survivin的siRNA靶向干扰膀胱癌的实验研究

目的实验研究靶向转染survivin的小分子干扰RNA（small interfering RNA，siRNA）对膀胱癌生物学行为的影响。方法设计1对survivin编码基因序列特异的siRNA，用脂质体转染T24膀胱癌细胞株。采用流式细胞术检测转染效率和细胞凋亡；荧光实时定量PCR检测干扰前后survivin的mRNA表达；用survivin的siRNA片段插入空载体后建立重组载体pRNAT-U6、1／Neo-survivin。结果脂质体转染T24细胞后的转染效率为92．3％；survivin编码基因序列特异的siRNA能有效下调survivin基因的表达，呈时间和剂量依赖性，最大效应浓度为100nmol／L，此时survivin表达水平下调了75．91％，并显著地抑制了细胞生长，抑制率达55．29％，差异有统计学意义（P〈0．01）；细胞凋亡率亦增至45．70％，与对照相比差异有统计学意义（P〈0．01）。用限制性内切酶将空载体线性化，T4DNA连接酶将siRNA片段插入空载体中，对该重组表达载体进行鉴定，获得RNA干扰质粒载体pRNAT-U6．1／Neo-survivin。结论siRNA．survivin能显著下调膀胱癌细胞survivin基因表达水平，促进细胞凋亡，抑制肿瘤细胞增殖，可能成为膀胱癌基因治疗的新工具。成功构建的靶向survivin基因表达的RNA干扰质粒载体为进一步应用RNA干扰技术进行survivin基因功能研究奠定了基础。     

The distribution of metastases in the liver

Summary Seventy-five livers with metastases were cut sagitally into 1 cm thick slices. A total number of 11,581 metastases sections was exactly mapped. There was an average of 154 metastases sections per liver. The average diameter of the metastases was 1 cm. 40% of the metastases reached to the hepatic surface, and 60% were invisible due to their deposition in the internal parenchyma. In 8% of the livers there were only superficial metastases (average 3.2 metastases), and in 12% were only deep metastases detected (average 2.6). The total number of superficial metastases increased with increasing diameter of the secondary tumors.An approximately homogeneous distribution of hepatic metastases within the liver parenchyma has been demonstrated.     

Wolffian附件肿瘤的临床病理观察

目的 观察Wolffian附件肿瘤的临床病理特点，探讨其病理诊断与鉴别诊断。方法 对3例Wolffian附件肿瘤进行HE及免疫组化染色（EnVision法），并进行病理观察。结果 3例Wolffian附件肿瘤均为单侧，位于阔韧带或输卵管系膜。镜下肿瘤细胞呈弥漫实体状分布，可见成片的梭形或多边形细胞及排列紧密的管状结构。管腔内衬立方或柱状上皮，细胞无明显异型，核分裂象少见。管周有PAS阳性的基膜物质。免疫组化示肿瘤细胞vimentin、AE1／AE3、α-inhibin、calretinin和WT1均呈阳性，CD99呈灶性阳性，EMA和CK7呈阴性。结论 Wolffian附件肿瘤是具有独特发病部位和组织病理学特点的妇科肿瘤，需要和一系列其他妇科肿瘤鉴别。     

PTEN基因对人肝癌细胞系HHCC作用的研究

目的：研究PTEN基因对人肝癌细胞系HHCC细胞恶性表型及凋亡的影响。方法：将PTEN真核表达载体pBabe-PuroPTEN及空质粒pBabe-Puro，通过脂质体介导的基因转染方法，转入该基因表达缺失的HHCC细胞系中，嘌呤霉素筛选获得稳定表达PTEN基因的转染细胞。通过平板克隆形成试验、DNA凝胶电泳、相差显微镜和电镜，观察PTEN基因对人肝癌细胞系HHCC恶性表型及凋亡的影响。结果：转染后的细胞，经原位杂交、免疫组织化学检测证实，有PTEN mRNA及其蛋白的表达；平板克隆形成试验证实，转染PTEN基因后，细胞形成克隆的能力降低；转染PTEN的细胞在相差显微镜和电镜下可出现典型的凋亡形态学改变；DNA琼脂糖凝胶电泳呈现出明显的梯状条带。结论：外源性PTEN基因导入HHCC细胞后，可降低HHCC的细胞的恶性表型，并促进凋亡发生。     

