrosis Factor-α (TNF-α) produced by activated macrophages in the hepatic sinusoids regulates the development of massive liver necrosis, and also acts as a cytokine to induce liver regeneration. It is well known that TNF-α binding to its receptor activates caspase cascade resulting in apoptosis, whereas this apoptosis is inhibited by NFκB, an endogenous transcriptional factor activated by TNF-α. Moreover, inhibitor of apoptosis proteins (IAPs) are accelerated in the transcription by NFκB, and can bind to the caspases to inactivate caspase cascade. In mice given a small amount of TNF-α, liver injury is not induced, but massive liver necrosis occurs following hepatocyte apoptosis after TNF-α administration in mice pretreated with d-galactosamine (GalN), a transcription inhibitor. In the present investigation, the relation of this inhibitory action of TNF-α on hepatocyte apoptosis to IAPs was studied using this model and other mouse models.【Methods and Results】With the following models, the hepatic expressions of IAPs were evaluated as IAP-1, IAP-2, XIAP and survivin mRNAs by RT-PCR. Hepatocyte apoptosis was determined by TUNEL method. 1) When mice received TNF-α, IAP-1 and IAP-2 were up-regulated in the liver from 1 to 5 hr with their peaks at 1 hr, but the expressions of XIAP and survivin were at the control levels until 8 hr. However, pretreatment with GalN before 30 min showed apoptosis in half of hepatocytes at 8 hr after TNF-α administration, with IAP-1 and IAP-2 expressions at the control levels at 1 hr. 2) Serum TNF-α concentration was increased later than 1 hr after endotoxin dosing in mice pretreated with P. acnes, but hepatocyte apoptosis was absent until 8 hr. Hepatic expressions of IAP-1 and IAP-2 were increased forming their peaks at 2 hr, but hepatic expressions of XIAP and survivin were unchanged until 8 hr. 3) IAP-1, IAP-2 and XIAP expressions were not changed from 10 min to 5 days after 70％ liver resection, whereas survivin expression was increased at 36 and 48 hr after the operation.【Conclusions】The inhibitory action of TNF-α on hepatocyte apoptosis may be produced via IAP-1 and IAP-2. However, hepatocytes during liver regeneration may be protected from apoptosis through different mechanisms, especially by action of survivin.
Keywords:apoptosis . hepatocyte. TNF-α. IAPs. NFκB.

原 著
副腎腺腫のCT値とMRI信号評価の検討

湯浅　昌之，小澤　栄人，平敷　淳子
埼玉医科大学放射線科〔平成17年12月14日 受付〕

Evaluation of Adrenal Adenoma: Correlation between Pre Contrast CT Value and the Signal Intensity Loss on Dual Chemical Shift MRI
Masayuki Yuasa, Eito Kozawa, Atsuko Heshiki (Department of Radiology, Saitama Medical, School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

Purpose: The aim of our study is to assess pre-contrast CT value and signal intensity loss of adrenal adenoma on dual phase chemical shift MRI. Materials and Methods: From May 1997 to December 2004, 52 patients with adrenal adenoma were examined with pre-contrast CT and dual chemical shift MRI. Eleven of 52 were proven by surgery and the rest did not show any change for 12 months period. All patients were studied with CT (GE Hispeed Advantage SG, SP LightSpeed Qx/i-α) and 1.5 T MRI units (Magnetom Quantum and Sonata, Siemens, Germany) with a body-coil. CT was scanned with slice thickness of 3 mm, 5 mm or 10 mm, and pitch of 1. In additional to the MR routine sequences such as FLASH and HASTE, dual chemical shift sequence was scanned (TR/TE/FA＝140/2.3 and 5.2/90). Hounsfield units (HU) on pre contrast CT, signal intensity ratio (SIR), and signal noise ratio (SNR) on MRI were measured. Signal intensities were measured quantitatively in opposed-phase (OP), in-phase (IP) and background noise (N). SIRs (OP/IP) and SNRs　(OP-N)/(IP-N)) were calculated as well as mean and standard deviation. Using JMP　(commercial analysis software), correlation analysis was performed between HU and the parameters of SIR and SNR with linear regression method. Results: The mean and SD of CT value, SNR and SIR was 12.6±15.04, SNR＝0.51±0.28, SIR＝0.47±0.30, respectively. Correlation coefficient between CT value and SNR was 0.78(p＜0.001), and between CT vale and SIR was 0.76(p＜0.001). Discussion and conclusion: SNR and SIR of in-phase and opposed-phase study is an established method for assessing of fat and water components in the adrenal adenoma. However, CT value also has the power for the evaluation of fat element. In this study the CT vs. SIR and the CT vs. SNR were correlated well. These results suggest that CT value may show the amount of fat element in adrenal nodule. In conclusion, pre-contrast CT value could suggest a good index of a lipid component of an adrenal adenoma. The signal intensity ratios are the reliable evaluation method for diagnosis adrenal adenomas.
Keywords: adrenocortical adenoma, CT attenuation, dual chemical shift imaging.
J Saitama Med School 2006;33:19-23
(Received December 14, 2005)

