Influence of angiotensinogen gene silencing on differentiation of preadipocytes 3T3-L1 / 吉林大学学报(医学版)

Objective To construct the angiotensinogen (AGT)-targeting RNA interfering plasmids, and to explore the effect of AGT gene on the differentiation of preadipocytes 3T3-L1 in the mice.Methods The small hairpin RNA (AGT-shRNA)interference plasmids,with a scrambled Non-shRNA as control,were constructed on the basis of the activity of U6 promoter-driven expression vector psiRNA-U6.1/Neo and transfected into the mouse preadipocytes 3T3-L1.Two cell lines were generated from stable transfections.RT-PCR and SDS-PAGE were performed to monitor the mRNA and protein expressions of AGT gene,respectively.The effect of adipose AGT on the differentiation of 3T3-L1 was investigated through microscopic observation and profiling of adipocyte differentiation markers. Results Both AGT-shRNA and Non-shRNA interference vectors were successfully constructed;the expression of AGT mRNA was inhibited (P < 0.05 ) and the expression of AGT protein was 43.86% of control.Both adipocyte differentiation markers,intracytoplasmic lipid levels and glycerol-3-phophate dehydrogenase (GPDH)activities were increased during the differentiation of preadipocytes 3T3-L1,but the data were lower than those in control group (P <0.05).Conclusion AGT gene silencing can significantly inhibit the differentiation of preadipocytes 3T3-L1,which demonstrates that there is important relationship between adipocyte metabolism and renin-angiotensin system (RAS)in adipose tissue.

Construction and identification of small interfering RNA expression plasmid target to angiotensinogen / 中国组织工程研究

BACKGROUND:In mammalian cells,introduction of double-stranded small interfering RNA(19-25 bp)can cleave and destroy the cognate RNA,which can result in suppression of gene expression.OBJECTIVE:To construct siRNA expression plasmid for interference angiotensinogen(AGT),thereby,to resist AGT expression in adipose cells.METHODs:The mRNA sequence of AGT gene was searched from NCBI(NM000029).Utilize of GenScript siRNA technology,AGT-siRNA oliaonucletides were chemically synthesized and inserted into pRNAT-U6 1/Neo vector after annealing,then transformed into TOP10.The recombinant plasmid was identified by restriction endonuclease and DNA sequencing.RESULTS AND CONCLUS1ON:The recombinant plasmid psiRNAT-U6.1/Neo-AGT was obtained by connecting 19 bp segment containing AGT-mRNA sequence to pRNAT-U6.1/Neo After EcoR Ⅰ and Hind Ⅲ digestion.351 bp segment was obtained from empty vector.and 397 bp fragment band was obtained form recombinant plasmid,which was coincidence to the expectation.DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the eukaryotic expression vector pRNAT-U6.1/Neo without base mutation.The interference vector psiRNAT-U6.1/Neo-AGT was successfully constructed.

Construction of recombinant eukaryotic expression vector pVAX-S_2S containing the gene of hepatitis B virus surface antigen and DNA-mediated immunization / 中国免疫学杂志

Objective:In order to obtain safer HBV DNA vaccine,recombinant kanamycin resistance eukaryotic expression plasmid containing the gene of HBV surface antigen was constructed and used to study humoral immune response in BALB/c mice.Methods:HBV antigen middle protein(preS2+S) encoding gene fragment was isolated from the recombinant eukaryotic expression plasmid pcDNA-S 2S;subcloned into eukaryotic expression vector pVAX1.After confirmed the presence of the gene by restriction endonuclease analysis,it was used to immunize the healthy BALB/c mice at different doses,and the levels of anti-HBs in serum was determined by ELISA.Results:Restriction endonuclease analysis confirmed recombinant plasmid pVAX-S 2S was what was expected.Serum anti-HBs was detected at the 2nd week after DNA vaccination at all three doses(100 ?g,50 ?g,10 ?g per mice)and their titers were increased with time.Quantitative comparison of the serum anti-HBs levels revealed significant(P

Recent advances in anti-basic fibroblast growth factor antibody / 中国病理生理杂志

Basic fibroblast growth factor (bFGF) has been used in wound healing, bone healing, vascular grafting, lens regeneration and limp regeneration. Anti-bFGF antibody is thought to be an major important reagent for bFGF research. This review summarizes the development of anti-bFGF antibody in recent years including preparation, screening, identification and application in order to provide reference to the studies of this field in our country.

Interaction between human augmenter of liver regeneration and Na~+-K~+-ATPase in vitro / 中国病理生理杂志

AIM:To check the physical interaction between GST-Na+-K+-ATPase domain and recombinant human augmenter of liver regeneration (rhALR) by GST pull down assay. METHODS: With PCR and genetic recombinant techniques, the coding region of ? subunit of Na+-K+-ATPase was cloned into expressing plasmid pGEX-4T and identified by endonuclease digestion and sequencing methods. Under the inducing of 0.1 mmol/L IPTG, the fusion protein GST-Na+-K+-ATPase domain was highly expressed by E.coli DH-5?. After hypersound quassating, the GST-Na+-K+-ATPase domain was purified by glutathione agarose beads and the physical interaction with rhALR was checked by GST pull down assay. RESULTS: Analysis by SDS-PAGE showed the rhALRs of monomer and dimmer in GST-Na+-K+-ATPase domain lane. The Western blotting of the GST-pull down assay showed the same results as well. CONCLUSION: The Na+-K+-ATPase domain is associated with rhALR specifically in vitro.