儿童横纹肌肉瘤中Myogenin 蛋白的检测及其诊断价值

目的：横纹肌肉瘤是儿童期最常见的软组织肉瘤，因其组织学常呈现低分化状态，故与其它儿童小圆细胞瘤的鉴别诊断十分困难。本实验初步探讨应用Western blotting方法检测生肌转录因子基因家族成员之一—Myogenin蛋白以作辅助诊断的可行性。方法：提取8例横纹肌肉瘤及20例其它儿童实体肿瘤新鲜组织中的蛋白质，进行SDS—PAGE电泳，然后将蛋白转移至硝酸纤维膜，作Western blotting蛋白质杂交，对印迹进行分析。结果：8例横纹肌肉瘤均有明显的Myogenin蛋白印迹，在1例肾母细胞瘤中呈假阳性；用Western blotting方法可检测到仅5μg组织蛋白中Myogenin蛋白的表达。结论：用Western blotting方法检测Myogenin蛋白对于横纹肌肉瘤具有极高的灵敏度和较好的特异性，因而有一定诊断应用价值。     

豇豆胰蛋白酶抑制剂单克隆抗体的制备及初步应用

目的　检测转豇豆胰蛋白酶抑制剂 (CpTI)基因水稻叶片中CpTI蛋白 ,来对转CpTI基因作物的检测建立初步的方法。方法　目前国际上主要通过基因检测的方法 ,而基因的检测可能存在假阳性的结果 ,而且基因的导入并不一定代表功能蛋白的表达 ,表达含量也为未可知。因此 ,通过检测转基因蛋白含量的多少是最直接和明确的方法。通过体外基因重组技术制备的CpTI蛋白来免疫动物得到单克隆抗体 ,就可通过westernblotting法检测转基因作物中的CpTI。结果　得到三株抗CpTI的单克隆抗体 ,利用这三种单克隆抗体的混合物对转CpTI蛋白的水稻叶片进行初步检测 ,得到了较为满意的结果。结论　利用抗CpTI的单克隆抗体混合物对转CpTI的水稻叶片进行westernblotting的检测方法较好     

Strong association of HLA-B27 heavy chain with beta 2-microglobulin

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with β₂-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with β₂-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/β₂-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.     

Gel chromatographic characterization of proteins in mucous and serous middle ear effusions of patients with otitis media in comparison to serum proteins

Otitis media with effusion (OME) is an inflammatory disease of the middle ear cavity that is associated with middle ear effusions (MEEs), which are frequently mucous and serous for pediatric and adult patients exhibiting low and high responsiveness to medical treatment, respectively. To assess the pathological outcomes in mucous and serous MEEs, their protein compositions were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting in comparison with those in the same patients’ sera. A mucin, which is immunochemically identical with nasal mucin, was a characteristic consituent of mucous MEEs (n = 25), being present at the concentration of 59.4 mg/ml and comprising about 60% of the total proteins, but it was not detected in serous MEEs (n = 30) or sera. Serum proteins with molecular weights of less than 260 kDa were detected in serous and mucous MEEs, in which albumin was the major protein. Albumin, IgM and α1-acid glycoprotein, and lysozyme, IgA and IgG in MEEs were present at lower and higher concentrations than in sera, respectively. The ratios of IgA, IgG, IgM and α1-acid glycoprotein to albumin in mucous MEEs were 4-, 3-, 1.4- and 1.0-times higher than those in the respective pediatric sera, and those in serous MEEs were 1.7-, 1.7-, 0.6- and 0.3-times higher than those in adult sera. Also, the concentrations of lysozyme in mucous and serous MEEs were 19 and 3 μg/ml, but those in pediatric and adult sera were negligible. These results indicate that the contents of these proteins, in comparison to albumin, might be useful criteria for assessing the inflammation level in MEEs.     

不同膳食构成对葡萄糖转运因子-4影响

目的探讨不同饲料构成对大鼠骨骼肌葡萄糖转运因子-4的影响。方法利用不同饲料（包括基础饲料、高蛋白、高脂肪、高能1与高能2饲料）喂养雄性Wistar大鼠9周,观察不同饲料构成对大鼠体重、脂体比、血糖及胰岛素的影响,并应用蛋白印迹法测定大鼠骨骼肌葡萄糖转运因子-4含量。结果高能饲料1组与高能饲料2组的体重、脂体比、血糖、胰岛素均显著高于基础组、高蛋白组与高脂肪组（P〈0.05）,而后3组体重、脂体比、血糖、胰岛素比较差异无统计学意义（P〉0.05）。高能饲料1组与高能饲料2组葡萄糖转运因子-4含量减少,能量相同的基础组、高蛋白组与高脂肪组含量相近。结论高能饲料可影响机体的葡萄糖代谢。     

