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Objective To construct recombinant eukaryotic expression plasmid encoding Swedish and Flemish mutations of amyloid precursor protein (APP) fused with fluorescent protein and to investigate the APP cleavage progress. Methods The last 300 bases of APP (named as C99 containing Flemish mutation), together with cyan and yellow fluorescence sequence (named as CFP and YFP,respectively) were obtained by polymerase chain reaction (PCR). The 54 bases in the middle of APP sequence were synthesized (named as 54 bp containing Swedish mutation). The 4 fragments mentioned above (CFP, YFP, C99 as well as 54 bp) were inserted into the vector pcDNA3.0. By genetic engineering, the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 was constructed and identified by enzyme digestion, PCR and sequencing. Then the plasmid was transfected into SH-SY5Y cells. Its expression was examined by fluorescence confocal microscopy and the fluorescence resonance energy transfer (FRET) signal was collected. The amyloid beta (A) deposition was detected by immunocytochemistry. Results (1) DNA sequencing showed the sequence of the constructed recombinant plasmid was correct. (2) FRET and two types of fluorescence could be seen by the spectrum confocal fluorescence microscopy. (3) The expression product of fusion gene was correct and cleaved by and secretases. (4) The A deposition was detected in the cell membrane, cytoplasma and intercellular space. Conclusion (1) The fusion protein can generate A by and γproteolytic processing. (2) It is for the first time to observe the APP cleavage by FRET. (3) It is also the first time to find that APP may be cleaved during its transportation from cell plasma to cell membrane. (4) C99 is very important for the correct cleavage of APP. Our test data strongly suggest that C99 may function as the signal peptide. It might guide and direct the APP to the right location for the cleavage.
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Objective To observe the effect of movement exercise combined with electroaeupuneture on the expression of Nestin in the hippocampus dentate gyrus (DG) after cerebral ischemia-repeffusion.Methods Fifty- four Wistar rats were used and randomly divided into a control group (Group A),an exercise training group (Group B),and an exercise training combined with electroacupuncture group (Group C).The middle cerebral arteries (MCA) of all the rats were occluded for 1 h,followed by reperfusion for 7,14 and 21 davs.Immunohistochemistry method was used to detect the expression of Nestin in the hippoeampus dentate gyrus.Results The number of Nes- tin-positive cells peaked in DG in all groups on the 7th day after cerebral isehemia-reperfusion.The number of Nes- tin-positive cells in DG ipsilateral to the ischemia-reperfusion lesion were significantly more than those in the opposite side at various time points (P
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Objective To investigate the role of synapsin-I in the differentiation of the embryonic stem cells (ESC) into the neuron,and to seek a controllable point for the ESC neural differentiation in vitro.Methods Neural differentiation of ESC was induced with the "five step approach",synapsin-I antisense oligonucleotides (AS ONs) was employed to inhibit the synapsin-I expression at different stages. At different time points,morphology,differentiation efficiency and neural specific markers were compared among the normal group and the transfected groups.A tumor cell line called PC12 cell was compared with the ESC at the same time.Results After the synapsin-I AS ONs were used in ESC differentiation, considerable decreases of neurite growth rate and neural precursor cells (nestin (+)) percentage were observed at Stage 3(68.5%?4.2% vs 76.2%?5.1% and 75.8%?4.9%,P