ABSTRACT
Objective:To construct recombinant lentiviral vectors harboring interference RNA ( RNAi ) targetting murine TNF-αgene,so as to lay the foundation on the RNAi gene therapy.Methods: Three small interfering RNA ( siRNA) sequences targeting murine TNF-αgene ( siRNA1,siRNA2,siRNA3) and negative-control siRNA were designed and synthesized.The inhibition effects of siRNAs on TNF-α,IL-1βand IL-6 secretion of LPS-stimulated RAW264.7 macrophages were observed using real-time PCR and ELISA methods.DNA oligo was designed and synthesized according to the most effective siRNA 2 sequence.The recombinant lentiviral shuttle plasmid expressing short hairpin RNA ( shRNA) was constructed and sequenced.The lentiviral shuttle plasmids with packaging plasmids were transfected into 293T cells to produce lentiviral particles.Results: ①The TNF-αmRNA relative expression levels of siRNA1, siRNA2 and siRNA3 were 0.24±0.01,0.16±0.02,0.19±0.01 respectively,significantly lower than that of negative control (0.95± 0.02) (F=531.3,P0.05).②The TNF-αprotein expression levels of siRNA1,siRNA2 and siRNA3 were (23.95±1.21),(17.27±1.46),(19.07± 1.57)ng/ml respectively,significantly lower than that of negative control (35.37±2.93)ng/ml (F=18.1,P=0.000 6<0.001).The inhibition rates of protein expression were 32.29%, 51.16%, 46.08%, respectively comparing with negative control.③The PCR product electrophoresis showed that recombinant vectors yielded 343 bp fragments,non-constructed vectors yielded 306 bp fragments.DNA sequencing partially showed insertion sequence.④Lentiviral particles were obtained by transfecting 293T cells with recombinant lentiviral shuttle plasmids and lentiviral packaging plasmids.Cells grew well during virus production with strong fluorescence expression.The titer of concentrated virus was 2×106 TU/μl.Conclusion:The lentiviral vector harboring RNAi targeting murine TNF-αgene has been successfully constructed.
ABSTRACT
ObjectiveTo investigate the possible pathogenesis of EB virus (EBV) latent membrane protein 1 in inducing systemic lupus erythematosus (SLE).MethodsThe mRNA expression levels of LMP1 and apoptosis-related genes bcl-2,bax in SLE patients and healthy controls were detected by real-time fluorescence quantitative polymerase chain reaction (PCR).The serum BAFF levels of SLE patients and normal healthy controls were detected by ELISA.2 test was used for positive rate analysis,2-△△Ct method was used for comparing the gene expression level,and Student-Newman-Kqeuls method was used for pair-wise comparison between the means.Results① The positive rate of LMP1 expression in 67 SLE cases was 25％,which was significantly higher than the 11％ in 65 healthy controls (P＜0.05).② The 2-△Ct value of bcl-2 mRNA expression level of SLE patients was 0.0257,1.41 times to that (0.0183) of healthy controls and the difference was statistically significant.③ The 2-△Ct value of bcl-2 mRNA expression level of LMP1 positive SLE patients was 0.0427,1.98 times to that of LMP1 negative SLE patients (0.0217),the difference was statistically significant.④ The serum BAFF levels of LMP1 positive SLE patients,LMP1 negative SLE patients,LMP1 positive healthy controls and LMP 1 negative healthy controls were ( 106± 15 ),(82± 19),( 68±19),(64±17) μg/L,respectively.There were significant differences between serum BAFF levels of LMPl-positive SLE patients and other groups(P＜0.0l ).There were significant difference between serum BAFF levels of LMP1-negative SLE patients and the control groups (P＜0.01).ConclusionEBV may induce and/or promote SLE by LMP1 through apoptosis-related genes bcl-2 expression and induction of B lymphocytes that produce BAFF,all these mechanisms can prolong the infected auto-reactive B lymphocytes survival.