SA脂质体介导GFP／bFGF基因在豚鼠耳蜗中的表达

目的 :观察天然碱性脂 (Stearylamine,SA)脂质体介导绿色荧光蛋白 /碱性成纤维细胞生长因子(GFP/bFGF)基因于不同时间段豚鼠耳蜗中的表达 ,为进一步研究耳聋的基因治疗提供实验基础。方法 :取豚鼠 1 6只 ,分成 4组 ,每组 4只。其中 3只右耳圆窗内注入SA -GFP/bFGF复合物 ,1只同法注入生理盐水作为对照。分别于术后第 2、7、1 4、2 1天取材。在荧光显微镜下观察GFP的表达 ,用免疫组化法检测bFGF的转导情况。结果 :荧光显微镜下见双侧耳蜗于术后第 2天开始部分细胞发出绿色荧光 ,第 7天达到高峰 ,支持细胞及内外毛细胞均显荧光 ,细胞轮廓清晰 ;第 1 4天开始减弱 ,第 2 1天消失。免疫组化染色显示 ,除血管纹外 ,耳蜗各回Corti器、螺旋韧带、螺旋缘及螺旋神经节细胞均有高浓度的表达产物 ,对照动物呈阴性表达。结论 :SA脂质体介导的GFP/bFGF基因单耳给药双侧耳蜗均有高效表达 ,为进一步研究基因治疗耳聋提供了可能。     

Characterization of gp 50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody

Several cell lines secreting monoclonal antibodies (Mabs) against a major forebrain synaptic membrane (SM) glycoprotein, gp 50, have been raised. Western blots show that the Mabs react with a polypeptide doublet of Mrs 49 and 45 kDa. These polypeptides exist solely in a concanavalin A (Con A) binding form. Removal of the Con A receptors by digestion with endo-beta-N-acetylglucosaminidase H (endo H) lowers the Mrs of the glycoprotein doublet to 36.5 and 34 kDa. Western blots of 2D polyacrylamide gels indicate that gp 50 exists in several isoforms. Solid phase radioimmunoassay (RIA) and Western blots of brain subcellular fractions show the antigenic material to be concentrated in the SM fraction, but to be present in much lower amounts in synaptic junctions and postsynaptic densities. Gp 50 appears to be brain specific. Regional distribution studies show that it is present in all brain regions but is two-fold concentrated in cerebellum, brainstem and midbrain compared to forebrain. Immunocytochemical studies of several brain regions show that gp 50-like immunoreactivity is neuron specific and is concentrated in selected neuronal species, particularly granule cells. In both cerebellar and hippocampal granule cells gp 50-like immunoreactivity is localized in the perikarya and primary dendrites. Though immunocytochemistry did not show staining of synaptic regions this may be due to masking of the reactive epitope. The results are discussed in terms of the molecular properties of gp 50 and its subcellular localization in brain tissue.     

人血浆作为真皮支架对自体皮肤增殖的影响：组织学和免疫组织化学试验●☆

背景：组织工程是一个多学科研究的交叉学科，其目标是使人体损伤的组织和器官再生，通过这种假设，几乎所有的动物组织都可以在实验室进行培养。一般的方法是从需要移植的患者身上提取干细胞，在一定的支持条件下允许其生长、增殖、生产为可替换的组织。另一方面，寻找细胞能够互相联结并形成分层结构的合适的支持条件，如基质或支架等非常必要。目前用于烧烫伤治疗的材料有很多种，如胶原，透明质酸、纤维蛋白和聚乳酸及其共聚物。 目的：讨论以新型的生物材料自体血浆为支架对自体成纤维细胞和角质化细胞生长、扩张、增殖的影响。 设计：建立一种真皮再生的方法，将自体成纤维细胞浸于人血浆基质中，排除各种影响样本安全性和排斥反应的问题，应用同样的方法在新的真皮上获取角质化细胞。 时间及地点：实验于2008年在意大利曼多瓦C. Poma医院动物工厂完成。 材料：人角质化细胞和成纤维细胞取自1例58岁乳房切除患者的皮肤碎片。实验得到C. Poma医院独立伦理委员会批准，患者知情同意。 方法：从自体活组织皮肤样本中分离人角质化细胞和成纤维细胞，置于培养瓶中增殖，随后将自体血浆作为皮肤移植形成构造的支架，免疫和免疫组织化学特征显示与正常皮肤相似。 主要观察指标：将血浆样本用甲醛固定，埋入石蜡，苏木精-伊红染色，用显微镜进行细胞计数。所有样本均经免疫组织化学评估。 结果：在血浆基质上获得了多层规则形状的角质化细胞和成纤维细胞，基底膜的形成说明自体血浆是角质化细胞和成纤维细胞的生长、分化、扩展的良好支架，真皮和表皮细胞联结重建皮肤移植与正常皮肤无异。 结论：血浆作为角质化细胞和成纤维细胞分化、扩展的支架表现出良好的性能，同时实验还特别发现成纤维细胞浓缩血浆可以暂时替代皮肤，也是角质化细胞生长的强有力的支架。此外血浆还具有廉价，易于制备等优点。自体角质化细胞和成纤维细胞及自体血浆的应用，可以避免血种类型的免疫排斥反应。这种治疗方法给有慢性缺损并且需要连续移植的患者带来了希望。 doi:10.3969/j.issn.1673-8225.2009.47.001     