原 著
妊娠中毒症の分娩後の血圧および妊娠中毒症既往女性の腎病理組織学的特徴

有馬　博
埼玉医科大学腎臓内科〔平成18年2月10日 受付〕

Recent and Remote Prognosis of Toxemia
Hiroshi Arima (Department of Nephrology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　Pregnancy-induced toxemia is named as pregnancy-induced hypertension syndrome and its definition is based on hypertension but not in proteinuria and edema. Previously pregnancy-induced toxemia is composed of three symptoms of hypertension, proteinuria and edema. In the present study, two studies were carried out. The first study examined renal patho-histology in 30 proteinuric patients who had a past history of toxemia. Ten patients had IgA nephropathy, 10 did glomerulosclerosis and 10 did focal glomerulosclerosis. The factors, such as the age of pregnancy, the levels of blood pressure and the degree of proteinuria during pregnancy, were not significantly different among three groups. However, the levels of serum creatinine in patients with focal glomerulosclerosis were significantly lower than those with IgA nephropathy and /or with glomerulosclerosis. In addition, there was no significantly difference of the two factors between the latter two groups. These data suggest that patients who were previously defined as having pregnancy-induced toxemia were composed at least of three types of nephropathy. From this study, patients who had proteinuria during pregnancy should be carefully followed up and if proteinuria persisted more than a year renal biopsy for diagnosis of nephropathy should be considered. In the second study, 52 patients who were diagnosed as having pregnancy-induced hypertension were followed up for a year after delivery. In 9 patients, high blood pressure more than 140 mmHg systolic or 90 mmHg diastolic was maintained beyond one year. A comparison between the patients whose blood pressure became less than 140 and 90 mmHg within a year and those did not revealed a significant difference in systolic blood pressure at the presentation of pregnancy-induced hypertension syndrome although any other factors such as age, the levels of proteinuria and so on. did not show any significant differences between the two groups. From the second study, the levels of systolic blood pressure might be predictable index for development of hypertension after delivery in patients with pregnancy-induced hypertension syndrome. Combining these two studies, it is concluded that if proteinuria or high blood pressure persists more than one year after delivery more cautious follow-up would be preferable.
Keywords: Toxemia, Pregnancy-induced hypertension, IgA nephropahy, Nephrosclerosis, Focal glomerulosclerosis
J Saitama Med School 2006;33:25-32
(Received February 10, 2006)

Thesis
男性糖尿病患者における足部Peripheral Quantitative Computed Tomography（pQCT）の低下と糖尿病性腎症との関連

竹尾　浩紀
埼玉医科大学内科学消化器・肝臓内科部門
（指導　藤原　研司　教授）

医学博士　乙第972号　平成17年3月25日 （埼玉医科大学）

The Relation Peripheral Quantitative Computed Tomogrophy (pQCT) and Diabetic Nephropathy in Male Diabetic Patients
Hiroki Takeo (Gastroenterology and Hepatology, Department of Internal Medicine, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

