Objective To express in prokaryotic system and to analyze the antigenic specificity of EB virus(EBV) latent membrane protein 2(LMP2) multi-epitopes gene rich of T cell and B cell epitopes.Methods Using on-line prediction service, T cell epitopes and B cell epitopes of EB virus latent membrane protein 2 were predicted. The genes rich of CTL and th cell epitopes were selected as the candidate gene sequences, while B cell epitopes around them were taken into account. The finial selected multi-epitope gene was synthesized after being optimized according to prokaryotic codon bias and inserted into prokaryotic expression vector pET32a( + ) to get the recombinant plasmid: pET32a( + )/EBV-LMP2 multi-epitopes. After transformed into E. coli BL21 (DE3) and induced by IPTG, the target multi-epitopes gene can be expressed as Trx-His fusion protein. The expression products can be identified by SDS-PAGE and Western blot. Moreover, rabbit serum antibody to EBV membrane protein and nasopharyngeal carcinoma(NPC) patient serum were used respectively to detect the antigenic specificity of the multi-epitopes. Meanwhile, 6-8 weeks female BALB/c mice were immunized with EBV-LMP2 multi-epitope at 2 week intervals, three times in all, Trx-His protein and PBS were set as the control groups. At the second week after the last immunization, the mice were sacrificed. LDH and indirect ELISA were taken to detect the specific spleen CTL activityand specific IgG in serum, which reflected the immunogenicity of the EBV-LMP2 multi-epitope. Results Two amino acid sequences which locate at the LMP2 (aa195 -232 ) and LMP2 (aa419-436 ) were selected and connected in series to be the target gene. The recombinant plasmid containing EBV-LMP2 multi-epitope gene successfully constructed and the target protein was expressed in E. coli BL21 ( DE3 ). The relative molecular mass(Mr) of The expression products is about 27 × 103 , which matches up to the expected Mr. The antigenic specificity of the multi-epitopes protein was identified by Western blot and the multi-epitopes protein also can be detected by rabbit serum antibody to EBV membrane protein and NPC patient serum respectively. In the result of the animal experiment, EBV-LMP2 multi-epitope was able to induce the specific CTL activity in BALB/c mice. With the increasing of the effector: target ( E: T) 1: 5,1: 10, 1: 25, the CTL activity was also increased wih( 12.52% + 2.59% ), (21.80% + 1.08% ), (23.68% + 3.74% ) respectively; EBV-LMP2 multi-epitope was able to induce LMP2-specific antibody response(A490 =0.258 +0.040) as compared with Trx-His protein(A490 =0.095 +0.011) and PBS(A490 =0.068 +0.014,P＜0.05＝. Conclusion The EBV-LMP2 multi-epitopes gene was designed successfully and expressed precisely in prokaryotic expression system with good antigenicity and immunogenicity.

Objective To construct recombinant plasmids of SIK2 cDNA and its truncated mutants and induce its expression in E.coil.Methods We designed primers of SIK2 and its truncated mutants.The gene fragments of SIK2,SIK2-Δ1 (280-926),SIK2-Δ2 (400-926),SIK2-Δ3 (1-400),and SIK2-Δ4 (700-926)were amplified by polymerase chain reaction (PCR)and cloned into pGEX-4T-2 vector to construct recombinant plasmids with GST. The plasmids were transformed into E.coil BL2 1 respectively,and induced with IPTG to express fusion protein. The results were confirmed by Coomassie blue staining and Western blot.Results We successfully constructed recombinant plasmid of SIK2 cDNA and its truncated mutants.Coomassie blue staining and Western blot resutls showed that these plasmids were induced to be expressed in E.coil BL21.Conclusion SIK2 cDNA and its truncated mutants were overexpressed in E.coil BL2 1 ,which lays expereimental foundation for further study on the function of each domain of SIK2 .

