Up-regulation of the transient A-type K+ current (IA) in the differentiation of neural stem cells of the early postnatal rat hippocampus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neural stem cells (NSCs) not only are essential to cell replacement therapy and transplantation in clinical settings, but also provide a unique model for the research into neurogenesis and epigenesis. However, little attention has been paid to the electrophysiological characterization of NSC development. This work aimed to identify whether the morphological neuronal differentiation process in NSCs included changes in the electrophysiological properties of transient A-type K(+) currents (I(A)).</p><p><b>METHODS</b>NSCs were isolated from early postnatal rat hippocampus and were multiplied in basic serum-free medium containing basic fibroblast growth factor. Potassium currents were investigated and compared using whole-cell patch-clamp techniques and one-way analysis of variance (ANOVA), respectively.</p><p><b>RESULTS</b>Compared with NSC-derived neurons, cloned NSCs (cNSCs) had a more positive resting membrane potential, a higher input resistance, and a lower membrane capacitance. Part of cNSCs and NSC-derived neurons possessed both delayed-rectifier K(+) currents (I(DR)) and I(A), steady-state activation of I(A) in cNSCs (half-maximal activation at (21.34 +/- 4.37) mV) occurred at a more positive voltage than in NSC-derived neurons at 1-6 days in vitro (half-maximal activation at (12.85 +/- 4.19) mV).</p><p><b>CONCLUSIONS</b>Our research revealed a developmental up-regulation of the I(A) component during differentiation of postnatal NSCs. Together with the marked developmental up-regulation of I(DR) in vitro neuronal differentiation we have previously found, the voltage-gated potassium channels may participate in neuronal maturation process.</p>

Prokaryotic expression, purification of human LINGO-1(aa76-319) and preparation of its polyclonal antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).</p><p><b>METHODS</b>The 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.</p><p><b>RESULTS</b>The prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.</p><p><b>CONCLUSION</b>The fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.</p>

Culture and identification of neural stem cells derived from the subventricular zone of adult mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method for culturing and identifying neural stem cells (NSCs) derived from the subventricular zone (SVZ) in adult mice.</p><p><b>METHODS</b>NSCs were isolated from the SVZ of adult mouse brain and cultured in serum-free medium. Cell cloning and BrdU incorporation were performed to identify the self-renewal and proliferative capacity of the NSCs. Fluorescence immunocytochemistry was used to examine the expressions of the NSC markers nestin and SOX2, neuronal marker Tuj1, astrocyte marker GFAP and oligodendrocyte marker NG2. The expressions of nestin and SOX2 were further examined by Western blotting and RT-PCR.</p><p><b>RESULTS</b>NSCs with self-renewal and proliferative capacity were obtained from the SVZ of adult mice and grown as floating neurospheres. The NSCs expressed nestin and SOX2 and could differentiated into Tuj1-positive neurons, GFAP-positive astrocytes and NG2-positive oligodendrocytes.</p><p><b>CONCLUSION</b>This method allows simple and stable culture of NSCs from the SVZ of adult mice.</p>

Construction of eukaryotic expression vector with brain-derived neurotrophic factor receptor trkB gene / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene.</p><p><b>METHODS</b>Using the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR.</p><p><b>RESULTS</b>The amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2.</p><p><b>CONCLUSIONS</b>The trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.</p>