The horseradish peroxidase labeled affinity purified mice anti-UEA (Urea soluable egg antigen of Schistosoma japonicum) antibody was used in enzyme-linked immunoe ectrotransfer blot (EITB) te monitor the changes after UEA being processed by M?.The immune respon-sive peptides were delected in the culture supernatant and homogenate of M? pulsed with UEA in vitro (M?+).After processing by M? the high molecular weight UEA was cleaved into low molecular weight peptides,as shown,by the reactive bands.They markedly differed from that native UEA or trypsin-digested UEA; The bands of M? supernatant and homogenate showed similarity with certain quantitative differences.According to the result described above,we considered: 1.UEA could be processed into smaller pieces by M?,the style of processing is cleavage.2.The processed peptides might be released to extracellular environment.

Indirect ELISA was employed to monitor the serum anti-UEA(urea soluble egg antigen of Schistosoma japonicum )antibody level of mice immunized by a. UEA pulsed macrophage (M + ); b. Cultural supernatant of M+; c. paraformaldehyde fixed M(P -M)pulsed with UEA; d. Ammonium chloride treated M0 (NH4Cl-M0) pulsed with UEA, e. P -M0 pulsed with trypsin digested UEA (T-UEA ); f. NH4C1-M pulsed with T-UEA. The normal M its supernatant and the culture media RPMI 1640 acted as the negative control.The results showed : 1. Serum anti-UEA antibody levels of mice immunized by a and b raised markedly, indicating that the immunogenicity of UEA might be kept up after M processing and the antigenic message could be transferred either by the M+ or by its supernatant; 2. Mice immunized by c and d gave similar results, but the anti-UEA antibody level of the former was higher than that of the latter, suggesting that polyformaldehyde could not alter the UEA binding site on the surface of M; 3. In the case of mice immunized by e and f , the antibody levels were much lower than that of mice immunized by c and d, suggesting that UEA binding sites on M surface as well as UEA immunogenicity could be changed by trypsin.

Objective To increase the water solubility of curcumin constituents by three kinds of food grade surfactants, and to study the effect of surfactants on the quality standard and stability and anti-hepatic fibrosis in vivo of curcumin solution. Methods The high-speed dispersion method was used to prepare the solution of curcuminoids-surfactants. The qualitative analysis of curcuminoids was confirmed by LC-MS/MS. HPLC-DAD fingerprinting was established and quantitative analysis was conducted. And the effects on anti-liver fibrosis of different concentrations curcumin ratio solution were evaluated by rat model. Results The maximum water solubility of curcumin constituents was 1.387 mg/mL. LC-MS/MS analysis showed that the structure of curcuminoids were stable; The results of fingerprinting analysis demonstrated that the similarity of 18 batches of samples were higher than 99%. The contents of curcumin, demethoxycurcumin and bisdemethoxycurcumin in different batches of samples were stable with the RSD of 1.60%, 2.24%, and 3.74%. The rat model of hepatic fibrosis showed that the contents of AST and ALT in free curcumin suspension group were (64.1 ± 20.3) and (45.1 ± 13.9) U/L, respectively, while that solution of curcuminoids-surfactants group was (19.4 ± 8.7) and (11.8 ± 4.9) U/L. Conclusion The solubility of curcuminoids was increased 517 times compared with the free state (2.677 μg/mL) after solubilization with surfactants, and repeated tests indicated that the structure and contents of curcuminoids were stable; the solubilized curcuminoids exhibited better anti-liver fibrosis effects than the free.

Objective To observe the immunological effect of tumor cell vaccines to mouse hepatoma A(HepA) treated by Haematoxylin. Methods HepA cell was treated by 0.1% Haematoxylin and made into three vaccines: HepA vaccine with complete Freund' s adjuvant, HepA vaccine with incomplete Freund' s adjuvant; and HepA vaccine without any adjuvants. Five times of immunization were given the grouped mice with the above three vaccines, then active HepA cells (1×105 for each mouse) were inoculated by intraperitoneal injection to attack them; reckoning the mean survival time (MST) of the grouped mice, comparing the immunoprotective action of the three vaccines to the tmnor-bearing mice. Those mice only receiving normal saline (equal volume to the vaccine) were as a control group. Results MST of control group was (23.30±1.24) day; MST of mice receiving H22 vaccine with complete adjuvant was (43.90±15.20) day (P<0.02); MST of mice receiving H22 vaccine with incomplete adjuvant was (39.60±13.77) day (P<0.05); and MST of mice receiving HepA vaccine without any adjuvant was (38.40±12.54) day (P<0.05); As compared with the control group, the three treated groups showed a life-lengthening rate (LLR) 88.41%, 69.96 % and 64.81% respectively. Conclusion The vaccines treated by Haematoxylin give a marked immunoprotective action to those tumor-bearing mice. The HepA vaccine' s immunological effect is increased by the Freund' s adjuvant (complete or incomplete).

