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P53蛋白的原核表达及纯化

中国生物制品学杂志 2009年第6期22卷 563-566页

作者：钱冠华蒲淑萍郭风劲 tsutomu mori Hideo KOCHI 段昌柱重庆医科大学细胞生物学与遗传学教研室重庆医科大学分子医学与肿瘤研究中心重庆400016 日本福岛县立医科大学生化学第一讲座

目的构建P53基因重组原核表达质粒,在大肠杆菌中表达融合蛋白并进行纯化。方法以pBabe-P53R质粒为模板,PCR扩增P53基因全序列,克隆至pGEX-4T-1载体中,构建重组原核表达质粒pGEX-4T-1-P53,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物... 详细信息

目的构建P53基因重组原核表达质粒,在大肠杆菌中表达融合蛋白并进行纯化。方法以pBabe-P53R质粒为模板,PCR扩增P53基因全序列,克隆至pGEX-4T-1载体中,构建重组原核表达质粒pGEX-4T-1-P53,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经不同配比的去垢剂复性后,用GlutathioneSepharose4Bgel亲和层析纯化。结果重组原核表达质粒pGEX-4T-1-P53经双酶切鉴定,表明构建正确。表达的GST-P53融合蛋白相对分子质量约80000,IPTG诱导时间以1h为宜。1%TritonX-100和1mmol/LEDTA为蛋白复性的最佳去垢剂。纯化的GST-P53融合蛋白可与羊抗人GST抗体和鼠抗人P53抗体发生特异性反应。500ml菌液可获得1mg纯化蛋白。结论已成功构建了P53基因重组原核表达质粒,表达并纯化了GST-P53融合蛋白,为研究NIRF与P53基因之间的相互关系奠定了基础。

关键词： P53基因原核表达纯化 GST-pulldown

Novel concept of endoscopic device delivery station system for rapid and tight attachment of polyglycolic acid sheet

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World Journal of Gastroenterology 2018年第2期24卷 211-215页

作者： Hirohito mori Hideki Kobara Noriko Nishiyama tsutomu Masaki Departments of Gastroenterology and Neurology Kagawa University

AIM To evaluate appropriate and rapid polyglycolic acid sheet(PGAs) covering time using device delivery station system(DDSS).METHODS This pilot basic study was conducted to evaluate the potential of accurate and rapid PGAs delivery using DDSS. Three 11-month-old female Beagle dogs were used in this study. Two endoscopic submucosal dissections(ESDs) 4 cm in diameter were performed in lesser curvature of middle gastric body and greater curvature of antrum(total 6 ESDs performed). DDSS(3 cm length, 12 mm in outer diameter) has 2 chambers which 16 cm^2 large 2 PGAs were stored, and DDSS was attached post ESD ulcers, respectively. Beriplast P?(CSL Behring K.K., Tokyo, Japan)(combination of fibringlue and thrombin) was applied equally to the artificial ulcer, and tight attachment of 2 PGAs with DDSS were completed. The evaluation items were covering times, post ESD bleeding and perforation during *** The covering time of PGAs(defined as the duration from the beginning of endoscope insertion into the mouth to the end of the fibrin glue coating process) was 6.07(4.86-8.29) min. There was no postESD bleeding(1-7 d after ESD), and there was no perforation during *** DDSS was very useful for rapid delivering and tight attachment of PGAs, and has potentials of multipurpose delivery station system.

关键词： Post-endoscopic submucosal dissection ulcers Bleeding Polyglycolic acid sheet Rapidly delivery Tightly attachment

Bleeding duodenal hemangioma: Morphological changes and endoscopic mucosal resection

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World Journal of Gastroenterology 2012年第22期18卷 2872-2876页

作者： Noriko Nishiyama Hirohito mori Hideki Kobara Shintarou Fujihara Takako Nomura Mitsuyoshi Kobayashi tsutomu Masaki Department of Gastroenterology and Neurology Faculty of Medicine Kagawa Medical University

