Antisense drug targeting with VEGF mRNA designed by computer aid and inhibitory effects on K562 cell line in vitro

AIM:The effective antisense sequences targeted VEGF mRNA with computer software would be screened and designed, and effect of them on growth K562 cells and protein expression of VEGF were studied with experiments.METHODS:Seven antisense sequences were selected and synthesized, which consisted of 18-20 deoxynucleotide acid and were modified with phosphorothioate, according to principle of low free energy of overall △G₃₇ Overall. Cell growth was assayed by trypan blue dye exclusion assay and level of VEGF protien in the media was determined by ELISA.RESULTS:Six of seven sequences were capable of inhibing growth of K562 cells and downregulating the VEGF protein expression significantly, compared with Scrambed control group. It was found that there was a close correlation between low level of overall △G₃₇ and antisense effectiveness (r=0.887,P<0.01).CONCLUSION:VEGF mRNA antisense oliogdeoxynucleotides, which were designed by computer software of RNAstructure, were able to inhibit growth of K562 and its protein expression. The VEGF mRNA may be new target attached by drugs. At same time, the computer aided design is useful methods to obtain the effective antisense.     

植物源农用杀菌剂银泰对作物刺激生长作用

本文通过应用植物源农用杀菌剂银泰的不同浓度处理草莓和菠菜，测定绿色面积指数、叶面积指数、根冠比、叶绿素含量、硝酸还原本科活性、生物产量、经济产量。结果表明，银泰对草莓和菠菜均具有明显的刺激生长作用。     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

Study on relationship between expression of PKCα in glomeruli and development of nephropathy in diabetic rats

AIM:To observe the dynamic changes of expression of PKCα, TGF-β₁ and α-SMA in glomeruli of diabetic rats induced by the alloxon and to invesitigate their roles in the diabetic nephropathy(DN).METHODS:Rats were randomly divided into four groups: normal control group (group A), diabetic group of one week (group B), diabetic group of one month (group C), diabetic group of two months (group D). Immunohistochemistry and Western blotting were used to detect the expression of PKCα, TGF-β₁ and α-SMA in renal tissue of all groups. Blood glucose, triglycerides, cholesterol, creatinine and urine protein were analysed by chemical methods. The morphological changes of renal tissue were checked through microscopy.RESULTS:The expression of PKCα and TGF-β₁ in renal tissue of diabetic groups were increased comparing with those of nomal control group(P<0.05). The mesangial cells expressed α-SMA in two months group. Chronologically the expression of PKCα, TGF-β₁ and α-SMA were positively correlative with each other and the impairment of kidney was also observed.CONCLUSIONS: During the DN process the expression of PKCα increased. PKCα raised GFR and the permeability of glomerular filtration membrane which enhanced urinary albumin excretion. PKCα also increased expression of TGF-β and therefore to induce the expression of α-SMA. The appearance of α-SMA was a marker of the phenotypic transform of renal 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.     

The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.     

Therapeutic effects of recombinant human growth hormone on rat sepsis

AIM:To investigate therapeutic effects of recombinant human growth hormone(rhGH) on rat sepsis and its possible mechanisms.METHODS:Mean arterial pressure (MAP), levels of plasma TNFα, IL-1β and endotoxin, leukocyte count and survival rate within 1 week were determined after E. coli injection among control group, sepsis group and sepsis+rhGH group.RESULTS:(1)rhGH diminished the decrease of MAP, reduced plasma endotoxin and TNFα levels and increased neutrophil ratio in total leukocytes in sepsis rat. rhGH increased survival rate within 1 week on sepsis rat. (2)No changes were found in IL-1β level among the three groups.CONCLUSION:rhGH showed desirable beneficial effects on rat sepsis, which may attribute to: improving circulatory function;maintaining intestinal mucosa barrier, attenuating bacteria/endotoxin translocation and inhibiting the production and release of TNFα.     

Serum contents of E-selectin,sICAM-1, sVCAM-1 in patients with acute coronary syndrome

AIM:To explore the serum levels of certain adhesion molecules and its significance in acute coronary syndrome(ACS). METHODS:The subjects included 40 patients with acute myocardial infarction(AMI) and 40 patients with unstable angina pectoris (UAP). Among the 80 patients, 60 patients accepted a follow-up 4 months. At the same time we selected 40 controls from people who attended a routine health check in the university. Serum levels of E-selectin, sICAM-1, sVCAM-1 were measured by ELISA.RESULTS:Serum levels of E-selectin, sICAM-1, sVCAM-1 were significantly higher in the ACS group(AMI or UAP) than in the control group. Four months later, the levels of E-selectin, sICAM-1 became significantly lower in the follow-up group than in the ACS group, while sVCAM-1 showed no significant difference. CONCLUSION:Serum levels of E-selectin, sICAM-1 may have certain diagnostic value for ACS, and can be a useful marker reflecting the stability of the disease.     

Effect of tea-polyphenols on the activation of NF-κB and the expression of TGF-β1 mRNA in THP-1 cells incubated with VLDL,LDL or ox-LDL

AIM:To investigate the effect of tea-polyphenols (TP) on the activation of NF-κB and the expression of TGF-β₁ mRNA in THP-1 cells (a human acute monocytic leukemia cell line). METHODS:THP-1 cells were incubated with the different concentrations of TP, VLDL, LDL or ox-LDL. In the THP-1 cellls, the nuclear malposition rate of NF-κB was detected with immunohistochemistry technique, the positive index of the TGF-β₁ mRNA expression was detected by hybridization in situ, and accumulation of total cholesterol (TC) in cells incubated with 0.4-40 μg/L TP was determined with oxidase assay. RESULTS:The nuclear malposition rate of NF-κB, the positive index of the TGF-β₁ mRNA expression and TC in THP-1 cells incubated with 0.4-40 μg/L of TP were lower than those with 0 μg/L of TP in TP-V group, TP-L group and TP-O (P＜0.05). The differences of these markers in THP-1 cells incubated with more than 40 μg/L TP in TP-V group, TP-L group and TP-O were not statistically significant, compared with TP-C group (P＞0.05). CONCLUSION:TP inhibited the activation of NF-κB, the expression of TGF-β₁ mRNA and the foam cell formation in the mono-macrophage.