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.

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

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.

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.

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.

Objective:To observe the effects of acupuncture and moxibustion on interleukin(IL)-9/IL-9 receptor(IL-9R)in the colon tissue of rats with ulcerative colitis(UC)and investigate the protective mechanism of acupuncture and moxibustion on the intestinal mucosal barrier in UC rats. Methods:Male Sprague-Dawley rats were randomly divided into a normal control(NC)group and a modeling group.UC models were prepared by giving 4%dextran sulfate sodium(DSS)water for 7 d.After the successful construction of the UC rat model,the modeling group was randomly divided into a UC group,a herb-insulated moxibustion(HM)group,and an electroacupuncture(EA)group.HM and EA interventions at bilateral Tianshu(ST25)were performed once a day for 7 d.Hematoxylin-eosin(HE)staining was used to observe the histopathological changes in the colon.The serum concentrations of IL-9,IL-6,IL-1β,and hemoglobin-H(HbH)were determined by enzyme-linked immunosorbent assay.The protein expression levels of IL-9,IL-9R,claudin-2,zonula occludens-1(ZO-1),and occludin in the colon tissue were measured by Western blotting or immuno-histochemistry.Immunofluorescence was used to detect the co-expression of PU.1 and CD4 with the IL-9 protein. Results:Compared with the NC group,the colon tissue of UC rats was severely damaged and ulcerated with congestion and edema,and the colonic histopathological score increased significantly(P<0.01).The serum HbH concentration decreased significantly(P<0.01),while the serum concentrations of IL-9,IL-6,and IL-1β increased(P<0.01).The protein expression of colonic ZO-1 and occludin decreased significantly(P<0.01),while the protein expression of colonic IL-9 and IL-9R increased(P<0.05).The positive co-expression levels of IL-9/PU.1 and IL-9/CD4 increased in the colon tissue(P<0.05).Compared with the UC group,the colonic mucosal structures were gradually repaired in both HM group and EA group,and healed ulcers could be observed,the colonic histopathological score decreased significantly(P<0.05).The serum concentration of HbH increased(P<0.01),while the serum concentrations of IL-9,IL-6,and IL-1β decreased(P<0.05).The protein expression levels of ZO-1 and occludin increased(P<0.05),while the protein expression levels of IL-9 and IL-9R decreased(P<0.01).The positive co-expression levels of IL-9/PU.1 and IL-9/CD4 decreased in the colon tissue(P<0.05). Conclusion:Both HM and EA can inhibit the protein expression levels of IL-9 and IL-9R in the UC colon by regulating the transcription factor PU.1,promote the repair of intestinal mucosal barrier,and down-regulate protein contents of proinflammatory factors IL-9,IL-6,and IL-1β in the serum,which may be one of the key mechanisms of acupuncture and moxibustion in reducing the inflammation of UC colonic mucosa and protecting the intestinal mucosal barrier.