Objective To study the changes in the expressions of CXCR4 of leucocytes as modulated by erythrocytes from cord blood. Methods 0.2ml suspension of whole blood cells (or 0.2ml suspension of leucocytes) was added to 0.3ml of auto plasma, and 0.2ml suspension of S180 (5?10~6/ml) was added as stimuli. The suspension was incubated at 37 ℃ for 1 hour. Normal saline instead of S180 was used as control The expression changes in CD35 on erythrocytes and CXCR4 on leucocytes were determined with flow cytometry assay. The data could be grouped under four heads: (1) cancer cells 0.2ml were added to whole blood cells 0.2ml and plasma 0.3ml; (2) NS 0.2ml were added to whole blood cells 0.2ml and plasma 0.3ml; (3) cancer cells 0.2ml were added to white blood cells 0.2ml and plasma 0.3ml; (4) NS 0.2ml were added to white blood cells and plasma 0.3ml. Results When activated by S180, the expression of CD35 on cord erythrocytes was decline not so low as that of adult′; while the expression of CXCR4 on cord leucocytes was increased not so high as that of adults′. When activated by S180, the expression of CD35 on adult erythrocytes was significantly decreased than that of group of not activating (40.21%?11.52% and 28.65%?8.08% respectively), while the expression of CXCR4 on adult erythrocytes was significantly increased (40.62%?17.89% and 64.18%?11.69% respectively, P

Objective To evaluate of red blood cell as giving instruction in whole white blood cell immunological activity by new nature experimental system of hemaimmune reaction rood map. Methods Plasma 0.3ml were added to whole blood cells (including: red blood cell and white blood cells) or white blood cells 0.2ml, and incubated for 1h at 37℃. The content of IL-8 and IL-12 was determined by enzyme linked immunadsorbent assay (ELISA) method. The expression level of CD4, CD8, CD35 and CXCR4 on white blood cells was determined by method of Flow Cytometry. Results The content of IL-8 (5.96?4.26) and IL-12 (9.84?2.23) in whole blood and plasma nature group was significantly lower than that (13.59?3.69?B?pg~ -1 ?ml~ -1 ) and (15.09?9.86?B?pg~ -1 ?ml~ -1 ) in white blood cell and plasma isolation group (P

Objective To investigate the effect of hypoxic preconditioning on anti-inflammatory responses of bone marrow mesenchymal stem cells (BMSCs) in rats through in vitro and in vivo experiments.Methods In vitro experiment The isolated rat BMSCs were cultured by whole bone marrow adherence method.The cells at passage 3 were seeded in 24-well plates at a density of 1 × 106 cells/ml and randomly divided into 5 groups (n =8 wells each) using a random number table:control group (group C),normoxia-incubated group (group N),hypoxic preconditioning group (group H),hypoxia preconditioning + STAT3 inhibitor Stattic group (group HS) and hypoxia preconditioning + anti-IL-10 monoclonal antibody group (group HA).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to21％ O2-74％ N2-5.0％ CO2 for48 h.In group H,BMSCs were exposed to 0.5％ O2-94.5％ N2-5.0％ CO2 for 24 h followed by 24 h exposure to normoxia.In HS and HA groups,500 μg/ml Stattic and 100 μg/rnl anti-IL-10 monoclonal antibody were added to the culture medium before hypoxia preconditioning,respectively.The expression of phosphorylated STAT3 (p-STAT3) and IL-10 was determined by Western blot.In vivo experiment Healthy male Sprague-Dawley rats,weighing 300-350 g,in which intrathecal catheters were successfully implanted without complications,underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Three hundred rats with spinal cord I/R injury were randomly divided into C,N,H,HS and HA groups (n =60 each) using a random number table.Immediately after onset of reperfusion,DMEM medium 300 μl was injected intrathecally in group C,and BMSC suspension 300 μl (1 × 106 cells/ml) was injected intrathecally in N,H,HS and HA groups.Neurological function was scored before ischemia and at 4,12,24 and 48 h of reperfusion (T0-,).The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the content of IL-10,TNF-α,IL-1β,IL-6,monocyte chemotactic protein-1 (MCP-1),and macrophage inflammatory protein-1 α (MIP-1 α) (by ELISA) and the number of activated microglia (by immuno-histochemistry).Results Compared with C and N groups,the expression of pSTATβ and IL-10 was significantly up-regulated,the neurological function score and IL-10 content were increased,the content of TNF-α,IL-1β,IL-6,MCP-1 and MIP-1α and the number of activated microglia were decreased in group H (P ＜ 0.05).Compared with group H,the expression of p-STAT3 and IL-10 in group HS and expression of IL-10 in group HA was significantly down-regulated,and the neurological function score and IL-10 content were decreased,and the content of TNF-α,IL-13,IL-6,MCP-1 and MIP-1α and the number of activated microglia were increased in HS and HA groups (P ＜ 0.05).Conclusion Hypoxic preconditioning can enhance anti-inflammatory effects of BMSCs,thus increasing BMSCs-induced reduction of spinal cord ischemia-reperfusion injury in rats.