Recently, the development of endoscopic procedures has increased the availability of minimally invasive treatments; however, there have been few case reports of duodenal hemangioma treated by endoscopic mucosal resection. The present report describes a case of duodenal hemangioma that showed various endoscopic changes over time and was treated by endoscopic mucosal resection. An 80-year-old woman presented with tarry stools and a loss of appetite. An examination of her blood revealed severe anemia, and her hemoglobin level was 4.2 g/dL. An emergency upper gastrointestinal endoscopy was performed. A red, protrusive, semipedunculated tumor (approximately 20 mm in diameter) with spontaneous bleeding on its surface was found in the superior duodenal angle. Given the semipedunculated appearance of the tumor, it was suspected to be an epithelial tumor with a differential diagnosis of hyperplastic polyp. The biopsy results suggested a telangiectatic hemangioma. Because this lesion was considered to be responsible for her anemia, endoscopic mucosal resection was performed for diagnostic and treatment purposes after informed consent was obtained. A histopathological examination of the resected specimen revealed dilated and proliferated capillary lumens of various sizes, which confirmed the final diagnosis of duodenal hemangioma. Neither anemia nor tumor recurrence has been observed since the endoscopic mucosal resection (approximately 1 year). Duodenal hemangiomas can be treated endoscopically provided that sufficient consideration is given to all of the possible treatment strategies. Interestingly, duodenal hemangiomas show morphological changes that are influenced by various factors, such as mechanical stimuli.

关键词： Duodenal hemangioma Endoscopic muco-sal resection Gastrointestinal bleeding Morphologicalchanges Capillary hemangioma

Difficulty in differentiating two cases of sigmoid stenosis by diverticulitis from cancer

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World Journal of Gastroenterology 2012年第27期18卷 3623-3626页

作者： Noriko Nishiyama Hirohito mori Hideki Kobara Kazi Rafiq Shintarou Fujihara Mitsuyoshi Kobayashi tsutomu Masaki Department of Gastroenterology and Neurology Faculty of MedicineKagawa UniversityKagawa 761-0793Japan Department of Pharmacology Faculty of MedicineKagawa UniversityKagawa 761-0793Japan

The incidence of colonic diverticulosis with or without diverticulitis has increased in the Japanese population due to the modernization of food and aging. The rate of diverticulitis in colon diverticulosis ranges from 8.1% to 9.6%. However, few cases of stenosis due to diverticulitis have been reported. These reports suggest that the differentiation between sigmoid diverticulitis and colon cancer is difficult. This report describes two cases of colon stenosis due to diverticulitis that were difficult to differentiate from colon cancer. Case i was a 70-year-old woman with narrowed stools for 1 month who underwent colonofiberscopy （CFS）. CFS revealed a diverticulum and circumferential stenosis in the sigmoid colon. Barium enema revealed a marked, hourglass- shaped, 2-cm circumferential stenosis in the sigmoid colon. Fluorodeoxyglucose （FDG）-positron emission tomography computed tomography （CT） revealed an increased FDG uptake at the affected portion of the sigmoid colon. Sigmoid colon cancer was suspected,and laparoscopic sigmoidectomy was performed. Patho- logical examination demonstrated active inflammation with no evidence of malignancy. Case 2 was a 50-year- old man who presented to a nearby clinic with reduced stool output despite the urge to defecate. CFS detected severe stenosis in the sigmoid colon approximately 25 cm from the dentate line. Contrast-enhanced abdomi- nal CT revealed multiple diverticula, wall thickening, and swelling of the lymph nodes around the peritoneal aorta and the inferior mesenteric artery. A partial sig- moidectomy was performed. Pathological examination of the resected specimen revealed no changes in the mucosal epithelial surface, but a marked infiltration of inflammatory cells was observed.

关键词： Diverticulosis Colon cancer Colon stenosisPositron emission tomography-computed tomographyMagnetic resonance imaging

Oval mucosal opening bloc biopsy after incision and widening by ring thread traction for submucosal tumor

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World Journal of Gastroenterology 2017年第39期23卷 7185-7190页

作者： Hirohito mori Hideki Kobara Yu Guan Yasuhiro Goda Nobuya Kobayashi Noriko Nishiyama tsutomu Masaki Department of Gastroenterology and Neurology Kagawa University Departments of Pharmacology Kagawa University

