ABSTRACT
<p><b>BACKGROUND</b>Human interleukin-10 (hIL-10) is a cytokine synthesis inhibitory factor, which is involved in various immune responses. The purpose of this study was to construct an adenoviral vector carrying the hIL-10 gene for expression of biologically active hIL-10 in rat bone marrow mesenchymal stem cells (rMSCs).</p><p><b>METHODS</b>A pSNAV2.0-hIL10 plasmid was used as a template to obtain a hIL-10 cDNA fragment that was subcloned by restriction enzyme digestion and ligation into a pDC316-IRES-EGFP-lacZ alpha plasmid carrying an enhanced green fluorescent protein (EGFP) marker gene. The pDC316-hIL-10-IRES-EGFP plasmid was linearized by PmeI digestion and used to transfect HEK293 packaging cells using the adenovirus packaging system AdMax. Virus particles were amplified by repeatedly infecting HEK293 cells with the seed virus and then purified by ion exchange. After the number of virus particles and titer was determined, rMSCs were infected with the adenoviral vector. The infection rate was determined by fluorescence microscopy and flow cytometry, and hIL-10 protein expression in rMSCs was measured by Western blotting.</p><p><b>RESULTS</b>The virus particle concentration, OD260/280 value and virus titer of the amplified and purified recombinant adenovirus were 3.2 × 10(11) VP/ml, approximately 2.0, and 1.1 × 10(10) TCID50/ml, respectively. Bright green fluorescence was observed by fluorescence microscopy and flow cytometry in the recombinant adenovirus-infected rMSCs. GFP expression was considered the multiplicity of infection (MOI) and was time-dependent. The infection rate was 92.9% at 100 MOI.</p><p><b>CONCLUSIONS</b>A bicistronic recombinant adenoviral vector for hIL-10 and EGFP gene expression were successfully constructed. The infection rate of rMSCs by the adenovirus was high (92.9% at 100 MOI) and the target gene hIL-10 was highly expressed in cells. The present study provides an experimental basis for further research of immunosuppressive therapy using hIL-10. The expression level of hIL-10 protein as detected by Western blotting was also MOI- and time-dependent.</p>
Subject(s)
Animals , Humans , Male , Rats , Adenoviridae , Genetics , Cell Line , Cells, Cultured , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Metabolism , Interleukin-10 , Genetics , Metabolism , Mesenchymal Stem Cells , MetabolismABSTRACT
Objective: To investigate the effects of preventive high thoracic epidural block (PHTEB) for different periods on cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH) in rabbits. Methods: T6-7 epidural space was opened and an epidural pipe was introduced to establish PHTEB model. Ropivacaine(1 g • L-1) was injected continuously through the pipe at 1 ml/h to block T1-5 sympathetic nerves. Sixty HTEB rabbits were assigned to 6 groups (n = 10 each): the normal 1 day group (N1), the normal 7 day group(N7), SAH 1 day group (S1), SAH 7 day group (S7), PHTEB plus SAH 1 day group (HS1), and PHTEB plus SAH 7 day group (HS7). CVS was developed by injecting non-anticoagulant autologous arterial blood (0.5 ml/kg) into the cisterna magna. Transcranial Doppler (TCD) ultrasonography was used to determine Vm of the basilar artery. The lesion and spasm of the basilar artery were observed under optical microscope. Results: Compared with group N1, Vm was significantly increased in group S1 (P0.05). Vm in group S1 was significantly increased compared with group S7 (P<0.05); and that in group HS1 was significantly increased compared with group HS7 (P<0.05). Pathological observation showed no obvious changes in group N1 and N7. The vessel wall was shrunk and the lumina became narrow in group S1 and S7. The pathologic changes in group HS1 and HS7 were slighter than those in group S1 and S7;and Group HS7 was slighter than group HS1. Conclusion: PHTEB can relieve CVS in SAH rabbits, and the relieving effect increases with time of PHTEB.