CTLA4Ig基因修饰的骨髓基质细胞抑制淋巴细胞活化的实验研究

目的:研究CTLA4Ig基因修饰的骨髓基质细胞(BMSCs)对淋巴细胞活化的抑制效应。方法:CTLA4Ig重组腺病毒载体转染BMSCs,以RT-PCR、免疫细胞化学、蛋白印迹法检测CTLA4Ig的表达情况,MTT法、ELISA法观察转染上清对混合淋巴细胞反应(MLR)体系中淋巴细胞活化、增殖的抑制效应。结果:转染组及转染后传两代BMSCs中检测到CTLA4Ig基因的转录,CTLA4Ig弥漫表达于细胞浆;转染后检测培养上清CTLA4Ig即呈阳性,72h达高峰,传一代培养上清中仍可检测到;转染上清对淋巴细胞的增殖、IL-2分泌有明显的抑制作用。结论:CTLA4Ig重组腺病毒能有效介导CTLA4Ig基因在BMSCs中的表达。且CTLA4Ig能有效抑制淋巴细胞的活化、增殖。     

腺病毒介导CTLA4Ig及CTLA4双基因在骨髓间充质干细胞中的表达

目的制备CTLA4Ig及CTLA4双基因共表达腺病毒载体,检测重组腺病毒在骨髓间充质干细胞(BMMSCs)中的表达。方法构建大鼠CTLA4Ig融合基因,将CTLA4Ig及CTLA4基因经IRES2连接,通过同源重组获得重组腺病毒表达载体,并在293细胞中进行病毒包装和扩增。检测CTLA4Ig和CTLA4在BMMSCs中的共表达情况及其免疫抑制功能。结果通过同源重组获得携带CTLA4Ig-IRES2-CTLA4的重组腺病毒表达载体,PacⅠ酶切鉴定正确,与脂质体共转染293细胞获得重组腺病毒。用重组腺病毒感染的BMMSCs可同时表达CTLA4Ig及CTLA4,且此类细胞具有明显抑制淋巴细胞反应的功能。结论重组腺病毒感染的BMMSCs可同时表达分泌型CTLA4Ig和膜结合型CTLA4,通过阻断协同刺激通路方式进一步增强BMMSCs的免疫抑制功能。     

CTLA—4在防治自身免疫性疾病上的作用

CTLA－4和CD28的结构相似,与抗原提呈细胞（APC）表面B7分子[B7－1（CD80）,B7－2（CD86）]有很高的亲和力,主要与抗原提呈细胞表面B7－1分子形成受体／配体对,相互作用可以抑制T细胞的增殖、活化,起着负性共刺激分子的作用,以维持机体淋巴细胞动态平衡,防止自身反应性T细胞过度激活,从而在防治自身免疫性疾病方面起重要作用。     

肿瘤相关抗原MART-1蛋白原核表达载体的构建、表达纯化和活性鉴定

目的构建编码肿瘤相关抗原MART-1融合基因的原核表达载体,在大肠埃希菌中表达His-MART-1融合蛋白,并对蛋白进行纯化和免疫原性鉴定。方法采用PCR法扩增编码MART-1的基因片段,将该基因片段克隆到含有His标签的pET-28b原核表达载体上,重组质粒经双酶切、PCR及测序鉴定正确后转化大肠埃希菌,IPTG诱导融合蛋白表达,用镍离子亲和层析法纯化融合蛋白并进行脱盐,SDS-PAGE电泳和Western blotting法鉴定。通过ELISA法检测经树突状细胞提呈后的His-MART-1融合蛋白对特异性CD4+T细胞的刺激作用。结果重组质粒经双酶切、PCR和测序鉴定证明载体构建成功。His-MART-1融合蛋白经表达纯化后,分子量约13kD,与预期值相符。Western blotting证实该融合蛋白可与抗His单克隆抗体发生特异性结合。His-MART-1融合蛋白经树突状细胞提呈后能刺激MART-1特异性CD4+T细胞分泌IFN-γ。结论成功构建了编码MART-1基因原核表达载体pET-28b-MART-1,表达及纯化获得该融合蛋白,并证实其具有良好的免疫原性。     

HMGB1促进人骨髓间充质干细胞迁移的实验研究

目的：构建人高迁移率族蛋白（HMGB1）全长编码基因的原核表达载体，诱导和纯化重组蛋白，分析其对人骨髓间充质干细胞的迁移作用。方法：RT—PCR方法从人的单个核细胞中扩增出HMGB1全长编码基因，克隆于原核表达载体pET-24a—d（＋），经IPTG诱导表达，通过亲和层析方法纯化得到携带（His）。标签的HMGB1融合蛋白；RT—PCR方法检测间充质干细胞HMGB1受体的表达；观察HMGB1对人骨髓来源间充质干细胞的迁移作用。结果：构建了融合蛋白HMGB1的原核表达载体，获得了高度纯化的HMGB1融合蛋白。RT-PCR方法检测到间充质干细胞表达HMGB1的某些受体如RAGE和TLR-4。HMGB1在体外能够诱导人骨髓来源的间充质干细胞的迁移。结论：重组HMGB1蛋白能够诱导骨髓间充质干细胞的迁移，此作用有可能通过RAGE和（或）TLR-4介导。     

NK细胞免疫球蛋白样受体KIR3DL1胞外区的表达及纯化

目的 表达并纯化NK细胞免疫球蛋白样受体KIR3DL1胞外区.方法 以pUC57-KIR3DL1为模板,PCR扩增KIR3DL1胞外区序列,与pGEM-T载体进行A-T克隆;测序正确后,采用定向克隆的方法将KIR3DL11胞外区基因连接到pET28a-DsbA载体上,成功构建了pET28a-DsbA/KIR3DL1重组质粒.将重组质粒导入大肠杆菌BL21(DE3),IPTG诱导DsbA-KIR3DL1融合蛋白表达,溶解于8mol/L尿素中的包涵体经Ni-NTA琼脂糖亲和层析纯化,成功进行复性,再经Superdex 75凝胶层析柱进一步纯化,SDS-PAGE和Western blot鉴定目的 蛋白的表达及纯化.结果 表达产物呈部分可溶性表达,包涵体纯化后证实获得纯度95%以上的DsbA-KIR3DS1融合蛋白.结论 KIR3DL1胞外区的成功表达及纯化为进一步研究KIR3DL1与相应配体之间的相互作用奠定了基础.     

钩端螺旋体外膜脂蛋白LipL32的重组表达及纯化

目的：构建L32-pQE32重组质粒，诱导表达重组钩端螺旋体外膜脂蛋白LipL32，对重组蛋白进行纯化。方法：采用PCR法从钩端螺旋体DNA中获取编码LipL32的基因片段，构建重组克隆载体pGEM-T／L32和表达载体L32-pQE32，转化受体茵E.coli DH5α和E．coli M15，IPTG诱导表达重组LipL32蛋白。Ni-NTA亲和层析纯化重组LipL32蛋白。结果：扩增出约750bp的LipL32成熟蛋白基因，LipL32基因插入pQE32表达载体，表达产物6个组氨酸与LipL32蛋白的融合蛋白相对分子质量约为31000，与预期大小一致。Western印迹显示能与钩体抗血清特异结合。结论：LipL32蛋白能在大肠杆菌中表达，Ni-NTA亲和层析可有效纯化重组LipL32蛋白，重组LipL32蛋白具有结合活性。     

sTNFR1在昆虫细胞中的表达及鉴定

目的 :研究可溶性肿瘤坏死因子受体 (solubleTNFαreceptor1,sTNFR1)在昆虫细胞中的表达。方法 :应用BAC_TO_BAC杆状病毒表达系统 ,构建含有sTNFR1基因的杆状病毒穿梭载体Bacmid ,转染sf2 1昆虫细胞进行高效表达 ,利用TALON金属鏊合层析进行重组蛋白的纯化。免疫印迹和细胞测活方法鉴定重组sTNFR1的生物学活性。结果 :从无血清培养上清中可纯化得到约 3mg L的重组蛋白。免疫印迹反应和细胞活性实验表明 ,重组蛋白具有良好的抗原结合能力和抑制TNF对L92 9细胞的细胞毒作用。结论 :重组sTNFR1在昆虫细胞中表达稳定 ,易于纯化 ,并具有良好的生物学活性     

人自噬相关基因LC3 B原核表达载体的构建及活性检测

目的：构建带GST标签的人LC3B基因原核表达载体,得到GST-LC3B重组质粒并纯化出GST-LC3B融合蛋白,体外检测并证实该蛋白的生物学活性。方法利用PCR技术从人乳腺文库中扩增出LC3B基因的编码序列,将该序列插入到pGEX-KG载体中,得到GST-LC3B重组质粒,转化大肠杆菌Rossate,经小量诱导后利用GST-Sepharose 4B亲和珠纯化GST-LC3B融合蛋白,通过SDS-PAGE电泳和Western印迹方法进行检测,GST pull-down技术证实其生物学活性。结果利用PCR技术从人乳腺文库中成功扩增得到大小约400 bp的目的基因片段,插入pGEX-KG载体中得到GST-LC3B质粒,经双酶切鉴定及测序结果表明重组质粒构建成功;转化Rossate菌并小量诱导,表达鉴定成功后纯化得到相对分子质量（ Mr）约为40×103的目的蛋白;GST pull-down技术检测出GST-LC3B融合蛋白可以和Atg4B蛋白在体外作用,具有较好的生物学活性。结论成功构建了人自噬相关基因LC3B的原核表达产物,为进一步研究LC3 B在自噬中的作用机制奠定了基础。     

微丝相关蛋白hHBRK1突变体的构建、表达及纯化

目的：构建微丝相关蛋白hHBRK1截短体和突变体原核表达载体，并表达及纯化融合蛋白。方法：采用常规PCR并结合定点突变技术，对hHBrkl基因进行缺失和点突变；利用限制性内切酶将PCR产物克隆至原核表达载体pGEX-4T；IPTG诱导融合蛋白表达，通过谷胱甘肽一琼脂糖纯化技术分离纯化GST融合蛋白。结果与结论：构建了包括氨基端缺失截短体hHBRK1-AN、羧基端缺失截短体hHBRK1-AC和点突变蛋白hHBRKl-S^56G^57在内的原核表达质粒，分离获得较高纯度的重组hHBRKl突变体融合蛋白，Western杂交证实纯化蛋白为GST融合蛋白。本实验为进一步研究hHBRK1的相互作用蛋白及其可能的结合位点提供了基础。     

Physiologic and pathologic calcifications and ossifications in the face and neck

The aim was to give a systematic presentation of physiologic and pathologic calcifications and ossifications in the face and neck with a special emphasis on clinical relevance. In a sometimes subacute setting one should recognize specific calcifications which often lead to important diagnoses such as fungal sinusitis or sclerosing labyrinthitis. In a more chronic situation intraocular calcifications in small children are pathognomonic for retinoblastoma. Juxtatumoral sclerosis of the laryngeal cartilage in laryngopharyngeal carcinoma is usually caused by tumor infiltration of the cartilage resulting in a higher tumor stage and, this way, has a major impact on the therapeutical strategy. Calcified lymph nodes are mainly unspecific but can be the result of tuberculosis or metastases of thyroid cancer. Cross-sectional imaging methods, most of all computed tomography, are ideally suited to reveal head and neck calcifications and ossifications, especially those which are clinically relevant.     

Head and neck infection and inflammation

This article discusses the imaging manifestations of infectious and inflammatory conditions of the head and neck. Special attention is paid to the sites, routes of spread, and complications of neck infections. Because the clinical signs and symptoms and the complications of these conditions are often determined by the precise anatomic site involved, anatomic considerations are stressed. Familiarity with the fascial layers, spaces of the neck, and the contents of each space is helpful for this discussion. The fascial layers of the neck are important barriers to infection, and once infection is established, the fascial layers play a part in directing its spread.     

Staging and surgical management of pancreatic and biliary cancer and inflammation

Management of benign and malignant diseases of the pancreas, liver, and biliary tract has made remarkable progress in the last two decades. Advances in minimally invasive surgery, interventional radiology, and diagnostic and therapeutic endoscopy have changed the treatment of common diseases such as cholelithiasis and more serious diseases such as pancreatic adenocarcinoma. Advances in biliary tract and pancreatic surgery have paralleled the advances in ultrasonographic imaging, CT, and MR imaging. This article outlines the surgeon's perspective on radiologic imaging and preoperative staging of benign and malignant biliary and pancreatic disease.     

