Objective To detect the expression and significance of serum miRNA-183 and TK1 in patients with colorectal cancer, and the mechanism thereof. Methods Fifty-two serum samples of colorectal cancer patients and paired health serum samples were collected. The expression of miRNA-183 was detected by real-time quantitative PCR, and TK1 was detected by Western blot enhanced chemiluminescence assay. The correlation between miRNA-183 and TK1 and their relations with the clinicpathologic characteristics were analyzed. Results The serum miRNA-183 expression was significantly higher in colorectal cancer group than that in normal control group (P<0.01). The expression of serum miRNA-183 was significantly higher in stage Ⅲ-Ⅳ group than that in stage Ⅰ-Ⅱ group (P < 0.01). There was more significant increase in serum miRNA-183 in lymphatic metastasis group than that without lymphatic metastasis group (P < 0.01). Receiver operating characteristic (ROC) curve showed that of there was a diagnostic value for serum miRNA-183 in colorectal cancer, with an optimal value of 1.15. The diagnostic sensitivity and specificity were 78.8%and 67.3%, and the positive predictive value and negative predictive value were 78.9%and 70.2%. The serum TK1 expression was also higher in colorectal cancer group compared with that of normal control group (P<0.01). And the TK1 expression was also higher in stageⅢ-Ⅳgroup than that in stageⅠ-Ⅱgroup (P<0.01). Furthermore, miRNA-183 expression was positively correlated with TK1 expression (rs=0.692, P<0.01). Conclusion The serum expression levels of miR-183 and TK1 may act as tumor markers for early colorectal cancer diagnosis, and also can be used to predict the malignant degree and prognosis of colorectal cancer.

Objective To investigate the effects of shRNA targeting a disintegrin and metalloproteinases 17(ADAM17)on the proliferation of human breast cancer MCF-7 cells in hypoxia environment.Methods The four specific ADAM17-shRNA se-quences aiming at ADAM17 were designed,transfected into MCF-7 cells by electroporation,and cultured in hypoxia environment. The experiment was divided into the control group (blank phosphate buffer solution,PBS),nonsense sequence group (transfected with ADAM17-shNC)and shRNA transfection group (transfected with ADAM17-shRNA,the highest silencing efficiency of shR-NA was selected for following experiments).Real-time PCR was used to detect the expression of ADAM17 mRNA.The prolifera-tion ability and cell cycle change of MCF-7 cells were detected by iCELLigence and flow cytometry (FCM),respectively.Results Compared with control group and nonsense sequence group,the four ADAM17-shRNA transfection groups all had the silence effect on ADAM17 gene expression (P <0.05 ),the difference was statistically significant(P <0.05 ),particularly shRNA1219 had the highest inhibitory rate (F =5.11,P <0.01 ).The cellular proliferation ability and cell growth speed in the shRNA transfection group were significantly decreased compared with the control group and nonsense sequence group (P <0.05).Most cells of shRNA transfection group remained in the G0/G1 phase (73.35 ± 2.45 ),which in the control group and nonsense sequence group was (62.56±2.35)and (62.68 ±1.20)respectively,the difference was statistically significant(P <0.05).The cell cycle progression was significantly delayed.Conclusion ADAM17-shRNA inhibits the proliferation of MCF-7 cells under hypoxic environment.

Objective:To discuss the effects of recombinant IFN-α-IL-18 fusion protein on chicken lymphocyte histamine induced and the NF-κB p65 activation and nucleo-cytoplasmic transport.Methods: The healthy chickens blood was sterile adopted with anticoagulant,then separation of the chicken peripheral blood lymphocyte and divided into 10 groups:The yeast expression and purification protein IFN-α-IL-18,IL-18,IFN-α were added with 250 ng/ml,500 ng/ml and 1 000 ng/ml respectively while the control was only added RPMI1640 with 3 repetitions for each group.Then the histidine decarboxylase activity,histamine,IFN-γ,PI3K,MAPK and NF-κB p65 in cell nucleus were detected.Results: The recombinant IFN-α-IL-18 and IL-18 could significantly promote the activity of histidine decarboxylase (P<0.01),increase the contents of histamine (P<0.01),induce IFN-γ (P<0.01),improve the contents of PI3K (P<0.01) and the NF-κB p65 levels in nucleus (P<0.01),and the higher concentration of IFN-α had a similar effect to lymphocytes.The effects of IFN-α-IL-18,IL-18 and IFN-α on MAPK was acratia.Conclusion: IFN-α-IL-18 and IL-18 can stimulate chicken peripheral blood lymphocyte populations increased histamine contents significantly and promote the induction of IFN-γ.IFN-α-IL-18,IL-18 and IFN-α increase PI3K expression in lymphocyte associated with the NF-κB activation and NF-κB p65 nucleo-cytoplasmic transport.The study built foundation for the function of IFN-α-IL-18 and the exploration of the mechanism of controlling epidemic diseases.

