垂体腺瘤生物学行为发生机制的临床病理学评价

垂体腺瘤细胞增殖和分化的核心过程是DNA复制和细胞分裂,此过程包含了染色体、端粒、DNA倍体、S期细胞比例、DNA片段多态性等的变化;变异的基因转录合成了具有生物活性的蛋白质、酶和(或)相应受体,如拓扑异构酶Ⅱ、Ki-67、Galectin-3、生长抑素受体、P27等,使垂体表型及细胞外基质发生了改变。其生物学行为表现为:垂体瘤细胞出现形态学改变(核分裂相),腺瘤内血窦生长丰富,起源细胞具有分泌特性,部分腺瘤细胞贴壁信赖性丧失而出现侵袭性行为,及腺瘤细胞大量增殖等临床病理学特点。本文就有关内容进行综述。     

慢病毒携带人Galectin-3基因RNAi的有效靶点筛选

OBJECTIVE: To construct a recombinant lentiviral U6 plasmids for RNA interference (RNAi) of galectin-3 gene and select the optimal target sequence of galectin-3 gene for RNAi. METHODS: Double-stranded oligo DNAs were designed and synthesized according to the sequence of galectin-3 gene, and ligated into linearized pGCL-GFP/U6 plasmid followed by transformation into competent DH5alpha cells. After PCR and sequence analysis for verification of the positive clones, the plasmid pGCL-GFP/U6 Gal-3shRNA-1 was extracted and transfected into CaCl(2)-treated 293T cells to obtain the viral vectors containing the RNAi sequence. MCF-7 cells were infected with pGCL-GFP/U6 Gal-3shDNA-1, and at the infection rate over 50%, the cells were harvested to extract the RNA. Real time-PCR was performed to determine the expression level of galectin-3 mRNA in the infected cells. RESULTS: The recombinant vector was successfully constructed as confirmed by sequence analysis. High titer of the virus was obtained, and after infection of MCF-7 cells, RNAi targeting the 1(#) and 3(#) sequences in galectin-3 gene resulted in suppression of galectin-3 mRNA expression by 95% and 85%, respectively. CONCLUSION: The recombinant lentiviral U6 plasmid for RNAi of Galectin-3 gene has been successfully constructed, which provides the basis for further study of the role of galectin-3 gene in tumor cells.     

体外培养人垂体无功能腺瘤来源细胞-F的超微结构观察

目的：研究体外培养人垂体无功能腺瘤来源细胞-F形态、结构特征及在体外条件下细胞的外分泌调节功能.方法：在体外建立人垂体无功能腺瘤来源细胞-F的培养体系前提下,采用光学显微镜、扫描电镜,透射电镜及免疫细胞化学法检测培养细胞.结果：培养的人垂体无功能腺瘤来源细胞-F,光镜下可见半贴壁状况,形态不规则,呈多角形或圆形,细胞核浆清晰可见,经7～8d的培养后,细胞生长数量增多,多相连成簇.扫描电镜下可见：细胞胞体大小不甚一致,呈类圆形或圆形,表面常隆起,核区隆起较高;细胞表面散布有微绒毛,排列较密,单一微绒毛呈杆状,顶端彭大,长不足10μm.透射电镜下可见：培养细胞胞体呈类圆形,整个胞膜外可见散布的微绒毛,并可见微绒毛向胞内延伸,与分泌颗粒关系密切;核周胞质内可见粗面内质网、高尔基体、线粒体灶性肿胀和空化,分泌颗粒稀疏、直径为150～220nm等分布,胞核增大,核仁明显,可见切迹,有核仁边集.抗人垂体催乳素mAb染色阳性面积大于60%,而其他激素抗体检测为阴性;连续测定培养细胞上清中催乳素分泌水平分别为：（2.97±0.08）,（2.91±0.97）,（0.92±0.30）,（1.12±0.50）,（0.62±0.17）,（0.40±0.06）ng/L.结论：体外培养的人垂体无功能腺瘤来源细胞-F能够在体外保持其生活状态及增殖分裂能力.     

接种卡介苗致寻常狼疮一例

<正> 患者女,16岁。4年前左上臂接种卡介苗10天后局部出现一黄豆大硬结,逐渐增大如贰分硬币。2个月后破溃,溢出白色稀薄脓液,不痛不痒,未治疗。4个月后溃疡逐渐缩小,部分形成瘢痕及肉芽肿,时轻时重。3年前左上臂溃疡处及其周围出现绿豆大小的棕褐色结节,结节逐渐增多并相互融合成大片状棕褐色浸润性     

非艾滋病相关隐球菌脑膜炎临床分析

目的探讨两性霉素B(AmB)联合三唑类抗真菌剂治疗非AIDS相关隐球菌脑膜炎的临床疗效和2007至2012年新型隐球菌对氟康唑(FCZ)的耐药情况。方法回顾性分析29例非AIDS相关隐球菌脑膜炎患者经AmB联合FCZ治疗(19例)和AmB联合伏立康唑(VRC)治疗(10例)的临床特点、疗效和转归。比较AmB联合FCZ治疗(19例)在2007至2009年(8例)和2010至2012年(11例)两个不同时间段的临床特点、疗效和转归。结果 (1)AmB联合FCZ组和AmB联合VRC组治疗1周后脑脊液葡萄糖含量,差异有统计学意义(P<0.05),余各项脑脊液生化指标和颅内压差异无统计学意义(P>0.05)。治疗结束后脑脊液蛋白质含量,差异有统计学意义(P<0.05),余各项脑脊液生化指标和颅内压差异无统计学意义(P>0.05)。AmB联合FCZ组和AmB联合VRC组有效率分别为73.7%和80.0%,差异无统计学意义(P>0.05)。(2)在2007至2009年和2010至2012年两个不同时间段经过AmB联合FCZ治疗后,颅内压和脑脊液各项生化指标比较,差异无统计学意义(P>0.05)。2007至2009年和2010至2012年两个时间段的有效率分别为75.0%和72.7%,差异无统计学意义(P>0.05)。结论目前,AmB联合FCZ可能是一种较好的治疗方案。     