ObjectiveTo explore the etiology and biochemical markers of acute liver failure (ALF) in children.Methods The cause and the biochemical markers of ALF in children who were treated in December 2014 to January 2011 were ana-lyzed retrospectively.ResultsA total of 67 children were enrolled, including 31 females and 36 males. According to the cause of the disease, the children were divided into non-genetic metabolic group, genetic metabolic group, and cryptogenic group. In the non-genetic metabolic group (29 cases, 43.28%) there were 12 cases of drug-induced ALF, 5 cases of Reye syndrome, 3 cases of hemophagocytic syndrome, 3 cases of herpes simplex virus infection, 2 cases of autoimmune hepatitis, one of case mushroom poisoning one case of hepatitis A virus infection, one case of cytomegalovirus infection and one case of sepsis respectively. In the genetic metabolic group (14 cases, 20.90%) there were 6 cases of Wilson’s disease, 2 case of glycogen storage disease, 2 of cas-es progressive familial intrahepatic cholestasis, 2 cases of neonatal intrahepatic cholestasis caused by citrin deifciency, one case of very long-chain acyl coenzyme A dehydrogenase deifciency and one case of primary carnitine deifciency. In the cryptogenic group there were 24 cases (35.82%). The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, blood glucose level and AST/ALT were statistically signiifcantly different in genetic metabolic group from in non-genetic me-tabolism disease group and cryptogenic group, (P<0.05). The genetic metabolic group had the lowest levels of serum ALT, AST, albumin and glucose while the genetic metabolic group had the highest ratio of AST/ALT.ConclusionsThe etiology of ALF in children are complex. Genetic metabolic disease should be considered when the child with ALF has no signiifcantly elevated ALT, extremely high ratio of AST/ALT, combined with hypoproteinemia and hypoglycemia.

OBJECTIVE To investigate the effect of subcutaneousimmunotherapy(SCIT) on levels of the serum human beta defensin-2 in children with allergic rhinitis. METHODS 30 cases of children with allergic rhinitis who were treated by SIT were selected as the treatment group, 20 cases of healthy children as the control group. Serum HBD-2 concentration of the control group was tested. Serum HBD-2 concentration of the treatment group was tested at three different time points: before SCIT, half a year after SCIT and one year after SCIT. And total nasal symptom scores(TNSS) and medication scores were recorded at each time point. RESULTS The serum HBD-2 concentration of the control group, that of the treatment group before SIT, half a year after SIT and one year after SIT were 4.62[4.08; 4.87], 3.74[3.37; 4.61], 4.62[4.13; 5.54], 4.79[4.45;6.19]ng/ml. The HBD-2 concentration gradually increased after SCIT. The TNSS of the treatment group before SCIT, half a year after SCIT and one year after SCIT were 7.43±2.15, 4.17±2.16, 4.20±1.92, The medication scores of the treatment group before SCIT, half a year after SCIT and one year after SCIT were 1.25[0.75; 1.38], 0.25[0; 0.75, 0.25[0; 0.75].There was no correlation (all P>0.05) between the serum HBD-2 concentration and TNSS or medication scores of the treatment group. CONCLUSION The serum levels of HBD-2 in patients with allergic rhinitis were lower than those in normal persons. The specific immunotherapy raised the serum HBD-2 levels of allergic rhinitis patients.

Objective:To dynamically observe the effect of N-acetylserotonin (NAS) on the expression of tumor necrosis factor-α (TNF-α) protein in retina of retinal ischemia reperfusion injury (RIRI) rats, and to explore the mechanism.Methods:By using random number table method, 90 healthy male Sprague-Dawley rats were divided into sham operation group ( n=10), RIRI group ( n=40), and NAS group ( n=40). The right eye was as the experimental eye. In the RIRI group and NAS group, the anterior chamber high intraocular pressure method was used to establish the RIRI model. In the NAS group, 10 mg/kg NAS was injected intraperitoneally before modeling and 30 minutes after modeling. At 6, 12, 24, 72 h after modeling, hematoxylin-eosin staining was used to observe the pathological changes of the retina, and the retinal ganglion cells (RGC) were counted. Each group was detected by immunohistochemical staining and Western blot about the relative expression of TNF-α, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) protein in the rat retina. Oneway analysis of variance was used for differences between groups. The general linear regression method was used to analyze the correlation between the relative expression changes of TNF-α protein and the changes of Nrf2 and HO-1 protein expression after NAS intervention. Results:Optical microscope observation revealed that the retinal edema of rats in the RIRI group was observed at 6, 12, and 24 h after modeling; the thickness of the retina in the NAS group was significantly thinner than that in the RIRI group, and the difference was statistically significant ( F=9.645, 477.150, 2.432; P<0.01). At 6, 12, 24, and 72 h after modeling, the retinal RGC counts in the NAS group were significantly higher than those in the RIRI group, and the difference was statistically significant ( F=12.225, 12.848, 117.655, 306.394; P<0.05). The results of immunohistochemical staining and Western blot showed that 6 h after modeling, the relative expression of TNF-α protein in the retina of the RIRI group increased significantly compared with that in the sham operation group, reaching a higher level at 12 h, and decreased at 24 and 72 h. But all were significantly higher than the sham operation group, the difference was statistically significant (immunohistochemical staining: F=105.893, 1 356.076, 434.026, 337.351; P<0.01; Western blot: F=92.906, 534.948, 327.600, 385.324; P<0.01). At different time points after modeling, the relative expression of TNF-α protein in the retina of the NAS group was significantly lower than that of the RIRI group (immunohistochemical staining: F=15.408, 570.482, 21.070, 13.767; P<0.05; Western blot: F=12.618, 115.735, 13.176, 111.108; P<0.05), but still higher than the sham operation group (immunohistochemical staining: F=40.709, 151.032, 156.321, 216.035; P<0.01; Western blot: F=33.943, 79.729, 74.057, 64.488; P<0.01), the difference was statistically significant; 12 h after modeling, Nrf2 in the retina of the NAS group (immunohistochemical staining: F=51.122, P<0.05; Western blot: F=33.972, P<0.05), HO-1 (immunohistochemical staining: F=30.750, P<0.05; Western blot: F=18.283, P<0.05) protein relative expression was significantly higher than that of RIRI group, and the differences were statistically significant. The results of linear regression analysis showed that the difference in the number of TNF-α + cells in the RIRI group and the NAS group was negatively correlated with the difference in the number of Nrf2 + and HO-1 + cells ( r 2=0.923, 0.936; P<0.01). Conclusions:NAS can inhibit the expression of TNF-α protein in the retina of RIRI rats and reduce RIRI. The mechanism may be related to the Nrf2/HO-1 pathway.

Objective To investigate the effect of pyrin domain 3 (NLRP3) inflammasome in the process of contrast induced human kidney cell apoptosis.Methods Human kidney 2 (HK-2) cells were cultured in DMEM-F12 medium with 5％ FBS.Cells were divided into control group,Contrast group (O group),NLRP3-siRNA+Iohexol group (si-NLRP3+O group),ASC-siRNA+Iohexol group (si-ASC+O group),and mannitol group (M group).Different concentrations of hypotonic contrast agent were added to HK-2 cell culture plates for 24,48 and 72 h.Flow cytometry was used to detect apoptosis.NLRP3 and ASC mRNA expressions were detected by RT-PCR.The expressions of NLRP3,ASC,caspase-8/cleaved caspase-8,Bcl-2/Bax,caspase-1/cleaved caspase-1,and caspase-3/cleaved caspase-3 protein were detected by Western blot.The levels of interleukin (IL) 1β and IL-18 in supernatant were detected by ELISA.Results Compared with the control group,the rate of apoptotic cells,as well as the expressions of NLRP3,ASC and cleaved caspase-1 proteins were increased in HK-2 cells of contrast group.The expressions of NLRP3 and ASC mRNA in the contrast group also increased,so did IL-1β and IL-18 levels (all P＜0.05),suggesting that NLRP3 inflammasome in HK-2 cells was activated by contrast.Compared with the control group,the expressions of cleaved caspase-8,Bax and cleaved caspase-3 protein were increased,and the expression of anti-apoptotic protein Bcl-2 was decreased (all P ＜ 0.05).Compared with the contrast group,the rate of apoptotic cells in the si-NLRP3 + contrast group and si-ASC + contrast group was significantly decreased;the expression of cleaved caspase-1 was decreased;the expressions of Bax and cleaved caspase-3 were decreased,and Bcl-2 level was increased.The expressions of IL-1β and IL-18 in the supernatant of cells were decreased (all P ＜ 0.05).Conclusion Contrast agent can activate the NLRP3 pathway in HK-2 cells and induce apoptosis,which could be reduced by blocking the NLRP3 pathway.

Background and Objectives: Psoriasis is a chronic inflammatory skin disease, which the mechanisms behind its initiation and development are related to many factors. DMSCs (dermal mesenchymal stem cells) represent an important member of the skin microenvironment and play an important role in the surrounding environment and in neighbouring cells, but they are also affected by the microenvironment. We studied the glucose metabolism of DMSCs in psoriasis patients and a control group to reveal the relationship among glucose metabolism, cell proliferation activity，and VEC (vascular endothelial cell) differentiation in vitro, we demonstrated the biological activity and molecular mechanisms of DMSCs in psoriasis. Methods: and Results: We found that the OCR of DMSCs in psoriatic lesions was higher than that in the control group, and mRNA of GLUT1 and HK2 were up-regulated compared with the control group. The proliferative activity of DMSCs in psoriasis was reduced at an early stage, and mRNA involved in proliferation, JUNB and FOS were expressed at lower levels than those in the control group. The number of blood vessels in psoriatic lesions was significantly higher than that in the control group (p＜0.05), which the mRNA of VEC differentiation, CXCL12, CXCR7, HEYL and RGS5 tended to be increased in psoriatic lesions compared to the control group, in addition to Notch3. Conclusions We speculated that DMSCs affected local psoriatic blood vessels through glucose metabolism, and the differentiation of VECs, which resulted in the pathophysiological process of psoriasis.