In order to develop a safer vaccine strain exploit Salmonella Pullorum vaccine strain as a live vaccine vector.Methods:AΔcrpΔasd mutant of S.pullorum C79-13 strain was constructed and it was developed E.coli donor strain mutant was generated through the two-step method introduced by χ7213 ( pREΔasd) was conjugated with the recipient of C 79-13Δcrp.The C79-13ΔcrpΔasd the transduction of recombinant suicide plasmid .Results:PCR and sequencing results showed that ΔsipBSL1344 was suc-cessfully constructed.The further study indicated that foreign DAP must be supplied for the ΔcrpΔasd mutant to grow,in addition,the asd gene was transmitted stably .Compared with C79-13 strain,the O antigens was identical to C79-13 strain,but the growth velocity was reduced significantly ,significantly reduced virulence .Conclusion: The ΔcrpΔasd mutant can accept asd+plasmid to construct host-vector balance lethal system , and this system can be used to express foreign gene efficiently and to develop potential oral multivalent vaccines.

Objective:To explore the function of the cya gene and the preliminary mechanism of attenuated strain.Methods:The biological characteristics of cya mutant in acid and alkali resistant,salt resistance,motility,biofilm components,poisonous to the cells of epithelial cell adhesion,invasion were analysis.Results:The mobility capabilities,acid and alkali resistance and salt tolerance of cya mutant were significantly lower than the parent strain;the composition testing revealed that the cya mutant did not produce cellulose,curli and biofilm;at the same time the adhesion and invasion to epithelial cells of cya mutant had a prominent depression,and the toxicity to HeLa cells was weaker than the parent strain.Conclusion:The function of cya gene is closely related to athletic ability, penetration of cell membrane, the formation biofilm and virulence.It will provide a theory reference to the functional research of Salmonella typhimurium cya gene and the mechanism of attenuated strain.This will contribute to the development of oral vaccine using attenuated Salmonella typhimurium as vector.

BACKGROUND:It is confirmed that the absence of hypoxia-inducible factor-1α (HIF-1α) accelerates the degenerative process in the intervertebral discs, and microRNAs have an important role in degeneration of the intervertebral discs.
OBJECTIVE:To evaluate the changes of microRNAs in the intervertebral discs of HIF-1α-deficient (HIF-1α-/-) mice which may mediate the signaling pathway of HIF-1α in the intervertebral discs.
METHODS: As previously reported, HIF-1α-/- mice were established. HIF-1α-/- mice and HIF-1αflox/flox mice (control mice) aged 4 weeks were used. MRI and histological staining were used to evaluate the degeneration of the intervertebral discs. Total RNAs were extracted from the intervertebral discs tissues by Trizol, and the differential expression profile of microRNAs was harvested by significance analysis of microarrays and Cluster, based on microarray screening. Real-time quantitative reverse transcription-PCR was applied to verify the reliability of microRNA array results.
RESULTS AND CONCLUSION:The number of nucleus pulposus cels in the intervertebral discs of HIF-1α-/- mice was decreased, the cels presented with smal size and the color deepened in the cytoplasm. Finaly, differential expression profile of microRNAs (n=10) was obtained, seven of which were upregulated and three were downregulated. In conclusion, the loss of HIF-1α may cause the imbalance of some important miRNAs, which may result in a large amount of dead nuclear pulposus cels and mediate disc degeneration in HIF-1α-/- mice.