Objective: Osteopenia can develop in diabetic patients. Its extent is generally evaluated in vertebral bones, but the relation to exercise is still unclear. We assessed the utility of peripheral quantitative computed tomography (pQCT) as a tool for detecting osteopenia of navicular bones in diabetic patients, including analysis of its relation to the clinical features. Patients and Methods: 158 diabetic male patients were divided into 3 groups based on 75 g oral glucose tolerance test (75gOGTT), and pQCT was evaluated in each group in relation to exercise practices by questionnaire survey, IMT thickness and plaque formation by cervical ultrasonography and urine albumin concentrations less than 30 mg/day (nl group), 30 to 300 mg/day (mi group) and more than 300 mg/day (MA group). Results: There were 10 normal, 31 impaired and 117 diabetic 75gOGTT patients (N, I and D groups, respectively). pQCT was not different between N and I groups (p=0.981), but significantly differ between N and D groups (p=0.014), and also between I and D groups (p=0.005). The abnormality by cervical ultrasonography was detected only in 1 patient by a specialist in the D group. In this group, there was no difference in pQCT between 53 patients who practiced exercise 3 times or more a week and the rest 64 patients (p=0.789). The significant difference was not found even in grouping these 117 patients by exercise practice or not (n=65 and 52, respectively, p=0.0622) and by existence of abnormal cervical findings or not (n=29 and 78, respectively, p=0.650). In patients with diabetic nephropathy, however, there was significant difference between nl and mi groups (n=86 and 24, respectively, p=0.002), and between nl and MA (n=7) groups (p=0.038), even though not significant between nl and mi groups or mi and MA groups (p=0.830). Conclusions: The present results suggest that osteopenia of navicular bones may be present in diabetic male patients with albuminuria more than 30 mg/day.
Keywords: diabetic nephropathy, albuminuria, osteopenia, IMT, exercise, pQCT

原 著
埼玉医科大学病院における前立腺癌に対する高線量率組織内照射の経験

黒田　功¹⁾，塚本　拓司¹⁾，中平　洋子¹⁾，矢内原　仁¹⁾，吉村　一良¹⁾，上野　宗久¹⁾，出口　修宏¹⁾，内野　三菜子²⁾，関根　広²⁾，土器屋　卓志²⁾
1) 埼玉医科大学泌尿器科　2) 埼玉医科大学放射線腫瘍科
〔平成18年11月24日 受付〕

HDR Brachytherapy for Prostate Cancer at Saitama Medical University : A Preliminary Report
¹⁾KURODA Isao,¹⁾TSUKAMOTO Takuji,¹⁾NAKAHIRA Youko,¹⁾YANAIHARA Hitoshi,¹⁾YOSHIMURA Ichirou,¹⁾UENO Munehisa,¹⁾DEGUCHI Nobuhiro,²⁾UCHINO Minako,²⁾SEKINE Hiroshi,²⁾DOKIYA Takushi (Department of Urology¹), Department of Radiology²), Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　We started radical radiotherapy for prostatic carcinoma since October 2003 besides surgical prostatectomy. The modality of radiotherapy was high dose rate brachytherapy(HDR) with external beam radiation therapy(EBRT). Assessment of the outcomes of HDR with EBRT was compared with that of retropubic radical prostatectomy (RRP) by serum PSA measurement after 6 months of the treatment. Curative ratio of HDR with EBRT and RRP were 79.5％ and 62％, respectively. Definitely the efficacy of radiotherapy for prostatic carcinoma was equal to that of surgical therapy.
Keywords: prostate cancer, brachytherapy, HDR
J Saitama Medical University 2006;33:41-45
(Received November 24, 2006)