Techniques for performing laparoscopic liver resection in various hepatic locations

Many studies have recently reported on laparoscopic liver resection, although its development has been slow compared to laparoscopy in other fields. The indications for the location of laparoscopic liver resection have previously been limited to easily accessible lesions. Performing laparoscopic liver resection in the posterior and superior parts of the liver has been considered difficult due to inadequate exposure, the poor operative field and the difficulty with parenchymal dissection. Flexible endoscopy, high definition imaging and various kinds of equipment for parenchymal transection have been introduced for clinical use. In addition, much experience with this procedure has been accumulated at many centers. Accordingly, there are an increasing number of reports on laparoscopic liver resection in difficult locations. At our institution, the location of the tumor is no longer a limitation to laparoscopic liver resection. However, for safer laparoscopic liver resection, the patient positioning and trocar placement should be individualized according to the tumor location. The type of resection also may depend on the remaining liver’s functional capacity. We describe here the technical considerations for performing laparoscopic liver resection, including the technical considerations for performing laparoscopic liver resection for lesions located in the postero-superior segments of the liver.     

Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Comparison between a swim-up and a Percoll gradient technique for the separation of human spermatozoa

Two methods of separating human sperm were compared using twenty-two semen samples. The sperm were separated by a swim-up technique or by self-migration on a Percoll gradient followed by medium change. After separation, the sperm obtained were assessed for progressive motility, ATP content, energy charge index ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) and morphology. In general, and especially for semen samples containing less than 20 X 10(6) sperm/ml, separation by Percoll gradient selected sperm that were superior to those separated by the swim-up technique. The relatively high energy charge index (greater than 0.8) showed that the sperm tolerated the separation conditions well. It is suggested that self-migration on a Percoll gradient should prove useful for obtaining sperm of high quality.     

免疫荧光检测人乳头状瘤病毒新技术

利用过氧化氢的氧化作用参与抗原抗体反应,使细胞的膜通透性增强,利于荧光标记抗体染液渗入,从而更快速、广泛地接触抗原,更有效的抗原抗体的嵌合交联。新技术的应用,增强了染色效果,缩短了实验时间,简化了操作,提高了阳性检出率,使实验准确性提高,为临床确诊、疗效观察及预后提供了可靠依据。     

孕足月胎膜早破与头位难产的相关性

目的：探讨足月妊娠胎膜早破与难产的关系。方法：选择262例足月妊娠胎膜早破患者作为观察组,另随机抽取本院同期262例无胎膜早破的健康孕妇作为对照组,比较两组难产率的发生情况。结果：观察组难产率为45.0%,明显高于对照组的22.9%（P〈0.01）。两组的难产因素差异无统计学意义（P〉0.05）。结论：胎膜早破与难产有相关性,应及早处理,防止母儿并发症的发生。     

病理技术质量控制的探讨

病理技术是病理学诊断不可分割的一部分,正确的染色结果是病理诊断的重要依据之一.病理技术的规范与否,制片质量的好坏很大程度上影响病理医生作出正确的病理诊断.