Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene

AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×10¹² phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.     

Selection and amplification of the liver stem cell subset from rat bone marrow cells with a medium containing cholestatic serum in vitro

AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.     

Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

细胞骨架解聚药物对小麦与叶锈菌互作诱发的细胞过敏性反应的影响

 小麦(Triticum aestivum)品种洛夫林10和叶锈菌小种366组成不亲和组合,小麦叶片发生过敏性坏死反应(HR)是小麦抵抗叶锈菌侵染的重要因素。在接种前给小麦叶片分别预注射微管解聚药物磺草硝(oryzalin)和微丝解聚药物细胞松弛素D (cytochalasin D,CD),结果表明2种药物注射使得寄主因叶锈菌侵染诱导的细胞过敏性坏死数目明显减少,并且注射药物的浓度越大,寄主细胞发生HR的数量越少。说明肌动蛋白和微管蛋白的聚合状态是诱发小麦叶片发生HR防卫反应所必需的,细胞骨架在小麦抵抗叶锈菌侵染过程中可能起着重要作用。     

红肉脐橙果肉中主要色素的定性及色素含量的变化

 运用薄层层析法及二极管阵列检测器与高效液相色谱联用的方法, 确定红肉脐橙果肉中含有番茄红素和β- 胡萝卜素两种主要色素, 它们在液相色谱图中占总峰面积(计溶剂峰) 的73. 57 %～77. 78 % , 番茄红素占55. 36 %～58. 49 % , β- 胡萝卜素占18. 21 %～19. 93 %。利用反相高效液相色谱仪以及番茄红素和β- 胡萝卜素标样制作标准曲线, 测定果实发育及室温贮存过程中色素的变化动态, 结果表明, 该脐橙果肉中的番茄红素在成熟期含量最高, 达109. 67μg·g^-1DM, β- 胡萝卜素此时也达到最高值(15. 52μg·g^-1DM) ; 番茄红素的含量在采收后基本呈下降趋势(但采后30 d 有显著升高) , 说明果肉中存在类胡萝卜素采后合成的现象; 贮存4 个月后, 番茄红素含量急剧降低至最高时的1/ 10。     

红肉脐橙(Citrus sinensis L.)果肉中特征色素提取方法探索

红肉脐橙是目前为止惟一果肉呈粉红色或红色的脐橙,其呈色色素尚未见报道。为深入研究其果肉中特征色素的构成及在果实生长发育和贮存过程中色素含量的变化,本研究在对该色素初步定性的基础上对提取方法进行了探索。在分析红肉脐橙果肉中色素萃取液的紫外可见吸收光谱时发现,该色素有类胡萝卜素的特征吸收峰,将萃取液进行薄层层析则进一步证实其内含有番茄红素。在色素提取的前处理方式上,本研究认为冷冻干燥样品的浸提效果较真空干燥的样品稳定(变异系数小),且浸提率相差无几,所以选择冷冻干燥作为合适的样品前处理方式。色素浸提条件的正交实验结果表明,最佳浸提条件为50℃、1.5h内浸提4次,浸提的料液比为1:60。此外,浸提温度、浸提时间、浸提料液比和浸提次数对浸提率都有显著影响,影响最大的因素是浸提温度,最佳温度为50℃,随温度降低浸提率显著降低。其次是侵提时间的长短,最佳浸提时间为1.5h,延长或缩短0.5h都会使浸提率下降。浸提次数对浸提率的影响是最小的。     

柑橘类果实汁胞的红色现象及其呈色色素

柑橘类果实中不乏汁胞为红色类型者,如红肉葡萄柚(Citrus paradisii Macf.)的粉红色是番茄红素所致,血橙(C.sinensis L.)的血红色来源于花青素,此外,还有红色的红江橙(C.sinensis L.)、橙红色的元红(C.reticulata Blan-co)和紫红色的五布红心柚(C.grandis L.)等,它们因色泽鲜艳而具备特殊的市场潜力,但呈色色素尚未见报道。在符合审美观的同时,红色汁胞品种还具有特殊的营养价值,如类黄酮、类胡萝卜素等色素富集可软化血管、抗癌、抗氧化及消除体内自由基等。综合有关文献,首次将国内外主要红色汁胞柑橘品种依宽皮柑橘、甜橙、柚、葡萄柚及杂柑等分类进行综述,分析可能的呈色色素,阐明导致红色性状产生的两类色素—花青素、类胡萝卜素生物合成途径     

The genetic susceptibility of HLA-DRB1 alleles in esophageal neoplasm of Hubei Han Chinese

AIM: To probe into the genetic susceptibility of HLA-DRB1 alleles to esophageal neoplasm in Hubei Han Chinese. METHODS: HLA-DRB1 gene polymorphism in 42 patients with esophageal neoplasm and 136 normal control subjects was studied by PCR and sequence. RESULTS: Allele frequency of HLA-DRB1 *0901 allele was significantly higher in esophageal cancer patients than those in normal controls(0.2500 vs 0.1397, P =0.028; the odds ratiO².053; etiologic fraction 0.1282).There were no association between the rested HLA-DRB1 alleles with patients. CONCLUSION: Individuals carrying HLA-DRB1 *0901 may be susceptible to esophagealo carcinoma, its nucleotide sepuence approachs to the corresponded allele sequence(exoN²)published in GenBank.     

达县分葱田甜菜夜蛾发生规律初步研究

采用系统调查方法研究了达县地区分葱田甜菜夜蛾的生活史、发生与环境的关系。甜菜夜蛾在达县分葱田1a发生6代,以三至五代为害最重,世代重叠。其数量消长与虫源、夏秋季气候、天敌、栽培管理等因素密切相关。通过调查分析提出本县初始虫源非本地虫源。     

昆虫病原线虫和共生细菌培养系统中噬菌体的检测

本文利用紫外照射、温度、pH、盐度诱导五株溶源性共生菌株Xenorhabdus和Photorhabdus同时测定斯氏和异小杆属线虫Steinernema carpocapsae A24,S.carpacapsae ALL,S.feltiae English,S.feltiae SN,Heterorhabditis bacterophora H06在体外培养过程中是否存在感染共生细菌的噬菌体。结果未发现噬菌斑，说明实验菌株在实验诱导条件下以及昆虫病原线虫固体培养系统中不可能诱发噬菌体的危害。