细胞自噬与肿瘤发生发展

自噬是真核生物中一种高度保守的胞内降解途径.其主要通过溶酶体或液泡进行饥饿状态下的营养动员,清除受损蛋白质、细胞器和胞内病原体.自噬主要包括巨自噬、分子伴侣介导自噬(CMA)和微自噬.自噬已被证实与多种人类疾病相关,其在肿瘤发生发展中具有重要意义.近年研究中,对于自噬和肿瘤关系有了进一步的认识,该文就自噬分子机制、调控通路以及与肿瘤发生发展关系的研究进展作一综述.     

The thyroid and parathyroid glands. CT and MR imaging and correlation with pathology and clinical findings

Thyroid imaging approach is based on the preliminary clinical evaluation. Lesions that are smaller than 2 cm should be assessed with US, which is capable of discriminating masses as small as 2 mm and distinguishing solid from cystic nodules. US-guided FNAB provides tissue for cytologic examination of thyroid nodules. CT and MR imaging are indicated for larger tumors (greater than 3 cm diameter) that extend outside the gland to adjoining structures, including the mediastinum, and retropharyngeal region. Metastatic lymph nodes in the neck and invasion of the aerodigestive tract are also in the realm of CT and MR imaging. Thyroid nodules are categorized on scintigraphy as hot or cold nodules. Hot nodules are rarely malignant, whereas cold nodules have an incidence of 10% to 20% of malignancy. Calcifications (amorphous, globular, nodular, and linear) occur in adenomas and carcinomas and have no differential diagnostic features except for psammomatous calcifications, which are a pathognomonic finding in papillary carcinomas and a small percentage of medullary carcinomas. Papillary carcinoma is the most common malignant tumor (80%) followed by follicular (20% to 25%); medullary (5%); undifferentiated; anaplastic carcinomas (< 5%); lymphoma (5%); and metastases. Lymph node metastases are common in papillary carcinoma, 50% at presentation, and less common in follicular carcinomas. The metastatic nodes in papillary carcinoma may enhance markedly (hypervascular); show increased signal intensity on T1-weighted images (increased thyroglobulin content or hemorrhage); and reveal punctate calcifications. Localized invasion of the larynx, trachea, and esophagus occurs predominantly in papillary and follicular carcinomas; the incidence is less than 5%. Ectopic thyroid tissue may be encountered in the tongue (foramen cecum); along the midline between posterior tongue and isthmus of thyroid gland; lateral neck; mediastinum; and oral cavity. Goiter and malignant tumors, notably papillary carcinoma, may develop in ectopic thyroid tissue. Carcinomas may also arise in thyroglossal duct cysts, which develop from duct remnants between the foramen cecum and thyroid isthmus. Infectious disease of the thyroid gland is not common and the CT and MR imaging findings are similar as described under neck infection. Other types of inflammatory disorders including Hashimoto's thyroiditis, granulomatous thyroiditis, and Riedel's struma display no specific imaging features. Imaging studies may, however, be indicated to confirm a suspected clinical diagnosis and assess compromise of the airway (Riedel's struma). HPT is a clinical diagnosis in which hypercalcemia is the most important finding. Parathyroid hyperplasia, adenoma, and carcinoma represent underlying lesions. To relieve the patient's symptoms surgical extirpation is indicated. The surgical success rate without imaging is 95%. The indications for imaging studies vary but it is generally agreed that reoperation after a previous failed surgical attempt and suspicion of an ectopic parathyroid adenoma should be investigated by imaging. These consist of US, nuclear medicine studies, CT and MR imaging. US and technetium sestamibi scanning have the highest accuracy rate for localizing an adenomatous gland at and near the thyroid gland. Ectopic adenomas, particularly if they are located in the mediastinum, are preferrably investigated with CT and MR imaging with gadolinium and fat suppression. Carcinomas and parathyroid cysts are optimally evaluated by CT and MR imaging. On MR imaging adenomas are low in signal intensity on T1-weighted images, high in signal intensity on T2-weighted images, and enhance post introduction of gadolinium.