Thesis
マウスおよびラットの骨格筋筋小胞体に対するセリバスタチンのCa²⁺放出作用

井上　亮拓
埼玉医科大学薬理学教室
（指導：丸山　敬 教授）

医学博士　乙第1022号　平成18年4月28日 （埼玉医科大学）

Ca²⁺-Releasing Effect of Cerivastatin on the Sarcoplasmic Reticulum of Mouse and Rat Skeletal Muscle Fibers
Ryotaku Inoue (Department of Pharmacology, Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　Ryanodine receptors (RyRs) play an important role as intracellular Ca²⁺-release channels in a wide variety of cells. Previously, our group reported that clofibric acid, an antihyperlipidemic agent, caused Ca²⁺release from the sarcoplasmic reticulum (SR) in a unique mode of action on RyR-Ca²⁺release channels in skinned skeletal muscle fibers. On the other hand, it was reported that simvastatin, a different type of antihyperlipidemic agent, increased the cytoplasmic Ca²⁺concentration via the release of stored Ca²⁺in L6 rat myoblasts and resulted in cell injury, although the exact mechanism is still debated. These results raise the possibility that both types of antihyperlipidemic agent may have a common ability to activate the RyR-Ca²⁺release channel.
　In this study, the effect of cerivastatin (Cer), classified as an HMG-CoA reductase inhibitor as is simvastatin, on Ca²⁺release from the SR was analyzed using chemically skinned skeletal muscle fibers from the mouse and the rat. The Ca²⁺released from the SR of the skinned fibers was quantified using Fura-2, and the activity of Ca²⁺release was expressed in terms of decay rate constants, based on the amount of Ca²⁺remaining in the SR. Cer (>20 μM) released Ca²⁺from the SR, while pravastatin showed only a little effect. The rates of Ca²⁺release were increased by Cer not only at pCa 7-4.5 but also at pCa >8. Cer-induced Ca²⁺release in the presence of Ca²⁺was affected by adenosine monophosphate, Mg²⁺and procaine in essentially the same way as for caffeine-induced Ca²⁺release. The Ca²⁺-uptake capacity of the SR was reduced after co-treatment with ryanodine and Cer at pCa 6.0 to a much grater extent than with ryanodine alone. Thus, Cer-induced Ca²⁺release in the presence of Ca²⁺must be a result of the activation of the Ca²⁺-induced Ca²⁺release (CICR) mechanism of the ryanodine receptor. However, even under the condition that CICR was maximally inhibited by Mg²⁺and procaine, or in the practical absence of Ca²⁺(pCa >8), Cer still caused Ca²⁺release. These results indicate that Cer causes Ca²⁺release also by activating some other mechanism(s) in addition to the activation of CICR. Either or both of these effects might be related to its adverse effect, rhabdomyolysis.
Keywords: Calcium release, cerivastatin, ryanodine receptor, sarcoplasmic reticulum, skeletal muscle

Thesis
介護老人保健施設・特別養護老人ホームにおける高齢者のアクチグラフによる睡眠・覚醒アセスメント

遠藤　順朗
埼玉医科大学・生理学教室

医学博士　乙第1006号　平成18年2月17日 （埼玉医科大学）

The Sleep-Wakefulness Assessments of Very Old Residents in Nursing Homes
Yoriaki Endo (Department of Physiology, Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　The aim of this study is to assess the sleep/wake activity of very old residents (over 90 years old) in nursing homes by using actigraphy. We have divided them following three groups, Group 1: non-demented and good ADL (activity of daily living) 16 people (mean age, 93.7 years old). Group 2: severely demented but good ADL 16 people (94.9 years old), and Group 3: severely demented and poor ADL (with severe health problems) 16 people (93.9 years old). Group 1 showed good circadian rhythmicity of sleep/wake activity, but frequent naps (the mean numbers of sleep episodes with over 5 minutes are 7.7, significantly higher than group 2) in their active time zone and several brief awakenings in their sleeping time zone (bed interval). Group 2 also showed good circadian rhythmicity of sleep/wake activity, less naps in active time zone than Group 1, but more frequent awakenings ( the numbers of awakening episodes are 7.7) and the fragmentation of sleep in bed interval. Group 3 showed the disruption of circadian rhythmicity, 65％ sleeping time in 24 hours and the fragmentation of sleep in 24 hours. From these results, it is suggested that it is necessary to concern the level of both dementia and ADL including individual health problems for the assessment of sleep/wake activity in nursing home residents and actigraphy is suitable as an assessment of sleep/wake activity for the nursing home residents.
Keywords: Sleep/wake activity, actigraphy, over 90 years old, circadian rhythm, nursing home, ADL

Thesis
Pax6によるNFATc1を介した破骨細胞分化を制御する新たな転写調節機構

甲川　昌和
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1031号　平成18年9月22日 （埼玉医科大学）

　骨の機能は破骨細胞による骨吸収と骨芽細胞による骨形成により厳密に制御され恒常性が維持されている．しかし骨吸収の亢進が起こるとそのバランスが崩壊して骨量が減少し，骨粗鬆症や関節リウマチのような病態をきたす．そのため骨吸収の中心的な役割を果たす破骨細胞の機能を解明することがこれらの問題を解決する重要な役割と考えられる．
　これまでNFATc1を中心とした転写因子群の協調的な働きは，破骨細胞マーカー遺伝子の効率的な発現誘導をもたらし破骨細胞のダイナミックな骨吸収に寄与することが明らかになっている．一方転写因子であるPaxファミリーはさまざまな細胞分化に重要な働きをすることで知られていたが，今回初めて破骨細胞内で発現することが明らかとなった． RANKLによる破骨細胞誘導においてmRNAの発現解析の結果，Pax6が骨髄マクロファージ細胞に発現していてRANKL刺激後その発現が変動することが分かった．また破骨細胞のマーカー遺伝子であるTRAP遺伝子のプロモータ上の1853塩基の配列を調べPax6のDNA結合配列に類似した領域が存在することを見出した． そのDNAプローブを作製し破骨細胞抽出液を使ったゲルシフトアッセイによりDNA結合活性を調べたところPax6が特異的に結合していることがわかり，TRAP遺伝子がPax6の標的となることが示された． さらに破骨細胞の分化過程でPax6のDNA結合活性が変化することも明らかになった． 次にTRAP遺伝子のプロモーター領域を使ったレポータージーンアッセイにより，NFATc1により増強されるTRAP遺伝子のプロモーター活性をPax6は量依存的に抑制することが示された．またPaxファミリーと結合し転写抑制に関わる共役因子として知られるコリプレッサーTleファミリーのなかで，Tle6が破骨細胞内で強く発現することを見出し， Pax6と協調しTRAP遺伝子のプロモーター活性を抑制することおよびNFATc1と直接結合することを明らかにした． さらにレトロウイルスを用いてPax6を破骨細胞内へ強制発現した結果，TRAP陽性の多核の融合巨細胞数がコントロールに比べ有意に減少した．このことはPax6が破骨細胞の分化抑制に寄与することを強く示している．
　以上，本研究で我々はPax6が破骨細胞で発現することを初めて見出し，NFATc1による転写活性を抑制して破骨細胞の分化を調節する新たな制御因子であることを示した．
Keywords: Pax6，NFATc1，Tle6，TRAP遺伝子，骨粗鬆症，骨吸収

Thesis
モノクロタリン心不全モデルにおけるレニン－アンジオテンシン系遮断の効果

堀田　ゆりか
埼玉医科大学大学院臨床医学研究系　循環器内科学専攻
（指導　西村　重敬 教授）

医学博士　乙第1016号　平成18年3月24日 （埼玉医科大学）

Upregulated Neurohumoral Factors Play the Most Important in Left Ventricular Remodeling in Rats with Monocrotaline-Induced Pulmonary Hypertension
Yurika Hotta (Department of Cardiology, Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

　Left ventricular remodeling might occur in patients with right ventricular hypertrophy and/or failure due to pulmonary hypertension, although LV is not under mechanical stress. We used rats with monocrotaline-induced pulmonary hypertension whether upregulated neurohumoral factors act to induce left ventricular remodeling and/or contribute to prognosis. Morphologic studies revealed a significant increase in left ventricle/body weight and right ventricle/body weight ratio in monocrotaline rats, indicating biventricular hypertrophy. RNase protection analysis clearly demonstrated upregulated mRNA of β myosin heavy chain, skeletal α-actin, and brain type natriuretic peptide (BNP) and downregulated mRNA of α myosin heavy chain in the free wall of left ventricle as well as in right ventricle, while the expressions of SERCA-2 and Na-Ca exchanger were not significantly changed. Plasma angiotensin II (ANG II), noradrenaline, and BNP levels were significantly increased. And also plasma BNP level was positively and significantly correlated with heart weight/body weight and plasma ANG II in monocrotaline rats. Kaplan Meier survival curve with Loglank analysis revealed that valsartan (AT1 blocker) significantly improved the prognosis of monocrotaline rats while carvedilol (αβblocker) did not significantly.
　We concluded that upregulated neurohumoral factors not direct mechanical stress played the most important role in the pathogensis of left ventricular hypertrophy and remodeling in right ventricular pressure overload model. ANG II appears to be the number one contributor among the various upregulated neurohumoral factors in our mode.
Keywords: Monocrotaline, Ventricular Remodeling, Angiotensin II, BNP, Valsartan, Carvedilol