Objective:In order to develop an oral live vaccine vector of swine that can stably carry exogenous genes.Methods:Mutant ΔcrpΔcyaΔasdC78-1 was constructed by the method of suicide plasmid pREasd-mediated bacteria homologous recombination on the basis of attenuated Salmonella choleraesuisΔcrpΔcyaC78-1.Complementary plasmid pYA3493 with asd was electrotransformed into the mutant,and thenΔcrpΔcyaΔasdC78-1(pYA3493) host-vector balanced lethal system was constructed.Its biological characteristics were analyzed further.Results:The results of PCR and sequencing showed thatΔcrpΔcyaΔasdC78-1(pYA3493) was constructed suc-cessfully.Biological characteristics showed that the serotype of ΔcrpΔcyaΔasdC78-1(pYA3493) was identical to ΔcyaΔasdC78-1 and vaccine strain C500 and it can stably carry theΔasd gene in vitro;its growth speed was a little slower than ΔcrpΔcyaC78-1 strain,but both of their growth speeds were significantly slower than vaccine strain C500;the biochemical characteristics of ΔcrpΔcyaΔasdC78-1 ( pYA3493 ) were basically the same with ΔcrpΔcyaC78-1 strain.Oral virulence test in mice showed that the virulence ofΔcrpΔcyaΔasdC78-1 ( pYA3493 ) was similar with ΔcrpΔcyaC78-1, but its median lethal dose is 412 times of vaccine strain C500.Conclusion:These results demonstrated that attenuated Salmonella choleraesuisΔcrpΔcyaΔasdC78-1(pYA3493) strain had the potential to be used as an oral live vaccine vector for expressing foreign genes efficiently.

Objective To analyze the biological characteristics of a mutant strain of Salmonella ty-phimurium SL1344 with sseK2-deletion (SL1344△sseK2) in order to provide reference for further study of safe and effective live vaccines. Methods The mutant strain SL1344△sseK2 with a deletion of 1047 bp in sseK2 gene was constructed through a two-step allelic exchange using recombinant suicide plasmid. Its com-plemented strain, SL1344C△sseK2, was also constructed. Biological and immunological characteristics of the mutant strain were detected. Results PCR, double-enzyme digestion and sequencing analysis showed that the mutant strain SL1344△sseK2 and the complemented strain SL1344C△sseK2 were successfully con-structed. The serotype of the mutant strain was 1,4,[5],12:i:1,2, identical to the parent strain SL1344. In addition, the mutant strain showed no significant change in biochemical characteristics or growth rate and was genetically stable in vitro. Compared with the parent strain SL1344, the virulence of SL1344△sseK2 was attenuated in BALB/ c mice. The median lethal dose of SL1344△sseK2 for 6-week-old BALB/ c mice was 3. 44×108 colony-forming units (CFU), which was 1620 times lower than that of SL1344. Oral immuniza-tion with SL1344△sseK2 protected 62. 5％ of the mice against challenge with wild Salmonella typhimurium strains on 17 d after vaccination. The levels of serum IgG antibody peaked on 14 d after immunization. No significant difference in biological characteristics was observed between the complemented and the parent strains, indicating that the mutant strain was basically complemented to the wild-type strain.Conclusions The mutant strain SL1344△sseK2 was constructed successfully and genetically stable with sig-nificantly attenuated virulence and good immunogenicity. This study suggested that sseK2 gene played an im-portant role in regulating the virulence of SL1344, which might provide reference for further study of its func-tion and for assessing its potential as a candidate live attenuated vaccine.

Objective: To develop an oral live vaccine vector which stably carries exogenous genes.Methods:SL1344ΔsipBΔasd host-vector balanced lethal system was constructed by the method of recombinant suicide plasmid-mediated allelic exchange on the basis of attenuated Salmonella typhinurium SL1344ΔsipB.Then,the biological characteristics of SL1344ΔsipBΔasd was analyzed.Results:The results showed that the mutant was stabile with the Δasd gene in vitro;the serotype and growth rate of SL1344ΔsipBΔasd strain was almost same as the parent SL1344ΔsipB and SL1344 strain.And the mutant strains remain swim ming zones.Virulence test in mice showed that the virulence of SL1344ΔsipBΔasd which carried complementary plasmid pYA3493 by electro-transformation decreased by 1.4%compared with SL1344.Conclusion: These results showed that the SL1344ΔsipBΔasd mutant was successfully constructed.It is likely that this mutant should be used as a live vector to express foreign genes.

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology