影响共聚焦荧光法检测单细胞中蛋白表达的因素探讨

采用免疫荧光技术联合共聚焦技术研究不同培养细胞中蛋白的表达及定位.结果发现:①同样以丙酮固定293细胞,不用0.5%Triton X-100处理,着丝粒蛋白E亚型(CENP-E)在分裂期细胞核周浓聚,核内未见;而用0.5%TritonX-100处理后,CENP-E呈散点状分布于分裂期细胞的核内.②分别以甲醇、95%乙醇和4%多聚甲醛固定并以1%TritonX-100处理k562细胞,不同固定剂处理组间结果无明显差异,信号转导与转录激活因子(STAT3)均定位于胞浆.共聚焦系统PMT=511时,显示胞核染色适中,核仁染色明显,而PMT=593时,整个核呈饱和染色状,微细结构不清楚.③以2%甲醛联合0.5%TritonX-100处理平滑肌细胞,固醇调节元件结合蛋白裂解激活蛋白(SCAP)在整个细胞中呈弥散性分布,核内含量较高,高尔基体上SCAP无特异性高表达.结论:是否使用TritonX-100处理对293细胞中核蛋白CENP-E的定位具有较大影响;k562细胞对醛类固定剂及醇类固定剂无特殊选择性,图片采集时共聚焦参数PMT对蛋白定位结果也有影响;醛类固定剂和Triton联用可准确定位SCAP在平滑肌细胞中的表达.

Abstract：

The expression and location of proteins in single cells cultured under different conditions were studied with a laser scanning confocal microscope. CENP-E appeared mainly around the nucleus in HEK293 cells by fixing with acetone without 0.5% Triton X-100 treatment, but localized mainly in the nucleus when the cells were fixed with acetone plus further 0.5 %Triton X-100-PBS solution and scattered in the nucleus with condensed points. Plus l% Triton treatment, K562 cells fixed with 4% paraformaldehyde, methanol or 95 % ethanol all showed that STAT3 protein mainly distributed in cytoplasm at the edge of the cell body, the nucleus occupied a greater part of the cell body with PI staining, nucleolus staining seemed stronger under PMT= 511, but seemed the same as the other part of the nucleus under PMT=593. SCAP (SREBP cleavage-activating protein) could be detected in cytoplasm and was more accumulated in nucleus in smooth muscle cells fixed with 2 % formaldehyde and 0.5 % Triton penetrating, and Golgi apparatus were found to scatter around the nucleus. In conclusion, it is important to locate CENP-E protein in HEK293 cells with TritonX-100 by immunofluorescence microscopy. SCAP in smooth muscle cells and STAT3 expression in K562 cell can be exactly located with the combination of formaldehyde and Triton penetrating. STAT3 expression in k562 cells fixed with formaldehyde or spirits fixative has no difference.The confocal system itself, such as PMT gain, can also affect the location of proteins in single cells.     

农村公路投融资现状及对策探讨

我国农村公路投融资的现状和存在的问题,分析了常用公路融资模式对农村公路的不适用性,结合我国的国情和农村公路建设的特点,提出了发行农村公路建设债券、建立农村公路专项资金、国家优惠政策和市场化运作等方面以拓宽农村公路融资渠道。     

云南产皱皮木瓜挥发物的热脱附-气相色谱/质谱联用分析

采用热脱附-气相色谱/质谱(TCT-GC/MS)联用技术,对动态顶空密闭循环吸附捕集绿熟期及完熟期的皱皮木瓜果皮和果肉挥发物分别进行检测。结果表明,皱皮木瓜果皮与果肉香气中富含酯类化合物,丁酸乙酯在果皮或果肉、绿熟或完熟期的含量达12%~15%,是含量最高的挥发成分之一。含量较高的酯还有2-甲基丁酸乙酯和己酸乙酯等。绿熟期果皮及果肉挥发物中均明显检出芳樟醇及其氧化物,而完熟期仅在果肉挥发物中检出少量芳樟醇。萜烯总体含量不高,除完熟期果皮挥发物中金合欢烯含量高于5%外,各部位及各成熟阶段的柠檬烯挥发成分含量均低于1%。挥发物中富含鸡蛋果素,其在绿熟期及完熟期、果皮及果肉挥发物中总含量约10%~17%,并均以鸡蛋果素异构体Ⅰ为主,而绿熟期果肉挥发物中未检测到其异构体Ⅱ。     

Screening for Monoclonal Antibodies Against the Membrane Proteins of Chicken Embryo Fibroblast Blocking the Infection of IBDV

This article aims to establish an efficient assay for screening monoclonal antibodies （McAbs） against the membrane proteins of chicken embryo fibroblast （CEF） for further studies of the cellular receptors of infectious bursal disease virus （IBDV）. McAbs against the membrane proteins of CEF were prepared by cell fusion. The monolayer CEF pre-incubated with the CEF-specific McAbs for 2 h were infected with IBDV and incubated with F22-EA6-biotin postinfection. Then, the cells were reacted with streptavidin-horseradish peroxidase （HRP） and finally stained by 3-amino-9-ethylcarbazole （AEC）. The inhibitive percentage of IBDV infection was calculated by counting the IBDV-infected cells to determine the inhibition efficiency of the CEF-specific McAbs. Compared with the control cells, the IBDV-infected cells pretreated with CEF-specific antibody significantly decreased; supernatant fluids of a total of 768 hybridomas were analyzed. The results of immunohistochemistry assays showed that six of them （1A5, 1H11, 2B 12, 3G1, 4D10, and 4B8） have the abilities to block the infection of IBDV to CEF, among which 4B8 can perfectly block the infection. This novel method is a sensitive and specific assay for the screening of CEF membrane protein-specific McAbs, which can block the infection of IBDV to CEF, and these McAbs can be used for the further investigations of the cellular receptors of IBDV.     

