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.     

Effects of phosphodiesterase inhibitor-induced insulin resistance on glucose and lipid metabolism in rats

AIM:To investigate the effects of milrinone (a selective phosphdiesterase III inhibitor PDE₃) on insulin secretion, blood glucose, plasma free fatty acids (FFA) and dose-response relationship, and assess possible effects of milrinone on glucose metabolism and insulin sensitivity in conscious rats.METHODS:The catheterized nonstressed rats were administered by the varying doses of milrinone (1, 5, 25 μmol/kg) and were compared with controls. A hyperinsulinaemic- euglycaemic clamp was established in awake rats, and milrinone(25 μmol/kg) and 25% dimethyl sulfoxide (DMSO, as a control) were given at 120 min during hyperinsulinaemic- euglycaemic clamp. Glucose turnover was decided by gas chromatograph mass spectrometer (GC-MS).RESULTS:After dosing, plasma FFA levels in 3 milrinone groups significantly increased compared with the controls and before dosing. The percentages of elevation of FFA by the different milrinone doses were very similar, 50%, 52%, 55% for 1, 5, 25 μmol/kg respectively at 2 min after dosing. Plasma insulin levels were significantly elevated in the 5 and 25 μmol/kg groups, and the effect of milrinone on glucose concentration was detectable only 25 μmol/kg group. During hyperinsulinaemic clamp, there were significant increase in plasma FFA (from 173.1±15.2 to 633.8±87.3 μEq/L) and hepatic glucose production (HGP), and a significant decrease in glucose infusion rates (GIR) (to about 21%).CONCLUSION:These data suggest that milrinone impaires the abilities of insulin to suppress lipolysis and HGP, and insulin-mediated glucose utilization in peripheral tissue. Therefore, milrinone administration may induce an acute insulin resistancein vivo.     

Biological characteristics of long passaging rat mesenchymal stem cells

AIM:To investigate multi-potential of rat bone marrow mesenchymal stem cells (rBMMSC) and mutation inclination, the rBMMSC were long passaged in vitro. METHODS:Cellular cycles of different passages were assayed by FACSan flow cytometry and karyotypes of passage 6, passage 25 and passage 45 were compared by G-binding analysis. RESULTS:The early passages and long-term passages all showed strong proliferation; passage 6, passage 25 and passage 45 all showed normal karyotype. CONCLUSION:Long-term culture and passage of rBMMSC still remains strong proliferation. With this capability, the mutation inclination is not enhanced.     

Expression of peroxisome proliferator-activated receptor α in rat fatty liver

AIM:To investigate the role of expression of peroxisome proliferator-activated receptor α(PPAR α) in pathogenesis of rat fatty liver.METHODS:The rats were treated with a low dose of carbon terachloride (CCl₄) and fed a high fat diet to produce fatty liver. We determined the concentrations of triglyceride (TG), total cholesterol (TC), free fatty acid (FFA) in liver and the alanine aminotransferase (ALT) activity, tumor necrosis factor-α (TNF-α), FFA in serum and the degree of hepatocytic steatosis. Total RNA of liver was extracted, and the expression of PPAR α were analyzed by semi-quantitative RT-PCR method.RESULTS:In model group, the hepatocytic PPAR α mRNA expression decreased to 0.41±0.28, compared to 1.41±0.29 in the control group (P＜0.01). The contents of TG, TC, FFA in model rat liver were (1.88±0.20) mmol·L^-1, (11.03±1.12) mmol·L^-1 and (1 260.38±151.27) μmol·L^-1, respectively, compared to (0.53±0.10) mmol·L^-1, (1.25±0.25) mmol·L^-1 and (334.30±27.09) μmol·L^-1 in the control group (P＜0.01). The activity of ALT, concentrations of TNF-α and FFA in serum were also increased remarkably in model group.CONCLUSION:Oxidation of fatty acid and utilization of lipids in liver are affected by reducing the expression of PPAR α, which result lipid accumulation in liver.     

Influence of ischemia-reperfusion on apoptosis of sinoatrial node cells in rabbits in vivo

AIM:To study influence of ischemia-reperfusion(IR) on apoptosis and expression of apoptosis-related genes Fas-L, Bax and Bcl-2 of sinoatrial node(SAN) cells in rabbits in vivo. METHODS:Ninety healthy adult rabbits were divided randomly into control group, ischemia groups (I_{10 min}, I_{30 min}, I_{60 min} and I_{120 min}) and IR groups (I_{10 min}R_4h, I_{30 min}R_4h, I_{60 min}R_4hand I_{120 min}R_4h). IR injury model of SAN was established by occluding and loosening the start section of right coronary artery. The apoptosis of SAN cells was detected by TUNEL staining. The expression of Fas-L, Bax and Bcl-2 of SAN cells was detected by immunohistochemistry. RESULTS:①No obvious apoptosis of SAN cells was observed in control group, I_{10 min} and I_{30 min} groups. Apoptosis of different degrees in SAN cells were found in 68.3%(41/60) rabbits in I_{60 min}, I_{120 min} and 4 subgroups of IR. ②The highest expression of Fas-L and Bax was observed in I_{120 min} group and that of Bcl-2 was in I_{60 min} group. ③The highest expression of Fas-L and Bax was observed in I_{60 min}R_4h group. The peak level of Bcl-2 was observed in I_{30 min}R_4h group. ④The expression of Fas-L and Bax was significant higher in IR group than that in ischemic group at the same time point. CONCLUSION:Ischemia and IR induced apoptosis of SAN cells in rabbit in vivo. Fas-L、Bax、Bcl-2 may participate in the regulation of apoptosis and the injury during IR aggravates the apoptosis of SAN cells.     