RNA干扰技术抑制血管内皮生长因子表达

目的：应用RNA干扰技术抑制血管内皮生长因子（VEGF）表达。方法：将VEGF基因作为RNA干扰的靶区，通过E—RNAi网上提供的服务，设计两个特异的RNA干扰序列，将其装入含U6启动子的载体上，构建成抗VEGF基因的小发夹样RNA（shRNA）表达载体，再转染人胚肾细胞HEK293、结肠癌细胞HT29、宫颈癌细胞Hela和肝癌细胞HepG2，通过RT—PCR、Northern杂交和免疫荧光观察VEGF表达受抑的程度。结果：成功构建了两种抗VEGF基因的shRNA表达载体，RT—PCR发现其在HEK293和HT29细胞系中，能明显抑制VEGF基因的表达，抑制率分别为72％和42％。但在Hela和HepG2细胞中的抑制率仅为28％和13％；Northern杂交显示，在HEK293细胞和HT29细胞中，VEGF mRNA明显受抑，抑制率达88％和89％；免疫荧光显示，在HT29细胞中，VEGF蛋白表达的受抑制率为73％。结论：针对VEGF基因的shRNA表达载体能够明显抑制VEGF基因的表达，但在不同细胞系中的作用强度存在差别。     

白藜芦醇合酶基因的克隆及丹参的转化

目的为探索利用分子生物学技术培育新型药用植物的新途径,将外源的白藜芦醇合酶(RS)基因导入到丹参中表达,以改善或增加丹参的效用。方法根据已公布的核苷酸序列,利用引物悬挂延伸法,经过3次扩增,最终从葡萄基因组DNA中获得完整的RScDNA基因序列;以质粒pBin438为基础,构建含有RS目的基因的组成型植物表达载体。在根癌农杆菌介导下,利用叶盘法转化丹参,进而通过PCR、PCR-Southern杂交对不同的转基因植株进行筛选与检测。结果总共得到了45棵卡那霉素抗性植株,经PCR和PCR-Southern杂交检测,确定葡萄RS基因已整合到部分转基因丹参苗的基因组中。结论成功建立了白藜芦醇合酶基因转化丹参的体系,并获得了转基因植株。     

Secondary reduction in calpain 3 expression in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy (primary dysferlinopathies)

Dysferlin is the protein product of the gene (DYSF) that is defective in patients with limb girdle muscular dystrophy type 2B and Miyoshi myopathy. Calpain 3 is the muscle-specific member of the calcium activated neutral protease family and primary mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A. The functions of both proteins remain speculative. Here we report a secondary reduction in calpain 3 expression in eight out of 16 patients with a primary dysferlinopathy and clinical features characteristic of limb girdle muscular dystrophy type 2B or Miyoshi myopathy. Previously CAPN3 analysis had been undertaken in three of these patients and two showed seemingly innocuous missense mutations, changing calpain 3 amino acids to those present in the sequences of calpains 1 and 2. These results suggest that there may be an association between dysferlin and calpain 3, and further analysis of both genes may elucidate a novel functional interaction. In addition, an association was found between prominent expression of smaller forms of the 80 kDa fragment of laminin 2 chain (merosin) and dysferlin-deficiency.     

Cloning and Secreted Expression of the Extracellular Domain of the Mumps Virus Fusion Protein in Pichia pastoris

The extracellular globular domain of the mumps virus fusion (F) protein (amino-acids 28-481) has been overexpressed from GS115 his4 Pichia pastoris cells following the generation of a recombinant clone. The heterologous protein was directed for secreted expression by in-frame cloning with the S.cerevisiae -factor secretion signal. The expressed protein was observed to secrete into the culture medium. An expressing clone was obtained initially by small-scale induction, metabolic labeling and immunoprecipitation. Expression analysis of the chosen clone was confirmed by western blotting with F protein specific polyclonal serum. The effects of culture volume, temperature and methanol concentration on the levels of expression, were studied. The results indicate that there is a balance required between the induction temperature and methanol concentration to achieve maximal expression. In addition, the presence of designated monomeric (47 K), dimeric (85–90 K) and trimeric (140 K) forms are dependent upon the induction conditions. Estimated secreted protein expression levels of > 1 mg/L were obtained in these studies. Further, the experiments demonstrate that the complete reconstruction of the KEX2 protease cleavage site is not necessary to facilitate secretion.     

shRNA干扰肝癌细胞Kir6.2基因表达对细胞内钙离子浓度的影响

目的研究shRNA干扰肝癌SK-Hep1细胞Kir6.2基因表达及对细胞内钙离子浓度的影响。方法脂质体转染法将shRNA质粒载体pGenesil-3共转染SK-Hep1细胞，实验分为SK组（未转染）、HK组（阴性对照）、K1组（干扰序列1）和K2组（干扰序列2）。经G418筛选出单克隆培养，RT-PCR和Western Blotting分别检测Kir6.2 mRNA和蛋白的表达，筛选出抑制Kir6.2表达的有效序列。Fluo4-AM孵育各组细胞，流式细胞仪检测荧光强度。结果RT-PCR结果显示K1、K2组Kir6.2 mRNA的表达与HK、SK组比较均降低。K1、K2组Kir6.2蛋白的量与HK、SK组比较均降低。K1和K2组钙离子浓度远远低于SK和HK组。结论shRNA抑制了肝癌细胞Kir6.2基因的表达，钙离子浓度显著降低。