Thesis
低血清刺激された神経芽細胞腫のプロテオーム解析

池田　理恵
埼玉医科大学　小児外科

医学博士　乙第1033号　平成18年9月22日 （埼玉医科大学）

（目的）腫瘍細胞になんらかの刺激(低酸素，温度，抗癌剤など)を与えると，これに応答して細胞内に生存，増殖に関する蛋白質の発現調節が起こると推測される．われわれは神経芽腫細胞株GOTO細胞に低血清刺激を与えた時に有意に発現する細胞内蛋白質を，近年開発された蛍光標識2次元デイファレンスゲル電気泳動システム(2D-DIGE)によるプロテオーム解析によって捕らえ，蛋白質レベルでどのような調節が行われるかを検討した．
（方法）GOTO細胞を10％ ウシ胎仔血清(FBS)入りRPMI-1640培地で70-80％の細胞密度まで培養後，1％ FBS入り同培地に置換し24時間培養した．10％FBS同培地で24時間培養したサンプルをコントロールとした．各群の蛋白質を抽出，蛍光標識し2次元電気泳動を行った．電気泳動後，ゲルイメージの発現蛋白質量の差異を解析した．有意な発現変化のあるスポットを切り出し，ゲル内でトリプシン消化，消化物を質量分析し，各スポットの蛋白質同定を行った．
（結果）10％FBS群に比べ1％FBS群でアポトーシス，増殖，分化に関する6つの蛋白質(cytochrome C, endoplasmic reticulum calcium-binding protein 55, laminin-binding protein, stathmin, pre-mRNA splicing factor SF2, heat shock cognate protein 70) の発現増加や減少が認められた．
（考察）同定した蛋白質は細胞増殖促進や抑制の相反する作用を持つ蛋白質だった．これらの蛋白質の発現増加および減少は低血清濃度という危機的環境において，生存や増殖の継続に向けた分子レベルでの調節機構が，GOTO細胞内で生じていることを示唆している．今回の様なストレス負荷時に発現する蛋白質の解析と同定は，神経芽腫の治療戦略上重要な情報を提供すると思われる．
Keywords: 神経芽細胞腫，プロテオーム解析，2D-DIGE

Proteomic Analysis of Proteins which Expressed in GOTO Cells Stressed by Low Serum Concentration
Rie Ikeda (Department of Pediatric Surgery, Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