OBJECTIVETo investigate the relationship between apoptosis-related proteins in gastric mucosa, p53 and Bax, and Helicobacter pylori (H. pylori) infection in children.

METHODSp53 and Bax expression in gastric mucosa were measured using immunohistochemical technique in 33 children with gastric mucosal lesions. Presence/absence of H. pylori infection was detected by the rapid urease and pathological tests.

RESULTSFifteen children (88%) showed positive expression of p53 in 17 children who were confirmed with H. pylori infection, compared with 9 (56%) in 16 H. pylori negative children. Thirteen children (76%) showed positive expression of Bax in the 17 children with H. pylori infection, compared with 6 (38%) in the 16 H. pylori negative children. The expression levels of p53 and Bax in the H. pylori positive group were significantly higher than those in the H. pylori negative group (p<0.05).

CONCLUSIONSH. pylori infection is associated with the over-expression of p53 and Bax proteins in gastric mucosa in children.

Child ; Female ; Gastric Mucosa ; chemistry ; Helicobacter Infections ; metabolism ; Helicobacter pylori ; Humans ; Immunohistochemistry ; Male ; Tumor Suppressor Protein p53 ; analysis ; bcl-2-Associated X Protein ; analysis

Objective: To evaluate the effect of penehyclidine hydrochloride (PHC) on Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway in non-ventilated lung injury in the patients undergoing radical operation for lung cancer. Methods: A total of 100 patients, aged 40-64 yr, with body mass index 18-27 kg/m², of American Society of Anesthesiology physical status Ⅱ or Ⅲ, undergoing radical operation for lung cancer, were divided into 2 groups (n=50 each) according to the random number table method: control group (group C) and PHC group.PHC 0.01 mg/kg was intravenously injected at 10 min before anesthesia induction in group PHC, while the equal volume of normal saline was given instead in group C. The peripheral tissues of the removed lung tissues were obtained for determination of wet/dry weight ratio (W/D ratio). The pathological changes and ultrastructure of lung tissues were observed under light microscope, and lung injury was assessed and scored.The expression of TLR4 and NF-κB protein and mRNA was detected by Western blot and real-time polymerase chain reaction, respectively.Before administration (T₀), at the onset of one-lung ventilation (T₁), at 60 min of one-lung ventilation (T₂), immediately after the end of one-lung ventilation (T₃), at the end of operation (T₄) and at 24 h after operation (T₅), blood samples were collected from the internal jugular vein for determination of serum tumor necrosis factor-alpha, interleukin-6 (IL-6) and IL-8 concentrations by enzyme-linked immunosorbent assay. Results: Compared with group C, the W/D ratio and lung injury scores were significantly decreased, the expression of TLR4 and NF-κВ protein and mRNA was down-regulated, and the concentrations of tumor necrosis factor-alpha, IL-6 and IL-8 were decreased at T₂-T₅ in group PHC (P<0.05). The pathological changes and damage to ultrastructure of lung tissues were significantly attenuated in group PHC as compared with group C. Conclusion The mechanism by which PHC attenuates non-ventilated lung injury is related to blocking TLR4/NF-κВ signaling pathway and reducing inflammatory responses in the patients undergoing radical operation for lung cancer.