延髓腹侧呼吸上皮源性囊肿一例

患者女,40岁.主因晕厥、咽部流液感、左耳听力下降7余年就诊.查体:神清语利,双瞳孔等大,对光反应灵敏,眼动充分,面纹对称,左耳听力下降,伸舌自如居中,舌肌未见萎缩,咽反射存在,颈软无抵抗,深浅反射存在,四肢活动自如,肌力肌张力正常,闭目难立征阳性,左侧巴氏征阳性.术前MRI示(图1a):延髓右前可见短T1长T2信号,不能强化.全麻下行右侧远外侧开颅肿瘤切除术,术中见肿瘤位于脑干腹侧偏右,灰白色,有包膜,囊性,内容物黏稠状,无血运,与周围组织边界清楚,和后组脑神经粘连,最终镜下全切肿瘤(图1b).术后给予脱水、止血、抑酸等治疗,出院时未见神经损害症状,痊愈出院.随访3个月未见异常.术后病理:胚胎残余囊肿,被覆纤毛柱状上皮及鳞状上皮,符合呼吸上皮源性囊肿(图2);免疫组化:CK和EMA阳性(图3).     

垂体催乳素腺瘤中半乳凝素-3-mRNA表达与生物学行为的关系

目的探讨垂体催乳素腺瘤中半乳凝素-3-mRNA(Galectin-3-mRNA)表达与腺瘤的临床生物学行为之间的关系。方法选择32例经蝶显微手术切除的垂体催乳素腺瘤患者标本,分侵袭组与非侵袭组,采用显色原位杂交法(CISH)检测石蜡切片标本中Galectin-3蛋白的mRNA。以非参数检验对两种方法检测结果的相关性进行分析。结果侵袭性与Galectin-3的mRNA表达呈显著性相关(相关系数=0.893 P<0.01);Galectin-3-mRNA与血浆PRL水平、腺瘤体积大小相关(相关系数=0.593,0.522 P<0.05)。结论Galectin-3-mRNA表达与对判断侵袭性催乳素腺瘤体积大小、侵袭、血清PRL水平可能有一定的参考价值。     

慢病毒携带人Galectin-3基因RNAi的有效靶点筛选

目的 构建携带人Galectin-3基因的RNAi慢病毒载体,测定感染滴度,并对不同靶点进行有效筛选.方法 根据Galectin-3基因设计4对,经退火制备的双链DNA oligo,连接经双酶切的线性化pGCL-GFP/U6载体,转化后经PCR鉴定,阳性克隆测序,质粒抽提,经CaCl2导入293T细胞,包装成慢病毒,收集病毒上清并检测其滴度.将含Galectin-3基因的RNAi病毒颗粒,感染靶细胞,荧光显微镜观察细胞荧光,感染率>50%时,收集细胞抽提RNA,RT-PCR检测Galectin-3 mRNA水平,评价干扰效果.结果 经测序、PCR分析证实目的 序列成功连接到载体上,载体构建成功,包装成高滴度慢病毒.感染MCF-7细胞系后,细胞荧光显示感染率>50%,定量RT-PCR检测结果发现:1#和3#靶点敲减Galectin-3基因效率达95%、85%.结论 成功构建并筛选了携带有人Galectin-3基因的RNAi慢病毒载体及有效靶点,为进一步研究Galectin-3在肿瘤细胞中的作用提供实验平台.     

在脑多形胶质母细胞瘤中异柠檬酸脱氢酶-1突变体的研究

Mutations in an active site A132Arg of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are associated with glioblastomas multiformes (GBMs), a common type of human brain tumors. A recent study showed these mutations in the site A132Arg impairs IDH1’s affinity for its substrate ICT and consequently reduce the enzyme products α-ketoglutarate (α-KG). The lack of cytosolic α-KG of normal cells leads to tumors through induction of the HIF-1 pathway. The roles of other active sites in substrate binding of IDH1 remain unclear. To investigate the mutation pattern of IDH1 and its influence on this enzyme function, we performed in silicon mutations on the total 10 catalytic active sites of IDH1 and calculated the affinity of these mutant IDH1s for its substrate ICT. We found that mutations in site A132Arg, A109Arg and B212Lys largely reduced IDH1’s affinity for ICT, indicating the central roles of these three active sites in substrate binding. In contrast, four mutations in site A77Thr, A94Ser and A275Asp significant increased IDH1’s affinity for ICT, which may contribute to enhanced catalytic activity of IDH1. These mutant IDH1s with higher catalytic activity than the wild-type IDH1 may provide a new method of gene therapy for GBMs.