ABSTRACT
BACKGROUND:Epidermal growth factor is an auxiliary growth factor,but its effect on the growth of bone marrow mesenchymal stem cells (BMSCs) is uncertain.OBJECTIVE:To establish a mature method for isolation,extraction and identification of rat BMSCs,to investigate the effects of epidermal growth factor (EGF) on the proliferation and migration ability of BMSCs and to explore its potential mechanisms at the same time.METHODS:Rat BMSCs were isolated and purified using the improved whole bone marrow adherence method.After the cells were subcultured to the third generation,we detected the expression of cells surface antigens CD29,CD45 and CD90 by flow cytometry.BMSCs were further identified by osteogenic and adipogenic differentiation.Meanwhile,the effect of EGF on the proliferation of passage 3 BMSCs was measured by cell counting kit-8 and clonogenic assay.And the migration of P3 cells was verified by Transwell chamber.In addition,we detected the expression of proteins related to PI3K/Akt and nuclear factor-κB signaling pathways by western blot assay.RESULTS AND CONCLUSION:The primary BMSCs were polygonal and spindle-shaped,and then gradually appeared to be spindle-shaped.The results of flow cytometry demonstrated that the passage 3 cells were positive for CD29 and CD90,but negative for CD45.Furthermore,we successfully induced the osteogenic and adipogenic differentiation of BMSCs in vitro.Additionally,our data demonstrated that EGF promoted the proliferation and migration of passage 3 BMSCs.The relative expression levels of p-Akt and Bcl-2 of PI3K/Akt signaling pathway was up-regulated and the expression of Bax was down-regulated.At the same time,the relative expression level of phosphorylated p65 of nuclear factor-κB signaling pathway was up-regulated and the expression of phosphorylated inhibitor κB was down-regulated.Moreover,the downstream protein of matrix metalloproteinase-9 was up-regulated.Those proteins were related to the migaration of BMSCs.In summary,our results suggest that EGF could promote the proliferation and migration of BMSCs.
ABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant adenovirus for carry tyrosine hydroxylase (TH) gene and expressing bioactive TH protein in the animal model of Parkinson disease.</p><p><b>METHODS</b>The TH gene was inserted into the shuttle plasmid, which was transformed into E.coli BJ-5183 for homologous recombination with the adenovirus genome. 293 cells were transfected with the recombinant adenovirus genome to obtain the recombinant virus, and the transcription and expression of TH were determined by RT-PCR and immunofluorescence assay, respectively. The production of L-DOPA in the in vitro reaction system was determined using capillary electrophoresis.</p><p><b>RESULTS</b>We have successfully constructed the recombinant adenovirus. The TH mRNA and the corresponding protein were detected by RT-PCR and immunofluoresence assay in 293 cells. L-DOPA was also detected in the reaction system.</p><p><b>CONCLUSION</b>The adenovirus constructed allows efficient expression of bioactive TH protein in vitro, which provides a basis for future study of gene therapy of Parkinson disease in animal models.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Metabolism , Cell Line , Electrophoresis, Capillary , Escherichia coli , Genetics , Metabolism , Genetic Therapy , Genetic Vectors , Genetics , Levodopa , Genetics , Parkinson Disease , Therapeutics , Recombinant Proteins , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Tyrosine 3-Monooxygenase , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation.</p><p><b>METHODS</b>The HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10.</p><p><b>RESULTS</b>Both groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response.</p><p><b>CONCLUSIONS</b>The adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.</p>
Subject(s)
Animals , Male , Mice , Adenoviruses, Human , Genetics , Hepatitis Antigens , Genetics , Allergy and Immunology , Immunoglobulin A , Allergy and Immunology , Immunoglobulin G , Allergy and Immunology , Mice, Inbred BALB C , Nasal Mucosa , Allergy and Immunology , Peritoneum , Allergy and Immunology , Recombinant Proteins , Genetics , Allergy and Immunology , Viral Hepatitis Vaccines , Viral Proteins , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta).</p><p><b>METHODS</b>Twelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR.</p><p><b>RESULTS</b>Hepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus.</p><p><b>CONCLUSIONS</b>The recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.</p>
Subject(s)
Animals , Antigens, Viral , Allergy and Immunology , Hepatitis E , Hepatitis E virus , Allergy and Immunology , Immunoglobulin G , Allergy and Immunology , Macaca mulatta , RNA, Viral , Blood , Recombinant Proteins , Allergy and Immunology , Vaccination , Viral Hepatitis Vaccines , Allergy and ImmunologyABSTRACT
Two plasmid constructs, pcE2 and pcE3, containing 3' fragment of open reading frame 2 (ORF2,1163 bp) of hepatitis E virus (HEV) and full-length ORF3 (369 bp), were injected into bilateral tibialis of Swiss mice respectively，for three times (0, 2nd and 4th weeks) and observed the HEV IgG by ELISA. HEV IgG was induced after the injection of pcE2 or pcE3 or both, and the percentage of seraconversion was 100% after two weeks of the third injection. Compared with injection of either construct, the antibody titers were higher in the group with combined injection of two constructs.