Objective To explore the application value of tissue dispersion quantitative analysis in rat liver fibrosis stages and provide a reference for clinical non-invasive diagnosis of liver fibrosis.Methods Seventy-two male Wistar rats were randomly divided into experimental group (n =64) and control group (n =8).The experimental groups of rats were gavaged with the volume fraction of 60％ CCl4 olive oil solution to form different stages of liver fibrosis,the control group did wvith normal saline,All the rat underwent tissue dispersion quantitative analysis to obtain 12 elastic parameters,the differences in above parameters were compared among rats with different liver fibrosis stages,then the correlation with pathological stages were analyzed.Results Except for COMP,ASM,CORR,there were significant differences in all parameters among rats of different liver fibrosis (P ＜0.05),which were correlated with pathological stages(P ＜0.05).Among these parameters,％ AREA had the highest correlation coefficient (r =0.891,P =0.001).The ROC curve was made by ％AREA to estimate the fibrosis stage.the area under ROC curve for ％ AREA was 0.914 (≥ the control group + S0),0.963 (≥early liver fibrosis S1 + S2),0.969 (≥middle liver fibrosis S3),0.948 (early cirrhosis S4),respectively.Conclusions The technique of tissue dispersion quantitative analysis has a good evaluation value for liver fibrosis stages,it is able to provide a preliminary reference for clinical non-invasive diagnosis of liver fibrosis.

BACKGROUND:The magnetic resonance molecular imaging used in the research of lumbar disc degeneration includes T2 mapping and T1ρtechnologies at present.
OBJECTIVE:To evaluate the feasibility of 1.5 T MR equipment on T2 mapping of New Zealand white rabbits lumbar disc.
METHODS:T2 mapping images of New Zealand white rabbit lumbar discs were obtained on 1.5 T MR equipment. The regions of interest T2 values of lumbar discs were measured with post-processing workstation.
RESULTS AND CONCLUSION:Sagittal and coronal T2 maps of 3-month rabbit discs were obtained
satisfactorily on 1.5 T MR equipment. The regions of interest T2 values of nucleus pulposus in L 4/5 , L 5/6 and L 6/7 discs were (104.6±14.0) ms, (109.1±13.8) ms and (109.5±15.1) ms respectively at Pfirrmann
regions of interest T2 values of anterior annulus fibrosus in L 4/5 , L 5/6 and L 6/7 discs were (82.1±9.5) ms, (80.4± 11.2) ms and (79.9±10.6) ms respectively, and T2 values of posterior annulus fibrosus in L 4/5 , L 5/6 and L 6/7 discs were (85.8±11.9) ms, (85.1±12.1) ms and (85.3±9.3) ms respectively. There were no significant differences in T2 values of nucleus pulposus, anterior annulus fibrosus and posterior annulus fibrosus in L 4/5 , L 5/6 and L 6/7 discs at PfirrmannⅠP>g 0ra.0d5e).( However, the T2 values of nucleus pulposus were higher than those of annulus
fibrosus in the same discs (P<0.01), and there were no significant differences in T2 values between the anterior and posterior annulus fibrosus (P>0.05). The T2 values of rabbit lumbar discs obtained on 1.5 T MR equipment can be used for quantitative assessment of intervertebral disc signal.

Shiga toxin 2 (Stx2) toxoid produced by formaldehyde treatment of purified toxin was used to immunize BALB/c mice for monoclonal antibody (MAb) production.The neutralizing activities of positive clones against Stx2 were screened by in vitro cytotoxicity assay.The isotype and specificity of resultant clone was determined,and its efficacy to neutralize the activity of purified Stx2 was evaluated by in vitro and in vivo toxicity model.Lastly,its spectrum of activity against Stx2 variants was also accessed by mouse toxicity model.It was demonstrated that one of the 12 positive MAb clones against Stx2,designating $2C4 had neutralizing activity.S2C4 belongs to the immunoglobulin G1 subclass and has a K light chain,and it reacts with the A subunit of Stx2 and does not bind to Stx2 B subunit or to Stx1.S2CA could efficiently neutralize the cytotoxicity of Stx2 to Veto cells and mice.It also protected mice against lethal doses of Stx2 variants challenge including Stx2c and Stx2vha.S2C4 is a promising candidate molecule in preventing the progression of hemolytic-uremic syndrome (HUS) mediated mainly by Stx2 in Stx-producing Escherichia coli(STEC)infection.

Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ ( StxⅡ) in shiga toxin-producing Escherichia coli ( STEC) infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡand its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.

