Objective To construct the wt-p53's eukaryotic expression vector pE6/p53/GFP that was controlled by the radiation induced promoter and research its functions.Methods Radiation response element E6 was synthesized by gene synthesis.The wt-p53 cDNA sequence was prepared from pcDNA3.1(+)/p53 plasmid by PCR.IRES2-EGFP report gene segment was prepared from double cistron expression vector IRES2-EGFP by enzyme digestion.After sequenced and identified,the recombinant plasmid was transformed into H1299(p53-/-)cell with Lipofectamine 2000,and the cell lines in stable expression was screened by G418.In the H1299(p53-/-)cell transfected with the recombinant plasmid or without,wt-p53 mRNA expression was analyzed by RT-PCR,the p53 expression by Western blot when exposed to 4 Gy 8 MV X ray for 0,3,8,12,24,36 h or when exposed to 0,1,2,4,8 Gy 8 MV X ray for 12 h.The cell cycle of H1299(p53-/-)cell transfected stably with the recombinant vector was analyzed by flow cytometry after exposed to 4 Gy 8 MV X ray.Results The recombinant pE6-p53/GFP plasmid had been constructed correctly and the expression of p53 gene in the transfected H1299 cell lines had been determined.After 4 Gy X ray radiation,the expression of wt-p53 protein had a significant rise.The transfected H1299 cell lines stopped in G1 stage after radiation and their cloning efficiency decreased notably.Conclusion We had constructed successfully the recombinant pE6/p53/GFP plasmid that was regulated by radiation induced response element E6.This provides experimental data for radiation-gene therapy of non-small lung cancer.

Objective · To evaluate the efficacy and safety of prophylactic phenylephrine in parturients prone to develop spinal hypotension.Methods · Fifty parturients undergoing elective cesarean delivery whose preoperative positional mean arterial pressure (MAP) change from supine to right lateral position were bigger than 8 mmHg (1 mmHg=0.133 kPa) were randomly allocated into 2 groups, i.e. high-risk prevented group (group A) and high-risk control group (group B). Another 25 parturients whose positional MAP change were smaller than 8 mmHg were allocated into low-risk prevented group (group C). After spinal anesthesia, phenylephrine (50 μg bolus and 50 μg/min infusion) was given immediately to group A and C, and the pump speed was adjusted to 25 μg/min 10 min later till fetuses were removed. Normal saline with the same volume and pump speed was given to group B. The incidences of hypotension, reactive hypertension, and bradycardia, the occurrence of nausea and vomiting, and Apgar scores at 1 min and 5 min of three groups were compared. Results · The incidence of hypotension in group A was 28%, 76% in group B, and 16% in group C. Group A and C were significantly lower than group B (P<0.01). The reactive hypertension rate was 4% in group A and 28% in group C. There was a difference between these two group (P=0.015). There were no significant differences among 3 groups in Apgar scores at 1 min and 5 min (P>0.05). Conclusion · Prophylactic phenylephrine in the paturients prone to develop spinal hypotension reduces the incidence of spinal hypotension without obvious adverse effects on the paturients and neonates.

Transcription factor cyclic AMP response element-binding protein (CREB) in embryonic cortical neurons is an important modulator of Brain-derived neurotrophic factor (BDNF) induced gene expression. Meanwhile, our early researches have indicated that BDNF possesses neuroprotective role for hypoxic neurons against hypoxia. In order todisclose whether the neuroprotective role of BDNF for embryonic rat cortical neurons against hypoxia is fulfilled via nucleoprotein CREB phosphorylation, we used western blotting method to detect the expression of CREB and phosphorylated CREB in experimental groups (with BDNF) and hypoxic control group (without BDNF) with the time changes of exposure to hypoxia. Results indicated that hypoxia and BDNF both could induce phosphorylation of CREB in embryonic cortical neurons. Phosphorylation of CREB in experimental group (with BDNF) was much higher than that in hypoxic control group at the same time points (P<0.01). The expression of phosphorylated CREB reached the highest level at the first hour after being exposed to hypoxia in experimental groups, then phosphorylated CREB decreased slowly and remained at the level for much longer time in experimental groups than in control group. The total amount of CREB in embryonic cortical neurons at the first 0-3 hours after being exposed to hypoxia in experimental groups were the same as that in hypoxic control group. CREB decreased more quickly in hypoxic control group at 5-6 hours after hypoxia. This in vitro research demonstrates that BDNF plays its neuroprotective role for embryonic rat cortical neurons against hypoxia via CREB phosphorylation.
Animals ; Brain-Derived Neurotrophic Factor ; chemistry ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Cerebral Cortex ; cytology ; Cyclic AMP Response Element-Binding Protein ; chemistry ; Embryo, Mammalian ; Neurons ; cytology ; Neuroprotective Agents ; chemistry ; pharmacology ; Phosphorylation ; Rats