Gastric submucosal tumors(SMTs) less than 2 cm are generally considered benign neoplasms, and endoscopic observation is recommended, but SMTs over 2 cm, 40% of which are gastrointestinal stromal tumors(GISTs), have malignant potential. Although the Japanese Guidelines for GIST recommend partial surgical resection for GIST over 2 cm with malignant potential as well as en bloc large tissue sample to obtain appropriate and large specimens of SMTs, several reports have been published on tissue sampling of SMTs, such as with endoscopic ultrasound sound fine needle aspiration, submucosal tunneling bloc biopsy, and the combination of bite biopsy and endoscopic mucosal resection. Because a simpler, more accurate method is needed for appropriate treatment, we developed oval mucosal opening bloc biopsy after incision and widening by ring thread traction for submucosal tumor(OMOB) approach. OMOB was simple and enabled us to obtain large samples under direct procedure view as well as allowed us to restore to original mucosa.

关键词： Gastric submucosal tumors Gastrointestinal stromal tumor Reversible opening biopsy Endoscopic ultrasonography Large sample

Rectal perforations and fistulae secondary to a glycerin enema:Closure by over-the-scope-clip

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World Journal of Gastroenterology 2012年第24期18卷 3177-3180页

作者： Hirohito mori Hideki Kobara Shintaro Fujihara Noriko Nishiyama Mitsuyoshi Kobayashi tsutomu Masaki Kunihiko Izuishi Yasuyuki Suzuki Department of Gastroenterology and Neurology Kagawa Medical University School of MedicineKagawa 761-0793Japan Department of Gastroenterological Surgery Kagawa Medical University School of MedicineKagawa 761-0793Japan

Rectal perforations due to glycerin enemas(GE) typically occur when the patient is in a seated or lordotic standing *** the perforation occurs and peritonitis results,death is usually *** describe two cases of rectal perforation and fistula caused by a *** 88-year-old woman presented with a large rectal perforation and a fistula just after receiving a *** case was further complicated by an abscess in the right rectal *** second patient was a 78-year-old woman who suffered from a rectovesical fistula after a *** both cases,we performed direct endoscopic abscess lavage with a saline solution and closed the fistula using an over-the-scope-clip(OTSC) *** procedures resulted in dramatic improvement in both *** endoscopic lavage and OTSC closure are very useful for pararectal abscess lavage and fistula closure,respectively,in elderly patients who are in poor general *** two cases are the first reports of the successful endoscopic closure of fistulae using double OTSCs after endoscopic lavage of the debris and an abscess of the rectum secondary to a GE.

关键词： Rectal perforation Glycerin enema Abscesslavage Fistula closure Over-the-scope-clip

Accurate hemostasis with a new endoscopic overtube for emergency endoscopy

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World Journal of Gastroenterology 2013年第17期19卷 2723-2726页

作者： Hirohito mori Hideki Kobara Shintaro Fujihara Noriko Nishiyama Makoto Oryu Kazi Rafiq tsutomu Masaki Departments of Gastroenterology and Neurology Kagawa Medical University School of Medicine Departments of Pharmacology Kagawa Medical University School of Medicine

Endoscopic hemostasis performed in the emergency room is difficult due to the presence of blood clots and food residue that makes obtaining a clear view of the bleeding vessel difficult. We experienced the efficacy of a newly developed inverted overtube to shorten the hemostatic time and obtain a clear endoscopic view with upper gastrointestinal bleeding patient who were transferred by ambulance car and required emergency endoscopy. The technique improved the endoscopic views and enabled us to perform the hemostatic procedures from the conventional standing position while freely and easily changing the patient's position. The presence of blood clots and food residue in the gastric fornix or upper gastric body makes identifying a bleeding exposed vessel impossible. This set-up significantly shortened the procedure time. The inverted overtube helped us obtain a clear view in patients who were laid in the right lateral position. Rapid identification of ex-posed vessels resulted in success of hemostasis.