Thesis
受容体ノックアウトマウスを活用したエストロゲンの初期卵巣作用に関する検討

大羽　沙弥佳
埼玉医科大学ゲノム医学研究センター遺伝子情報制御部門医学研究科 生物・医学研究系専攻 博士課程4年

医学博士　平成18年10月30日 （埼玉医科大学）

　女性ホルモンであるエストロゲンは，幅広い生物種で生殖器の形成過程，二次性徴の成長に不可欠である．その生理機能は，2種類のエストロゲンレセプター (ERαとERβ) を介し下流応答遺伝子群の転写を制御することで引き起こされる．ERαおよびERβは，各臓器での発現パターンの違いやノックアウト (KO) マウスにおける卵巣の表現型からも異なる機能を有すると想定されているが，その機能は未だ不明な点が多い．
　本研究では，ほ乳類におけるエストロゲンの卵巣作用に関する機能解析を行うために，受容体ノックアウトマウスERαKOおよびERβKOマウスを活用し，特にエストロゲンシグナルと初期卵巣作用に重点を置き研究を行った．ERαKOマウス卵巣では，未成熟3週齢卵巣において，顆粒膜細胞のTUNEL陽性細胞の減少，Caspase-3の発現局在の変化およびcyclin D2の発現減少が観察されたが，生後6日齢（P6齢）マウス卵巣ではcyclin D2の発現や局在に変化がなかった．一方，ERβKOマウス卵巣では，生殖細胞増殖因子c-kit抗体による免疫染色を指標とした生殖細胞数の変化を検討した結果，P6齢卵巣においてc-kit陽性細胞が有意に増加していた．さらに，P6齢雌マウスにエストロゲン付加した際のc-kit mRNAの発現を検討した結果，エストロゲン投与6時間後に，c-kitのmRNA量が減少していることを確認した．以上の結果から，エストロゲンの初期卵巣作用に関し，ERαは，KOマウス3週齢卵巣におけるアポトーシスの抑制と細胞増殖の減少に，直接または間接的に関与していることが示唆された．ERβは，出生前後の時期に原始生殖細胞の増殖を抑制する方向で，生殖細胞数を制御している可能性が示唆された．
Keywords: エストロゲン，ERα，ERβ，卵巣機能，アポトーシス，細胞増殖

Analysis of Estrogen Signaling on Early Ovarian Function Utilizing ERα and ERβ KO Mice
Sayaka Ohba (Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan)

Thesis
アンギオテンシンタイプ1受容体拮抗薬オルメサルタンのPPARγ2プロモータへの転写活性増加作用

金沢　健太¹⁾，井上　郁夫¹⁾，池田　正明²⁾，片山　茂裕^1)
1)埼玉医科大学　内科学内分泌・糖尿病内科部門　²⁾埼玉医科大学　内科学内分泌・生理学教室

医学博士　甲第1028号　平成17年6月23日 （埼玉医科大学）

Angiotensin Type 1 Receptor Blocker, Olmesartan Induce Peroxisome Proliferator-Activated Receptorγ mRNA Level Through Promoter of PPARγ2
Kenta Kanazawa¹⁾, Ikuo Inoue¹⁾, Masaaki Ikeda²⁾, Shigehiro Katayama¹⁾(Division of Endocrinology and Diabetes, Department of Internal medicine¹), Department of Phisiology²), Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan)

Background: Angiotensin type 1 receptor (AT₁R) blockers (ARBs) have been shown to reduce the incidence of type 2 diabetes mellitus by an unknown molecular mechanism. The peroxisome proliferator-activated receptor γ (PPAR γ) is the central regulator of insulin and glucose metabolism improving insulin sensitivity. We investigated the regulation of PPARγ function by ARB, olmesartan.
Methods and Results: The ARB, olmesartan dose dependently (from 0 to 50 nmol/L) significantly enhanced in mRNA expression of PPARγ in human kidney cell, human renal proximal tubule cells (RPTEC), by method of quantitative real-time polymerase chain reaction (olmesartan:2.15±0.25-fold induction). In addition, in transcription reporter assays, olmesartan (from 0 to 10 μmol/L) induced transcriptional activity of promoter of PPARγ2 by 1.29±0.22-fold (P＜0.05) and induced the gene expression level of PPARγ2 by 2.52±0.69-fold(P＜0.05). More over, olmesartan, dose dependently, induced transcriptional activity of promoter of cellular retinol-binding protein II (CRBP II) which is known as one of the target genes of PPARγ. Olmesartan induced PPARγ mRNA level through promoter of PPARγ2, resulting in stimulateing the target gene of PPARγ.
Conclusions: The present study demonstrates that olmesartan induces PPARγ activity through promoter of PPARγ2, resulting in enhancing of target of PPARγ. Our results demonstrate new pleiotropic actions of olmesartan, providing a potential mechanism for their insulin-sensitizing/antidiabetic effects.
Keywords: Olmesartan, Angiotensin type 1 receptor (AT₁R) blocker(ARB), Peroxisome proliferator-activated receptorγ (PPARγ), Cellular retionol- binding protein II (CRBPII)