Purpose:To identify the expression of the DNA v accine in mice muscles and detect its ability to induce special cellular and hum oral immune response to carcinoembryonic antigens. Methods:Using cloning technique,the cDNA fragments of CEA gene and mouse co-stimulatory molecule IL-2 gene were constructed into the mammali an co-expression plasmid vector pIRES.Tongue muscle of mice were injected with our plasmid. The expression of CEA molecules in muscle was identified by immunoh istochemitry technique .The humoral responses were detected by ELISA technique, the special celluar immuse responses were detected by CTL and NK cytotoxity assa y. Results:We identified that our plasmids can express the CEA and IL-2 molecules in muscles of mice.CEA-antibody and IL-4 molecules appear in blood serum of mice,CTL activities and NK cytotoxity were much stronger in immun ized mice. Conclusions:The constructed mammalian co-expression plasmid pI RES-CEA , pIRES-IL-2,pIRES-CEA- IL-2 can express CEA and IL-2 molecules i n muscles and can generate cellular and humoral immunological responses to CEA p ositive carcinoma.

Purpose:To construct the mammalian co expression plasmid pIRES CEA IM 2 and detect the expression of the plasmid in vitro. Methods:Using cloning technique,the cDNA fragments of CEA gene and mouse molecule IL 2 gene were constructed into the mammalian co expression plasmid vector pIRES.The inserted target genes in the mammalian co expression plasmid were verified by nucleotide sequencing.C 26 cell line was transfected with this mammalian co expression plasmid using lipofecarnin reagent. The expression of CEA and IL 2 molecules were detected by ELISA technique.Results:The mammalian co expression plasmid pIRES CEA IL 2 was obtained by cloning technique. The nucleotide sequences of CEA gene and molecule IL 2 gene in this mammalian co expression plasmid had high homology with CEA standard strain (99.8%) and mouse IL 2 (99.9%) respectively. After transfection with this mammalian co expression plasmid, the CEA and IL 2 molecules were expressed in C 26 cells. Conclusions:The constructed mammalian co expression plasmid pIRES CEA IL 2 can express CEA and IL 2 molecules in vitro at the same time.

Objective To explore changes of anti-HCV antibody and HCV-RNA in patients with HCV infection and its clinical significance in diagnosis and treatment of hepatitis C .Methods Serum samples from patients with HCV infection were collected . HCV antibodies were analyzed with a chemiluminescence micro-particle immunoassay method ,while a PCR-fluorescent probe meth-od was used to detect HCV-RNA .Concentrations of ALT and AST were also determined .Based on the concentrations of ALT , AST and HCV-RNA ,samples were divided into two groups respectively and the changes of different indicators were analyzed and compared .Meanwhile samples from 37 HCV-infected patients were collected continuously .Different indicators after treatment were compared with those before treatment .Results HCV-Ab and logarithm values of RNA load in the group with abnormal concentra-tions of ALT and AST were significantly higher than those in the normal group (P<0 .05) .HCV-Ab ,ALT and AST concentrations in HCV-RNA positive group were significantly higher than those in HCV-RNA negtive group(P<0 .05) .The areas under receiver operating characteristic curve of HCV-Ab and logarithm values of RNA load were 0 .990 and 0 .838 respectively .Concentrations of ALT ,AST and logarithm values of RNA load after treatment were significantly lower than those before treatment (P<0 .05) while there was no significance between HCV-Ab level after treatment and that before treatment (P>0 .05) .Conclusion The diagnostic performance of HCV-Ab is better than that of logarithm values of RNA load .Determination of ALT ,AST and HCV-RNA is of clinical importance in monitoring the effect of hepatitis C treatment .

Objective To evaluate the value of plasma soluble urokinase plasminogen activator receptor (suPAR) and serum pmcalcitonin (PCT) to investigate their assessment of disease severity and prognosis in patients with sepsis.Methods The levels of plasma suPAR and serum PCT were monitored in 77 patients with sepsis.The acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) and sequential organ failure assessment (SOFA) score were recorded.According to the disease severity and their prognosis,the value of plasma suPAR,serum PCT,APACHE Ⅱ and SOFA score on predict the disease severity and prognosis of septic patients were compared.Results The levels of plasma suPAR in septic patients [(7.9 ±6.5) ng/mL] were lower than severe sepsis patients [(8.4 ±4.5) ng/mL] and septic shock patients [(13.9 ± 8.0) ng/mL],allP ＜ 0.05.The levels of serum PCT in septic patients (6.3 ± 3.5) ng/mLwere lower than severe sepsis patients [(23.7 ± 3.9) ng/mL] and septic shock patients [(25.7 ±4.3) ng/mL],allP ＜0.05.But there was no significant difference in the levels of serum PCT between the severe sepsis group and the septic shock group.Receiver operator characteristic curve (ROC)of the level of plasma suPAR could distinguish survivors from non-survivors in septic patients,maximal area under curve (AUC) of plasma suPAR was 0.803.The best cut-off value of plasma suPAR to distinguish survivors from non-survivors was 9.905 ng/mL.And the AUC of serum PCT was 0.61 (P ＞ 0.05) ; the AUCofAPACHEⅡ score was 0.832 (P＜0.05); the AUC of SOFA score was 0.767 (P＜0.05).Conclusion Monitoring of the levels of plasma suPAR and the APACHE Ⅱ score can help to assess the severity and the prognosis of sepsis in the early stage.