关键词： Emergency endoscopic hemostasis Right lateral decubitus position Identification of exposed vessel Newly developed inverted overtube Clear endoscopic view

Efficacy and safety of over-the-scope clip: Including complications after endoscopic submucosal dissection

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World Journal of Gastroenterology 2013年第18期19卷 2752-2760页

作者： Noriko Nishiyama Hirohito mori Hideki Kobara Kazi Rafiq Shintarou Fujihara Mitsuyoshi Kobayashi Makoto Oryu tsutomu Masaki Department of Gastroenterology and Neurology Faculty of Medicine Kagawa University Kagawa 761-0796 Japan Department of Pharmacology Faculty of Medicine Kagawa University Kagawa 761-0796 Japan

AIM: To retrospectively review the results of over-thescope clip (OTSC) use in our hospital and to examine the feasibility of using the OTSC to treat perforations after endoscopic submucosal dissection (ESD). METHODS: We enrolled 23 patients who presented with gastrointestinal (GI) bleeding, fistulae and perforations and were treated with OTSCs (Ovesco Endoscopy GmbH, Tuebingen, Germany) between November 2011 and September 2012. Maximum lesion size was defined as lesion diameter. The number of OTSCs to be used per patient was not decided until the lesion was completely closed. We used a twin grasper (Ovesco Endoscopy GmbH, Tuebingen, Germany) as a grasping device for all the patients. A 9 mm OTSC was chosen for use in the esophagus and colon, and a 10 mm device was used for the stomach, duodenum and rectum. The overall success rate and complications were evaluated, with a particular emphasis on patients who had undergone ESD due to adenocarcinoma. In technical successful cases we included not only complete closing by using OTSCs, but also partial closing where complete closure with OTSCs is almost difficult. In overall clinical successful cases we included only complete closing by using only OTSCs perfectly. All the OTSCs were placed by 2 experienced endoscopists. The sites closed after ESD included not only the perforation site but also all defective ulcers ***: A total of 23 patients [mean age 77 years (range 64-98 years)] underwent OTSC placement during the study period. The indications for OTSC placement were GI bleeding (n = 9), perforation (n = 10), fistula (n = 4) and the prevention of post-ESD duodenal artificial ulcer perforation (n = 1). One patient had a perforation caused by a glycerin enema, after which a fistula formed. Lesion closure using the OTSC alone was successful in 19 out of 23 patients, and overall success rate was 82.6%. A large lesion size (greater than 20 mm) and a delayed diagnosis (more than 1 wk) were the major contributing fac

关键词： Over-the-scope clip Gastrointestinal bleed-ing Endoscopic submucosal dissection complications Gastrointestinal fistulae Gastrointestinal perforation

Management of a large mucosal defect after duodenal endoscopic resection

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World Journal of Gastroenterology 2016年第29期22卷 6595-6609页

作者： Shintaro Fujihara Hirohito mori Hideki Kobara Noriko Nishiyama Tae Matsunaga Maki Ayaki Tatsuo Yachida tsutomu Masaki Departments of Gastroenterology and Neurology Kagawa University Faculty of Medicine

Duodenal endoscopic resection is the most difficult type of endoscopic treatment in the gastrointestinal tract(GI) and is technically challenging because of anatomical specificities. In addition to these technical difficulties, this procedure is associated with a significantly higher rate of complication than endoscopic treatment in other parts of the GI tract. Postoperative delayed perforation and bleeding are hazardous complications, and emergency surgical intervention is sometimes required. Therefore, it is urgently necessary to establish a management protocol for preventing serious complications. For instance, the prophylactic closure of large mucosal defects after endoscopic resection may reduce the risk of hazardous complications. However, the size of mucosal defects after endoscopic submucosal dissection(ESD) is relatively large compared with the size after endoscopic mucosal resection, making it impossible to achieve complete closure using only conventional clips. The over-the-scope clip and polyglycolic acid sheets with fibrin gel make it possible to close large mucosal defects after duodenal ESD. In addition to the combination of laparoscopic surgery and endoscopic resection, endoscopic full-thickness resection holds therapeutic potential for difficult duodenal lesions and may overcome the disadvantages of endoscopic resection in the near future. This review aims to summarize the complications and closure techniques of large mucosal defects and to highlight some directions for management after duodenal endoscopic treatment.

关键词： Endoscopic mucosal resection Endoscopic submucosal dissection Duodenum Complication Bleeding Perforation Over-the-scope clip Clip Closure Endoscopic full-thickness resection