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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.
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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.
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Objective To construct an efficient eukaryotic expression recombinant vector of human interleukin-1O(hIL-lO),and observe its expression in rabbit synoviocytes(RSCs).Methods Total RNA was extracted from peripheral blood mononuclear cells(PBMCs)of a patient with drug allergy.Specific Drimers for full-length open reading frames(ORFs)of hIL-10 were designed according to GeneBank(NM 000572).Withtotal RNA as the template,full-length ORFs of hIL-10 were amplified by reverse transcription Dolymerase chain reaction(RT-PCR).RT-PCR products were digested by restrictive endonucleotidase.then inserted into plasmid pcDNA4/HisMaxA.Both restrictive endonucleotidase analysis and DNA sequencing Were carried out for inserts verification.RSCs were transfected with recombinant plasmid expression vector PcDNA4 HisMaxA-hiL10 by liposome-mediated gene transfer methods,then cultured in vitro.The supernatants were collected af-ter transfection for 12 hours,24 hours,48 hours,72 hours,7 days,14 days respectively for IL-10 measure-ment by enzyme linked immunosorbent assay(ELISA).Results Full-length ORFs of hIL-1o(0.54 kb)had been successfully cloned from PBMCs through RT-PCR.The inserts and insert location of pcDNA4 HisMaxA were in a fight way verified by enzyme analysis and DNA sequencing.ELISA results showed that exogenous hIL-10 gene had expressed in the transfected RSCs from 12 hours to 7 days after transfection,and hIL-10level of transfection group significantly higher than that of the control group.Conclusion pcDNA4 HisMaxA-hiL10,the hIL-10 efficient eukaryotic expression vectors,has been suecessfully constructed.
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BACKGROUND:Nuclear transcription factor(NTF) ?B is a transcription factor,which exists universally in eukaryocyte.It has been identified that its overacting is correlated to the ongoing and development of many diseases.Among the pathomechanism of rheumatoid arthritis,the abnormal activation of NTF ?B has a lot to do with the inflammatory hyperplasia of synovium and the erosion of tissue around articulation.OBJECTIVE:To review the recent progress of NTF ?B,pathogenesis and treatment of rheumatoid arthritis.RETRIEVAL STRATEGY:A computer-based online search of PubMed database was undertaken to identify the articles published in English from January 2000 to July 2007,with the Key words of "nuclear transcription factor ?B,rheumatoid arthritis,pathogenesis,treatment".Meanwhile,Wanfang database was searched for the related Chinese articles published between January 2000 and July 2007,with the same key words in Chinese.A total of 72 articles were finally selected for the first trial.Inclusive criteria:the articles focus on the NTF ?B,the pathogenesis and treatment of rheumatoid arthritis.Exclusive criteria:repeated experiments.LITERATURE EVALUATION:Among the 30 inclusive literatures,7 were related to reviews while the others were clinical or basic researches.DATA SYNTHESIS:①NTF ?B is a significant transcription factor,which participates in regulating many genes related to immune function and inflammation reaction.The promoters of many genes have the binding sites of NTF ?B.②It is indicated that the abnormal activation of NTF ?B plays an important role in the pathogenesis of rheumatoid arthritis.In recent years,many researches inhibiting its activation through different components of signaling pathways have been doing,which become very hot in anti-inflammatory and anti-rheumatism treatment.③In recent years,people have been making great progress in the therapy of rheumatoid arthritis aiming at cytokine and its receptor,however,interfering NTF ?B to treat rheumatoid arthritis is still in study stage.CONCLUSION:NTF ?B target therapy provides a new therapeutic strategy for the treatment of rheumatoid arthritis,which is a very exciting prospect indeed.