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.     

Effects of TGF-β1 and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells(HSPC)

AIM:To study the effect of TGF-β₁ and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells (HSPC). METHODS:CD₃₄⁺cells were purified from fresh umbilical cord blood by immunomagnetic beads, and mononuclear cells were purified from bone marrow by Ficoll-hypaque. The effects of TGF-β₁ and /or TNF-α antisense PS-ODNS on ex vivo expansion of CD₃₄⁺ cells、CFU-GEMM、CFU-GM、CFU-E and BFU-E were detected by using liquid and semi-solid culture systems.RESULTS:TGF-β₁ antisense PS-ODNS cooperated with cytokines increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E, which was as 4, 2.6, 2.7, 1.8, 2.1 times as that of the control (the cytokines combination), respectively. TNF-α antisense PS-ODNS cooperated with cytokines respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 4, 2.9, 2.6, 1.7, 1.8 times as that of the control. The above two antisense PS-ODNS cooperated with cytokines could respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 5.3, 2.1, 2.7, 1.9, 1.8 times as that of the control.CONCLUSION:Inhibition of endogenous TGF-β₁ and TNF-α by antisense PS-ODNS will be one of the effective methods to expand HSPC ex vivo.     

不同碳源对虎奶菇菌核蛋白质营养价值的影响

测定了四种碳源对虎奶菇菌核蛋白质营养价值的影响。结果表明，以玉米淀粉为碳源，虎奶薅菌蛋白质的化学评分，必需氨基酸指数，生物价和营养指数分别为11．63、69．80、64．40和9．98，均居四种参试碳源的第一位；氨基酸评分和氨基酸比值系数分分别为14．55和12．45，均居四种参试碳源的第二位，这些结果证实，以玉米淀粉为碳源，虎奶菇菌核蛋白质营养价值高，值得推广应用。     

姬松茸与双孢蘑菇不同菌株的RAPD扩增研究

以4个姬松茸菌株和2个双孢蘑菇菌株为材料，用20个随机引物对它们进行RAPD扩增，通过对供试菌株遗传相似系数的估算和系统聚类分析，构建姬松茸和双孢蘑菇遗传相关树状聚类图谱，结果表明，进行RAPD扩增的20个随机引物中，有12个能产生三种带型，当遗传相似系数升至0.8713时，这6个菌株被分为3类，第一类为菌株18和26，第二类为菌株13和12，第三类为菌株33和45。     

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。     

南方灭鼠应用稻谷与大米毒饵的比较

生物测定法测得谷壳对复方88-1灭鼠剂的阻滞率为54．3％，光谱分析法测得其对敌鼠钠盐的阻滞率在47．43％—57．00％之间。相同条件下，褐家鼠Rattus norvegicus取食敌鼠钠盐稻谷毒饵量为大米毒饵的2倍多。在南方潮湿的环境下，大米毒饵更易松散、霉变，使其适口性更低。综合考虑认为南方灭鼠使用稻谷毒饵更为合适。     

核桃树体内酚类物质含量的变化

通过对核桃酚类物质含量变化研究,结果表明,核桃枝、芽、叶中均有酚类物质存在,其平均含量范围为29.5～118.6 mg/g DW。核桃酚类物质分布随器官组织和季节的不同而有差异,即:枝条初皮部>叶片>芽体>枝条木质部;一年生韧皮部和芽体中酚类物质含量均于休眠达到高峰。不同核桃品种间酚类物质含量存在差异性,即:光皮绵核桃>白水核桃>辽核1号>中林5号。     

红汁乳菇多糖提取最佳工艺研究

对红汁乳菇多糖提取、纯化工艺进行了优化研究，结果表明在料液比1：30，浸提温度95％浸提时间2h,pH8．0，可获得最佳提取效果。在醇析浓度为70％，V(氯仿)：V(正丁醇)=1：0．2，V(氯仿 正丁醇)：V(样品)=1：l时，纯化含量为58％。同时用超声波法对红汁乳菇多糖的提取作了研究，其得率为6.99％，效果较佳。