Adiponectin is an adipocyte-derived secretory protein. It was found to be associated with insulin resistance, inflammation and arteriosclerosis. To further study the biological function and expression of adiponection in vivo, adipoenctin gene knock-out and LacZ gene knock-in mouse model was constructed. Gene targeting strategy was designed to replace part of exon 2 and exon 3 of adiponectin gene with full length LacZ gene in frame with remaining upstream ATG and signal peptide sequence of exon 2. The targeting vector (Adipo-LacZ-XpPNT) was constructed and verified by restriction enzyme digestion and sequencing. CJ7 ES cells were transfected with targeting vector linearized by NotⅠ digestion, selected in the medium containing both G418 and ganciclovoir. Resistant clones were screened by PCR and further confirmed by Southern blot for correct homologous recombinants. Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts. After mating, mice heterozygous and further homozygous for adiponectin knockout and LacZ gene knock-in were established. Expression of both endogenous adiponectin and exogenous LacZ gene in mouse tissues and sera were detected by RT-PCR, Northern-blot, Western blot and ELISA. The results show that adiponectin was disrupted at both mRNA and protein levels. LacZ gene is expressed exclusively in adipose tissue of mutant mice. Its expression profile is identical to endogenous adiponection. Unexpectedly, LacZ activity could not be detected in both adipose tissue and serum although LacZ protein can be detected in adipose tissue but not in serum of mutant mice. In conclusion, mice homozygous for adiponectin knockout and LacZ gene knock-in have been successfully constructed. Mutant mice display LacZ expression profile identical to endogenous adiponectin albeit neither LacZ activity nor protein can be detected in serum of mutant mice.

Objective:To evaluate the role of histone deacetylase 6 (HDAC6) in spinal dorsal horn in dexmedetomidine-induced reduction of neuropathic pain (NP) in rats.Methods:Forty clean-grade healthy male Sprague-Dawley rats, aged 7-9 weeks, weighing 190-240 g, were divided into 5 groups ( n=8 each) using a random number table method: control group (group C), sham operation group (group SH), group NP, dexmedetomidine group (group D), and specific HDAC6 inhibitor ACY-1215 plus dexmedetomidine group (group AD). The animals were commonly fed without any treatment in group C. The sciatic nerve was only isolated but not ligated in group SH.The animals were anesthetized with intraperitoneal 10% chloral hydrate 350 mg/kg.NP was induced by chronic constrictive injury (CCI). The right sciatic nerve was exposed, and 4 loose ligatures were placed on the sciatic nerve at 1-mm intervals with 4-0 silk thread in NP and D groups.In group D, dexmedetomidine 40 μg/kg was intraperitoneally injected once a day starting from the end of operation until the animals were sacrificed.In group AD, ACY-1215 25 mg/kg was intraperitoneally injected every day immediately before CCI, and dexmedetomidine 40 μg/kg was intraperitoneally injected daily after CCI until 15 days after CCI.The equal volume of solvent was given instead of dexmedetomidine in S and NP groups.The mechanical paw withdrawal threshold to von Frey filament stimulation (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before CCI (baseline, T 0) and 3, 6, 9, 12 and 15 days after CCI (T 1-5). The rats were then sacrificed, and the dorsal horn tissues of L 4-6 spinal cord were obtained for determination of the expression of myeloid differentiation factor 88 (MyD88) and nuclear factor kappa B (NF-κB) p65 by Western blot. Results:Compared with group C and group SH, the MWT was significantly decreased, and the TWL was shortened at T 1-5, and the expression of MyD88 and NF-κB p65 in the spinal dorsal horn was up-regulated in NP, D and AD groups ( P<0.05). Compared with group NP, the MWT was significantly increased, and the TWL was prolonged at T 1-5, the expression of MyD88 and NF-κB p65 in the spinal dorsal horn was down-regulated in group D ( P<0.05), and no significant change was found in the parameters mentioned above in group AD ( P>0.05). Compared with group D, MWT was significantly decreased, and TWL was shortened at T 1-5, and the expression of MyD88 and NF-κB p65 was up-regulated in the dorsal horn of the spinal cord in group AD ( P<0.05). Conclusion:HDAC6 in spinal dorsal horn is involved in dexmedetomidine-induced reduction of NP in rats, which is related to inhibiting MyD88/NF-κB signaling pathway.