基于Kruskal算法和主成分分析的农业经济信息评价

为了解县域单元内部乡镇之间农业经济信息的空间格局及结构,以山东省禹城市11个乡镇为研究对象,重点选取了反映农业经济信息的农业机械总动力等9项指标,运用主成分分析的统计方法,建立了农业经济信息的主成分载荷矩阵,计算了农业经济信息主成分综合得分,从计算机学科图论的角度人手,结合Kruskal求解最小生成树算法从定量的角度分析了禹城市11个乡镇农业经济信息的综合潜力状况,利用Kruskal算法和主成分结合的方法对乡镇单元尺度进行农业经济信息分析,确定最佳的区位优势、分类和组合,以便于农业的集约化和耕作的合理化,较好地配置农业的产业结构,为农业可持续发展和实现合理的农业地域分工提供科学依据。     

4种栽培玫瑰花粉生活力研究

采用离体培养的方法测定了4种栽培玫瑰花粉的生活力,并考察了不同浓度蔗糖溶液对花粉萌发率的影响以及不同冷藏时间下花粉活力的变化情况。结果表明,不同玫瑰品种的花粉萌发率差异显著,‘紫枝’、‘北京单瓣白’的花粉萌发率相对较高,最高萌发率超过40%;‘平阴一号’、‘西湖一号’的花粉萌发率较低,最高萌发率仅为10%～20%。培养基中蔗糖浓度对各品种萌发率的影响差异显著;冷藏花粉的生活力随冷藏时间的延长明显下降。     

黄山浮溪河流域鳞毛蕨科药用蕨类植物资源调查

[目的]调查研究黄山浮溪河流域的毛蕨科药用蕨类植物的种类、分布、药用价值及开发利用情况。[方法]通过对安徽黄山浮溪河流域的蕨类植物资源的初步调查研究,结合我国西南少数民族医药中有关药用蕨类植物资源与少数民族医药中蕨类资源利用情况,对该流域药用蕨类资源中鳞毛蕨科植物的主要种类及其分布情况进行了综合性评价,同时讨论了黄山蕨类植物资源保护及开发利用前景。[结果]黄山浮溪河流域具有药用价值的鳞毛蕨科植物有12种,隶属于3个属。[结论]该研究为进一步开发利用鳞毛蕨科药用蕨类植物资源,保证其药源供应和临床用药安全提供资料。     

高产单细胞蛋白酵母的诱变育种及培养条件优化

从啤酒废酵母泥中分离纯化出5株酿酒酵母,以其中单细胞蛋白(SCP)含量较高的SY-5为出发菌株,通过紫外诱变分生孢子,经过初筛和复筛,得到1株高产SCP的诱变菌株SY-5-7,比出发菌株高7.84%,达到48.67%。通过单因素试验对诱变菌株SY-5-7培养条件进行优化,结果表明,最适培养条件为培养基起始pH值5.0、培养温度26℃、接种量2%、发酵周期为70h,碳源为50g/L蔗糖、氮源为10g/L酵母膏。在此基础上进行培养,SY-5-7的生物量比初始条件提高了约35%。     

昆明裂腹鱼不同组织乳酸脱氢酶同工酶研究

采用不连续聚丙烯酰胺凝胶电泳法,对昆明裂腹鱼的眼球、心脏、肾脏、性腺、脾脏、肝胰脏、脑、鳃和肌肉等9种组织的乳酸脱氢酶同工酶(LDH)进行了测定。结果表明:昆明裂腹鱼LDH共有15条酶带表现出特异性,在不同组织中检出的LDH酶带数量不同,以肝胰脏中最多,共检出9条;在酶活性方面,LDH在脑、鳃、肌肉和肾脏等组织中活性最强,而在眼球组织中最弱。     

鸡传染性喉气管炎病毒PCR快速检测方法的建立

根据GenBank发表的传染性喉气管炎病毒(ILTV)TK基因序列(EF552578),设计合成了1对特异性引物,通过优化反应条件,成功地从ILTV疫苗株中扩增出329 bp特异性片段,而传染性支气管炎病毒、新城疫病毒及马立克病毒基因组均未扩增为出相应的片段。回收的PCR产物经EcoRⅠ酶切和测序鉴定,证实了该扩增片段的特异性。PCR检测ILTV DNA的最小检测量为30 pg。经临床初步应用表明,该方法的建立使传染性喉气管炎病毒的检测更为快速、简便、经济和实用。