软组织平滑肌肉瘤中p16基因的甲基化检测

目的 探讨软组织平滑肌肉瘤（ＬＭＳ）中p16基因ＩＮＫ４Ａ的甲基化状态及其与p16表达的关系。方法 应用ＭＳＰ法检测３８例软组织平滑肌肉瘤,１０例平滑肌瘤及５例正常平滑肌组织中p16基因ＩＮＫ４Ａ的甲基化状态,用免疫组织化学ＳＰ方法检测p16蛋白表达情况。结果 ３８例ＬＭＳ中９例发生异常甲基化,异常甲基化率为２３.7%（９／３８）。其中,７例p16蛋白表达阴性,２例p16蛋白弱阳性,在p16蛋白表达阴性的ＬＭＳ中,异常甲基化率为５０％（７／１４）。结论 p16基因第一外显子启动子区５‘CpG岛的异常甲基化是导致p16基因失活、蛋白缺如的重要基因外机制,并可能参与肿瘤的发生。     

Mediastinal lymphatic efferents from the diaphragm

The prognosis of non small cell lung cancer (NSCLC) invading the diaphragm is poor, probably due to the richness of the lymphatic drainage of the diaphragm. The aim of this study was to determine mediastinal lymphatic efferents from the diaphragm. The diaphragms of 20 adult cadavers (77-104 years) were injected with a dye (modified Gerota's medium) to permit the lymph vessels to be catheterised and then dissected. Each stage of the dissection was described and photographed: 23 injections on the right and 25 on the left. Diaphragmatic lymph vessels passed to one of three lymph centres: posterior (paraaortic nodes, n = 16), anterior (juxtasternal nodes, n = 16) and mediastinal (visceral nodes, n = 16). From these lymph centres arose ascending lymph pathways: posteriorly to the thoracic duct (8/16), anteriorly along the internal thoracic vessels (10/16) and in the mediastinum to the peritracheobronchial nodes (6/10). Lymphatics from the diaphragm are abundant and drain towards mediastinal node lymph centres connecting to the blood stream via the thoracic duct. These lymph pathways are common with those of the pulmonary segments. Poor prognosis of NSCLC invading the diaphragm may be explained by the common lymphatic drainage of both the lung and diaphragm.     

Assessment of local cellular immunity in lung cancer by bronchoalveolar lavage

Summary Small cell lung cancer (SCLC) is the most malignant of the pulmonary neoplasms and is associated with a poor local cellular immune response. 16 patients with non small cell lung cancer (NSCLC) and 11 patients with SCLC underwent bronchoalveolar lavage (BAL) in the lung which harbored the tumor in order to investigate the lymphocyte surface antigens utilizing the immunoperoxidase technique. Analysis of blood lymphocytes was performed in parallel. 8 patients with previous sarcoidosis in complete remission who underwent BAL and 10 normal blood donors served as controls.Among blood lymphocytes the CD3⁺, CD4⁺ and CD16⁺ cell populations were elevated significantly and the T4/T8 ratio was elevated in NSCLC patients, but only CD16⁺ were augmented in SCLC. Cell populations expressing the activation markers transferrin (TF) receptor, interleukin-2 (IL-2) receptor and the very late antigen VAL-1 were also increased in NSCLC, while SCLC was associated with antigen distributions similar to controls. No differences between the cohorts were seen in the expression of human leukocyte antigen (HLA)-DR. In BAL the population of CD3⁺ and CD4⁺ cells were reduced in SCLC and the T4/T8 ratio was diminished in contrast to controls and NSCLC patients, whereas these two latter groups did not differ from each other. The distribution pattern of CD16, TF receptor and IL-2 receptor in the study groups resembled that of cells of the blood stream, but CD16⁺ natural killer cells were additionally down regulated to control values in SCLC. No differences were seen in the distribution of VLA-1. HLA-DR⁺ cells were clearly elevated in both cancer groups.In general NSCLC was associated with a shift to higher relative numbers of immunocompetent and activated cells. This was most probably attributable to an immune response to neoplastic growth. This shift was largely lacking in SCLC. The analysis of lymphocytes from the periphery of the target organ emerged as a sensitive tool for the study of cellular immunity in lung cancer and showed many similarities to circulating blood cells. However, the analysis of natural killer cells and HLA-DR suggested a dissection of cellular immune response between blood and lung in pulmonary cancer. A depressive interaction between the tumor and the cellular host immune response may contribute to the exceptional malignancy of SCLC.Abbreviations BAL bronchoalveolar lavage - HLA-DR human leukocyte antigen-DR - IL-2 interleukin-2 - NSCLC non small cell lung cancer - SCLC small cell lung cancer - TF transferrin - VLA-1 very late antigen-1     