Objective To evaluate the effect of dexmedetomidine on retinal ischemia-reperfusion (I/R) injury in mice.Metbods Forty-eight C57BL/6 male mice,aged 8 weeks,weighing 20-24 g,were divided into 3 groups (n =16 each) using a random number table:control group (C group),I/R group and dexmedetomidine group (D group).The model of retinal I/R injury was established by elevating intraocular pressure for 60 min using anterior chamber cannulation followed by 24 h of reperfusion.At 15 min before ischemia and 5 min before reperfusion,dexmedetomidine 25 μg/kg was intraperitoneally injected in group D,and the equal volume of normal saline was intraperitoneally injected in C and I/R groups.Eight mice were sacrificed at 24 h of reperfusion,and the retina was removed for microscopic examination of pathologic changes (with light microscope) after haematoxylin and eosin staining and for determination of cell apoptosis in retinal tissues (by TUNEL).Apoptosis index (AI) was calculated.Eight mice were sacrificed at 24 h of reperfusion,and the retina was removed for determination of the expression of Bcl-2,Bax,caspase-3 and CCAAT/enhancer-binding protein homologous protein (CHOP) in retinal tissues (by Western blot).Bcl-2/Bax ratio was calculated.Results Compared with group C,AI was significantly increased,the expression of Bax,caspase-3 and CHOP was up-regulated,the expression of Bcl-2 was down-regulated,Bcl-2/Bax ratio was decreased (P＜0.05),and pathologic changes were found in retinal tissues in group I/R.Compared with group I/R,AI was significantly decreased,the expression of Bax,caspase-3 and CHOP was down-regulated,the expression of Bcl-2 was up-regulated,Bcl-2/Bax ratio was increased (P＜0.05),and pathologic changes of retinal tissues were significantly attenuated in group D (P＜0.05).Conclusion Dexmedetomidine can reduce retinal I/R injury,and the mechanism mav be related to inhibiting cell apoptosis in mice.

Objective To investigate the effect ofdexmedetomidine (Dex) on endoplasmic reticulum stress (ERS) and apoptosis in brain injury after asphyxiating cardiac arrest and cardiopulmonary resuscitation (CA/CPR) in rats.Methods A total of 60 clean male Sprague-Dawley rars were randomly divided into sham-operated group,CA/CPR group and Dex precondition group (n=20).Rats in the control group did not receive CA/CPR;and rats in the CA/CPR group and Dex precondition group were performed cardiac arrest induced by asphyxia,and then,CPR was performed.Dex with dose of 4.0 microgram/kg (body weight) was intravenously injected into rats in the Dex precondition group prior to 5 min of asphyxia.The same volume of saline by intravenous injection was given to rats in control group and CA/CPR group.Brain tissues were collected after the experiment,and wet to dry weight (W/D) ratio was tested.The mRNA expressions of CCAAT-enhancer binding protein homologous protein (CHOP),activation of transcription factor 4 (A TF4) and X-4 box binding protein 1 (XBP1) in the hippocampus were detected by reverse transcription-polymerase chain reaction (RT-PCR).The protein expressions of CHOP,B-cell lymphoma-2 (Bcl-2),Bcl-2 associated X protein (Bax) and cysteinyl aspartate specific proteinase 3 (caspase-3) in the hippocampus were measured by Western boltting.Neuronal apoptosis was detected by terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).Morphological and ultrastructural changes of brains of rats were observed by light microscopy and electron microscopy.Results As compared with the control group,CA/CPR group and Dex precondition group had significantly increased W/D ratio of brain tissues and mRNA expressions of XBP1,A TF4 and CHOP in the hippocampus,significantly higher protein expressions of CHOP,Bax and caspase-3,and statistically lower Bcl-2 expression (P＜0.05).As compared with the CA/CPR group,Dex precondition group had significantly decreased W/D ratio of brain tissues and mRNA expressions of XBP1,A TF4 and CHOP in the hippocampus,significantly lower protein expressions of CHOP,Bax and caspase-3,and statistically higher Bcl-2 expression (P＜0.05).TUNEL indicated that the neuronal apoptosis rate in the control group (7.49％±4.33％) was significantly lower than that in the CA/CPR group and Dex precondition group (29.73％±6.27％ and 16.82％±5.75％,P＜0.05);significant difference was noted between CA/CPR group and Dex precondition group in the neuronal apoptosis rate (P＜0.05).Changes in the morphology and ultramicrostructure injuries of brain tissues were more significant in CA/CPR group,while the changes were obviously alleviated in Dex precondition group.Conclusion Dex can alleviate brain injury after asphyxiating CA/CPR in rats,whose mechanism may be related to ERS and inhibition on apoptosis of nerve cells.