Objective To investigate the Toll like receptor-4 (TLR4) expression on pancreatic islet beta-cell of septic rat and its effects on glucose regulation.Methods SD male septic rats were made with LPS intra-abdominal injection in a dose of 5 mg/kg body weight and it repeated once 3 h later.Rats were randomly (random number) divided into four groups randomly (n =5 in each):normal control group,LPS group,LPS antibody group and PLS with LPS antibody group.The expression and protein level of TLR4 were measured by RT-PCR,Western-blot and immunochemistry analysis respectively.IVGTT (intra-venous glucose tolerant test) was used to measure the glucose and insulin levels 6 hours after LPS administration and as well as in control group,and then their AUC were calculated.Results The TLR4 protein and mRNA expressed on pancreatic islet beta-cell of normal rat were significantly up-regulated 6 hours after LPS administration,while its up-regulation could be inhibited when LPS antibody was used in advance (P ＜ 0.01).Rat blood glucose levels were higher at 10,30,60 and 120 min in LPS group and insulin levels were lower at 30,60,120 min compared with normal control (P ＜ 0.01).LPS antibody improved the insulin secretion and then blood glucose level distinctly decreased during 30-120 min period after LPS challenge proved by IVGTT test.Conclusions TLR4 expression up-regulated on pancreatic islet beta-cell of septic rat and LPS-TLR4 system might be a mechanism of stress hyperglycemia genesis.

Objective To investigate the role of silent mating type information regulation 2 homolog 1(SIRT-1) in lipopolysaccharide (LPS)-induced mitochondrial injury on INS-1(insulinoma cell lines-1)cells. Methods INS-1 cells were divided into 5 groups: control group, 1 mg/L LPS group, LPS group with 10 μmol/L RSV (resveratrol) pretreatment for 1 h, LPS group with 20 μmol/L EX527 pretreatment for 1 h, LPS group together with EX527 pretreatment for 1 h, then incubated these INS-1 cells with RSV and LPS for 24 h. The cell viability and ATP generation were detected, then total, cytoplasmic and mitochondrial protein were isolated from INS-1 cells. The protein expression of SIRT1, TLR4, acetylated FoxO1, and cytochrome C (CytC), Mfn1 (mitofusion1), Mfn2 (mitofusion2), and Fis1 (fission1) were tested by Western-blot. Mfn1, Mfn2, and Fis1 genes expression were detected by real-time PCR. The comparison of multiple groups was performed by one-way ANOVA. LSD-t method was used to compare between every two groups. Results After 1 mg/L LPS stimulation for 24 h, there was decreased cell viability (n=4, F=13.98, P<0.01) and ATP generation (n=4, F=27.92, P<0.05) in INS-1 cells. RSV pretreatment could maintain ATP production, but it could not reverse the EX527-induced ATP decrease (P>0.05). Furthermore, RSV upregulated gene and protein expression of SIRT1, Mfn1 and Mfn2, whereas decreased TLR4 and Fis1 expression. LPS-induced CytC released to cytoplasm was alleviated by RSV (P<0.01), but there was no significant change about FoxO1 protein expression (P>0.05). Conclusions RSV may regulate FoxO1 acetylation followed by its effects on SIRT1 activity, which may be partly the mechanism of mitochondrial damage induced by LPS on INS-1 cells.

In most insects, polyunsaturated fatty acids (PUFAs) are mainly polyunsaturated fatty acids with a carbon-chain length less than 18 carbon atoms, hardly any long-chain polyunsaturated fatty acids such as C20 and C22 that are more valuable and bioactive. This study, by using Drosophila melanogaster (Fruit fly) as a model organism, optimized the Δ6-fatty acid elongase enzyme Elovl5 gene from mice and transferred it to fruit flies for expression. Vectors containing Elovl5 gene were successfully injected into drosophila embryo through the microscopic injection. There were enhanced green fluorescent proteins expressed in the whole developmental stage of Drosophila be means of fluorescence microscope. At the same time, expression of Elovl5 gene significantly contributed to the transformation of fruit flies C18-polyunsaturated fatty acids in the body towards the biosynthesis of longer-chain polyunsaturated fatty acids. The transgenic fruit fly model rich in long-chain polyunsaturated fatty acids such as C20 and C22 were obtained, providing a basis for further research on biosynthesis of polyunsaturated fatty acids in fruit flies.
Acetyltransferases/genetics* ; Animals ; Drosophila melanogaster/genetics* ; Fatty Acid Elongases/metabolism* ; Fatty Acids/genetics* ; Gene Transfer Techniques ; Mice