Objective To purify Shiga toxin Ⅱ (STX Ⅱ) of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 by affinity chromatography, and characterize its biological function. Methods The immno-affinity chromatography column was prepared by STX Ⅱ A subunit-specific antibody S1D8 coupling to Sepharose 4B matrix. The purity and specificity of STX Ⅱ molecule secreted by EHEC O157:H7 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, respectively. The purified toxin was serially diluted and the toxic activities to Vero cell line and mice were observed. The 50% cytotoxic dose (CD50) for Vero cell line and 100% lethal dose (LD100) for mice were calculated. The protection effect of anti-STX Ⅱ polysera to the mice against the purified toxin challenge was also observed. Results STX Ⅱ was successfully purified from culture supernatant of EHEC O157:H7 using affinity chromatography scheme. The relative molecular weights of STX Ⅱ A and B subunits were 32 000 and 7 500 confirmed by SDS-PAGE, respectively. The purified toxin could react with monoclonal antibodies against STX Ⅱ A and B subunits, respectively.The toxin was cytotoxic to Vero cell with CD50 of 20 ng/L and lethal to mice with LD100 of 5 ng.The toxin could be neutralized by anti-STX Ⅱ polysera in vivo. Conclusion STX Ⅱ is successfully purified and its toxic effects are confirmed in both cell line and mouse model.

Objective To evaluate the practicability of using CRISPR/Cas9 genome editing tech-nology for inhibition of hepatitis B virus ( HBV) replication. Methods Two sgRNA targeting sites were de-signed for the S region of HBV genome. The CRISPR/Cas9 expression plasmids specific for HBV were con-structed and then transfected into a cell line expressing HBV genome(HepG2-N10). The cytotoxicity of cells transfected with different expression plasmids were detected by MTT assay. The levels of hepatitis B surface antigen ( HBsAg ) were determined by using chemiluminescent immunoassay ( CLIA ) . The expression of HBV at mRNA level was analyzed by quantitative real-time PCR ( qRT-PCR) . The qPCR was performed for the detection of extracellular and intracellular HBV DNA. The next-generation sequencing ( NGS) Illumina MiSeq Platform was used to analyze HBV genome editing. Results No significant cytotoxic effects were de-tected in HepG2-N10 cells transfected with different expression plasmids. Compared with the cells carrying pCas-Guide-GFP-Scramble, the levels of HBsAg in the supernatants of transfected cell culture harboring pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were decreased by 24. 2% (P<0. 05) and 16. 9% (P>0. 05), respectively. The levels of HBsAg in cells transfected with pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were respectively decreased by 16. 4% (P>0. 05) and 32. 1% (P>0. 05) as compared with that of pCas-Guide-GFP-Scramble transfected group. The expression of HBV at mRNA level was inhibited as indica-ted by the results of qRT-PCR. Moreover, the levels of extracellular HBV DNA were respectively suppressed by 23% (P>0. 05) and 35% (P<0. 05), and the levels of intracellular HBV DNA were respectively sup-pressed by 7. 2% (P>0. 05) and 18% (P>0. 05). Different types of insertion/deletion mutation were de-tected in HBV genome by high-throughput sequencing. Conclusion HBV-specific CRISPR/Cas9 system could inhibit the expression of HBV gene and the replication of virus. Therefore, the CRISPR/Cas9 genome editing technology might be used as a potential tool for the treatment of persistent HBV infection.

Acetaminophen (APAP) overdose is one of the most common causes of acute liver failure. The study aimed to investigate the protective effect of carnosic acid (CA) on APAP-induced acute hepatotoxicity and its underlying mechanism in mice. To induce hepatotoxicity, APAP solution (400 mg/kg) was administered into mice by intraperitoneal injection. Histological analysis revealed that CA treatment significantly ameliorated APAP-induced hepatic necrosis. The levels of both alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum were reduced by CA treatment. Moreover, CA treatment significantly inhibited APAP-induced hepatocytes necrosis and lactate dehydrogenase (LDH) releasing. Western blot analysis showed that CA abrogated APAP-induced cleaved caspase-3, Bax and phosphorylated JNK protein expression. Further results showed that CA treatment markedly inhibited APAP-induced pro-inflammatory cytokines TNF-alpha, IL-1beta, IL-6 and MCP-1 mRNA expression and the levels of phosphorylated IkappaBalpha and p65 protein in the liver. In addition, CA treatment reduced APAP- induced hepatic malondialdehyde (MDA) contents and reactive oxygen species (ROS) accumulation. Conversely, hepatic glutathione (GSH) level was increased by administration of CA in APAP-treated mice. Mechanistically, CA facilitated Nrf2 translocation into nuclear through blocking the interaction between Nrf2 and Keap1, which, in turn, upregulated anti-oxidant genes mRNA expression. Taken together, our results indicate that CA facilitates Nrf2 nuclear translocation, causing induction of Nrf2-dependent genes, which contributes to protection from acetaminophen hepatotoxicity.
Acetaminophen ; Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Blotting, Western ; Caspase 3 ; Cytokines ; Glutathione ; Hepatocytes ; Injections, Intraperitoneal ; Interleukin-6 ; L-Lactate Dehydrogenase ; Liver ; Liver Failure, Acute ; Malondialdehyde ; Mice* ; Necrosis ; Reactive Oxygen Species ; RNA, Messenger ; Tumor Necrosis Factor-alpha