端粒酶转录酶及其调节相关蛋白与增殖细胞核抗原在卵巢上皮性肿瘤中的表达及临床意义

目的　探讨端粒酶转录酶 (hTERT)及端粒酶调节相关蛋白 (TRAP)与增殖细胞核抗原(PCNA)在卵巢上皮性肿瘤中的表达和临床意义。方法　收集 10 6例卵巢上皮性肿瘤 (恶性 5 4例 ,交界性 33例 ,良性 19例 )的临床资料 ,行hTERT、TRAP和PCNA 3种抗体的免疫组织化学标记链霉素卵白素生物素 (LSAB)法染色。对 87例恶性和交界性患者进行了随访 ,4 5例获结果 ,随访时间为 2～6 0个月。结果　hTERT蛋白的表达在良性 (4/ 19)和交界性 (90 9% ,30 / 33)以及良性和恶性(94 4 % ,5 1/ 5 4 )间的差异均有显著性 (P均 <0 0 0 1) ,TRAP蛋白的表达在良性 (4/ 15 )和恶性(77 8% ,2 8/ 36 )间的差异有显著性 (P <0 0 0 1) ;hTERT和TRAP蛋白的表达在卵巢癌Ⅰ、Ⅱ期和Ⅲ、Ⅳ期两组病例中的差异均无显著性 (P >0 0 5 ,P >0 3)。PCNA的表达在良性 (6 9± 5 9) %和交界性 (2 6 4± 17 8) %、良性和恶性 (5 1 8± 2 2 1) %以及交界性和恶性间的差异均有显著性 (P <0 0 1,P <0 0 0 1,P <0 0 5 )。 33例交界性患者全部存活 ,5 4例恶性患者中 35例 (6 4 8% )有转移 (包括 5例淋巴结转移 ) ,4例 (7 4 % )死亡。结论　hTERT和TRAP蛋白的表达与卵巢上皮性肿瘤的良恶性有关 ,但与其临床分期无关 ;hTERT和TRAP蛋白的表达相似     

肺组织β-AR和GRK2与重症急性胰腺炎肺损伤的关系以及甲强龙的影响

目的：探讨重症急性胰腺炎（SAP）致急性肺损伤（ALI）大鼠肺组织β-AR和G蛋白偶联受体激酶2（GRK2）的变化规律以及甲强龙的影响。方法：SD大鼠36只，随机分为3组，每组12只。对照组，仅做十二指肠翻动；模型组，胰胆管逆行注射5%牛磺胆酸钠（1 mL/kg），建立SAP模型；干预组，建立SAP模型后1 h给予甲强龙（30 mg/kg）。于6 h和12 h分别处死大鼠6只取肺组织，放射配基结合实验测量肺组织β-AR最大结合容量B_max和平衡解离常数K_d，免疫荧光法检测肺组织GRK2表达。结果：模型组和干预组胰腺组织损伤评分显著高于对照组，模型制备成功；模型组和干预组肺组织损伤评分显著高于对照组，发生SAP肺损伤；模型组β-AR B_max显著低于对照组和干预组，K_d显著高于对照组和干预组；模型组GRK2的表达显著高于对照组和干预组。结论：SD大鼠肺组织有丰富的GRK2表达，SAP肺损伤大鼠肺组织GRK2表达显著增加，这可能是β-AR下调的重要机制。