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.     

Effect of cilazapril on pulmonary vascular endothelial dysfunction in hypoxic rats

AIM:To study the effect of cilazapril on pulmonary vascular endothelial dysfunction in hypoxic rats. METHODS:The structure and function of endothelium in hypoxic rats were studied by biochemical analysis, radioimmunoassay, transmission electron microscope and correlated with hemodynamic. RESULTS:1) The change and damage of ultrastructure in endothelial cell (EC) were obsevered in hypoxic rats. 2) The contents of plasma nitric oxide (NO) and superoxide dismutase (SOD) activity in blood as well as endothelial nitric oxide synthase (eNOS) activity in the lung tissue were significantly lower in the hypoxic rat than those in contral animals. The concentrations of plasma endothelin-1(ET-1) and angiotensin converting enzyme(ACE) as well as malondialdehyde(MDA) were significantly higher in the hypoxic rat than these in contral animals. The relaxing and contracting factors had a significant positive/negative correlation with mean pulmonary artery pressure (mPAP). 3) Cilazapril significantly decreased the level of ET-1 and ACE and significantly increased the level of NO and activity of eNOS and SOD. At the same time, cilazapril extenuated hypoxia-induced injuries of EC. CONCLUSION:The results indicate that damaging structure and dysfunction of EC existes in hypoxic rats. The cilazapril effectively preventes and treates the chronic hypoxic PH by relieving the injury and improving secretion in EC.     

20%多效唑·甲哌 FEE7 微乳剂防止小麦倒伏和增产机理研究

冬小麦二棱期喷施植物生长调节剂20%多效唑·甲哌鎓微乳剂375 mL/hm2,可以显著抑制茎秆基部节间伸长,增加各节间充实度,其中赤霉素(Gas)和生长素(IAA)降低,可显著增强小麦抗倒伏能力和降低田间倒伏率.处理还协调了穗数、穗粒数和粒重的关系,增产幅度6.2%～28.6%.增产原因可能在于促进籽粒灌浆强,增加籽粒中内源Gas、IAA、细胞分裂素(CTKs)的水平,增强了籽粒库活性,同时促进茎叶中干物质向籽粒运转.     

黑河干流中游地区耗水量变化的历史分析

黑河流域水资源极其匮乏 ,加之地区上的配置不均衡和中游地区水土资源的过度开发导致了黑河下游严重生态环境问题 ,已影响到我国北方广大地区生态环境质量 ,受到国家的高度重视和全社会的广泛关注。黑河水资源开发利用主要集中在中游地区 ,该区是全国重要的商品粮、蔬菜和经济作物生产基地之一 ,耗水量占流域耗水量的 85 %以上 ,是黑河的主要耗水区和径流利用区。本文利用黑河上游控制站莺落峡站和中游控制站正义峡站水文资料 ,分析了黑河中游耗水量的变化 ,结果表明 :2 0世纪 80年代以前黑河中游耗水量在 4.2 0× 10 8m3 左右 ,比较稳定 ;进入 2 0世纪 80年代以来耗水量明显增加 ,80年代耗水量比 40年代末期至 70年代增加了 2 .0× 10 8～ 2 .5×10 8m3 左右 ;90年代耗水量又比 80年代增加了 1.6 0× 10 8m3 左右。 80年代以来流域耗水量呈明显增加的趋势 ,这是以人类活动影响为主的结果     

城市绿地系统对经济发展提升作用的机制——与大连市实证研究

城市绿地系统具有生态、经济、社会等多重属性 ,在城市复合系统中占有特殊的位置 ,对城市经济发展亦具有直接、间接的提升作用 :( 1 )改善城市环境质量 ,创造环境优势 ,促进城市地价及布局其间的各种经济成分增值 ;集聚外来资金和高新产业发展 ,带动整个城市产业结构优化升级。( 2 )绿地建设作为城市经济产业的有机组成部分 ,形成环境经济产业链 ,拉动其相关经济产业的发展 ,促进经济增长。 ( 3 )提高城市知名度 ,促进城市旅游业发展 ,集聚高素质人才 ,促进城市经济发展 ,本文以北方海滨城市大连为例 ,在定性分析绿地经济提升作用机制的基础上 ,建立计量经济模型 ,定量分析城市绿地系统的经济提升作用