Study of brain-derived neurotrophic factor gene transgenic neural stem cells in the rat retina / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neural stem cells (NSCs) transplantation and gene therapy have been widely investigated for treating the cerebullar and myelonic injuries, however, studies on the ophthalmology are rare. The aim of this study was to investigate the migration and differentiation of brain-derived neurotrophic factor (BDNF) gene transgenic NSCs transplanted into the normal rat retinas.</p><p><b>METHODS</b>NSCs were cultured and purified in vitro and infected with recombinant retrovirus pLXSN-BDNF and pLXSN respectively, to obtain the BDNF overexpressed NSCs (BDNF-NSCs) and control cells (p-NSCs). The expression of BDNF genes in two transgenic NSCs and untreated NSCs were measured by fluorescent quantitative polymerase chain reaction (FQ-PCR) and enzyme-linked immunosorbent assay (ELISA). BDNF-NSCs and NSCs were infected with adeno-associated viruses-enhanced green fluorescent protein (AAV-EGFP) to track them in vivo and served as donor cells for transplantation into the subretinal space of normal rat retinas, phosphated buffer solution (PBS) served as pseudo transplantation for a negative control. Survival, migration, and differentiation of donor cells in host retinas were observed and analyzed with Heidelberg retina angiograph (HRA) and immunohistochemistry, respectively.</p><p><b>RESULTS</b>NSCs were purified successfully by limiting dilution assay. The expression of BDNF gene in BDNF-NSCs was the highest among three groups both at mRNA level tested by FQ-PCR (P < 0.05) and at protein level measured by ELISA (P < 0.05), which showed that BDNF was overexpressed in BDNF-NSCs. The results of HRA demonstrated that graft cells could survive well and migrate into the host retinas, while the immunohistochemical analysis revealed that transplanted BDNF-NSCs differentiated into neuron more efficiently compared with the control NSCs 2 months after transplantation.</p><p><b>CONCLUSIONS</b>The seed cells of NSCs highly secreting BDNF were established. BDNF can promote NSCs to migrate and differentiate into neural cells in the normal host retinas.</p>

Expression of human Id-2 gene in Escherichia coli and preparation of the antisera against human Id-2 / 南方医科大学学报

<p><b>OBJECTIVE</b>To express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2.</p><p><b>METHODS</b>The coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2.</p><p><b>RESULTS</b>PCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP).</p><p><b>CONCLUSION</b>The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.</p>

Identification of mimotope peptides which bind to the SARS-CoV spike protein specific monoclonal antibody 2C5 with phage-displayed peptides library / 生物工程学报

To identify the epitope of SARS-CoV spike protein specific neutralizing monoclonal antibody (MAb) 2C5. The antibody was used as target and three rounds of bio-panning were conducted with phage-display peptide library. After the third panning, 20 phage-plague clones were randomly picked and analyzed for the binding ability with the MAb 2C5 by ELISA. The display sequence analysis demonstrated that among the twenty phage clones, eight clones displayed the same seven-peptide TPEQQFT. All these eight phage-clones showed strongest binding activity with 2C5 in phage ELISA analysis. Furthermore, phages displaying peptide TPEQQFT could specifically inhibit the binding of MAb 2C5 with SARS-CoV spike protein. The results demonstrated that TPEQQFT is a mimic epitope peptide containing neutralizing MAb 2C5. This study may provide information for further structural and functional analysis of spike protein and development vaccine for severe acute respiratory syndrome.

Effect of Cilazapril on endothelial cell function and fibrinolysis system in the canine atrial fibrillation models / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effect of cilazapril on endothelial cell function and fibrinolysis system in the canine atrial fibrillation (AF) models.</p><p><b>METHODS</b>All canines were divided into three groups: (1) Control group, without atrial pacing; (2) Atrial pacing group, in which atrial fibrillation was established by rapid atrial pacing at 400 bpm for 6 weeks; (3) Atrial pacing together with cilazapril group, in which cilazapril was given before and after atrial pacing. Nitric oxide (NO) of atrial endocardium was measured with NO-specific microelectrode. The expression of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) protein in atrium was determined by Western Blot analysis and immunohistochemical staining. Plasma levels of von Willebrand Factor (vWF), PAI-1 and tPA were analyzed by enzyme-linked immunoadsorbent assay.</p><p><b>RESULTS</b>NO production from atrial endocardium was significantly increased in atrial pacing together with cilazapril group than atrial pacing group [(42.6 +/- 9.9) nmol/L vs (23.4 +/- 5.8) nmol/L, P < 0.05], whereas the plasma levels of vWF were decreased [(75.4 +/- 12.8)% vs (125.9 +/- 20.6)%, P < 0.05]. Compared to controls, the expression of atrium tPA protein in atrial pacing together with cilazapril group was significantly upregulated (4052 +/- 857 vs 1936 +/- 421, P < 0.05) and PAI-1 protein was downregulated (2487 +/- 542 vs 3164 +/- 827, P < 0.05). Cilazapril also significantly increased tPA antigen and decreased PAI-1 antigen in plasma.</p><p><b>CONCLUSION</b>Cilazapril can favorably improve endothelial function and resume the balance of fibrinolysis system in AF, which might be of beneficial to hypercoagulated state in AF.</p>