Objective To investigate the regulative role of N-Acetyl-D-glucosamine(GlcNAc) on the stressed mice macrophages function.Methods The stressed mice model was established by electric footshock method.The mice were divided into 5 groups:normal control group,stressed mice model group,low dose Glc-NAc treatment group(0.25 ml 15% GlcNAc),medium-dose GlcNAc treatment group(0.5 ml 15% GlcNAc) and high-dose GlcNAc treatment group(1 ml 15% GlcNAc).GlcNAc was intragastrically injected to corresponding mice 2 h before the electrical stimulation.Peritoneal macrophage(PM?) phagocytosis capability was detected by phagocytosis saccharomycete assay,and PM? energy metabolism was detected by MTT assay.Results Compared with normal control group,stressed mice PM? phagocytosis capability was significantly lower(P

Objective To evaluate the antitumor effect of oncolytic adenovirus expressing human IL-18 gene on malignant melanoma implanted in nude mice.Methods BALB/c nude mice were subcutaneously inoculated with A375 cells to establish a model of malignant melanoma.When the volume of implanted tumor reached 100-150 mm3,murine models were randomly divided into three groups to receive a 3-day intratumoral injection of IL-18 gene-expressing human oncolytic adenovirus named ZD55-IL-18,IL-18 gene-expressing adenovirus named Ad-IL-18,phosphate buffer saline(PBS),respectively.The tumor size was measured at an interval of 4 days for 9 weeks.Hematoxylin and eosin (HE)staining was performed to observe the morphological changes of tumor cells.The protein expression of IL-18 and E1A.microvessel density in tumor tissue,and apoptosis of tumor xenografts were detected by immuno fluorescence assay,immunohistochemistry and in situ end labeling technique (TUNEL).respectively.Results The treatment with ZD55-IL-18 significantly inhibited the growth of tumor.Forty-four days after the treatment,the mean tumor volume was 1039.378±29.67 mm3 in ZD55-IL-18-treated mice.significantly smaller than that in Ad-IL-18 treated mice(2900.46±62.65 mm3)and PBS-treated mice(3980.24±63.78 mm3).HE staining showed that the nuclei of tumor cells were heavily stained with few nucleoli in ZD55-IL-18-treated mice.Increased positivity rate of IL-18 was noticed in ZD55-IL-18-treated mice vs.AD55-IL-18-treated mice(83.4%±3.2%vs 24.4%±2.1%.P＜0.01).Moreover,immunofluorescence assay revealed the presence of E1A protein in tumor tissue.A decrease was found in the microvessel density in ZD55-IL-18-treated mice compared with the PBS-treated mice(P＜0.01).The apoptosis rate in tumor cells from high to low was 86.28%±3.25%in ZD55-IL-18-treated mice,43.67%±3.46%in Ad-IL-18-treated mice,and 10.73%±2.48%in PBS-treated mice;there was a significant difference between the three groups(all P＜0.05).Conclusion The oncolytic adenovirus expressing human IL-18 gene,ZD55-IL-18,has a significant inhibitory effect on the growth and metastasis of malignant melanoma implanted in nude mice.

Objective To study the effects of oncolytic adenoviruses ZD55 harboring IL-24 gene (ZD55-IL-24) on the apoptosis of human melanoma cell line A375. Methods The oncolytie adenoviruses ZD55-IL-24 were verified by PCR. Then, the viruses were propagated, purified, and titrated by HEK293 cell plaque assay. A375 cells were cultured, divided into three groups transfected with ZD55-1L-24, ZD55 fused with enhanced green fluorescent protein (ZD55-EGFP), and replication-deficient adenovirus ZD55 carrying IL-24 gene (AD-IL-24), respectively. The multiplicity of infection was 0.1, 1, 10 and 100, respectively.Subsequently, the eytotoxity of these viruses and proliferation of A375 cells were determined by crystal violet staining and methyl thiazolyl tetrazolium (MTT) assay, respectively. The expressions of EIA and IL-24 protein were detected by Western blot in A375 cells. Results PCR verified that the adenoviruses ZD55-IL-24 contained IL-24 gene without wild adenovirns contamination. Crystal violet staining revealed that ZD55-IL-24 had an obvious eytotoxic effect on A375 cells, and MTT assay indicated that ZD55-IL-24 inhibited the proliferation of A375 cells in a time-and concentration-dependent manner. As shown by Western blot analysis, ZD55-1L-24 expressed IL-24 and E1A protein in A375 cells with a high efficiency. Conclusions The oncolytic adenoviruses ZD55-IL-24 can efficiently express IL-24 gene, inhibit the proliferation of, and induce the apoptosis in A375 cells.

Adult ; Fracture Fixation, Internal ; Fractures, Bone ; complications ; surgery ; Humans ; Joint Dislocations ; surgery ; Male ; Middle Aged ; Talus ; injuries ; surgery ; Treatment Outcome ; Young Adult

Objective:To study the role of Jagged1 and Notch signaling pathway played in the differentiation and proliferation of RAW 264.7 cells.Methods: RAW 264.7 cells were divided into three groups to culture:The control group:RAW 264.7 cells were threated with culture and RANKL.The Jagged1 group:RAW 264.7 cells were threated with recombinant protein Jagged 1 besides the control group.The DAPT group:RAW 264.7 cells were threated with DAPT besides the Jagged 1 group.The mRNA expression of osteoclast markers(TRAP,CK,CTR) and Notch key target genes (HES-1 and HEY-1) were measured by real-time PCR.The formation of osteoclast , bone resorption , Notch expression and proliferation of RAW 264.7 cells were detected by TRAP staining , scanning electron microscope ,immunofluorescence and cell counting kit-8 ( CCK-8 ).Results: TRAP, CK, CTR , HES-1 and HEY-1 mRNA expression were significantly higher than the control group and DAPT group in Jadded 1 group ( P<0.05 ).TRAP+cell count ,osteolytic area was significantly increased in Jagged 1 group compared with control and DAPT group , and no significant difference observed between the last two groups.Immunofluorescence results showed high expression of N ICD in cell membrane and cytoplasm in all groups and additionally expressed in nucleus in Jadded 1 group.Cell proliferation was inhibited in Jagged 1 group also ( P<0.05 ).Conclusion:Jagged1 promotes RAW264.7 cells osteoclast differentiation and inhibits proliferation by activating Notch signaling pathway .

Abstract:Objective To investigate the protective effects of blocking CD40/CD40L interactions with human CD40-Ig fusion protein in a murine graft-versus-host disease model.Methods Human CD40 gene extracellular region was inserted into plasmid pIG1, which contains genomic human IgG1 Fc gene. A transient vector containing CD40-Fc fusion gene was transfected into COS-7 cells. The CD40-Ig fusion protein was detected through enzyme-linked immunosorbent assay (ELISA). A constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1 and transfecting it into CHO cells. CD40-Ig was purified by protein A affinity chromatography. SDS-PAGE, Western blot and ligand binding assay were used to identify the qualities of CD40-Ig. Murine acute graft-versus-host disease (GVHD) was induced by intravenous injection of C57BL/6J (H-2b) spleen cells into sub-lethally irradiated BALB/c (H-2d) mice. Protective effects against murine graft-versus-host disease by in vivo administration of CD40-Ig were evaluated.Results Mammalian expression vectors pIG/40Ig and p3.1/40Ig were constructed as described above. Chimeric proteins were expressed in COS-7 and CHO cell culture supernatant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion proteins had a disulfide-bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L. In vivo administration of CD40-Ig could prevent the development of GVHD and significantly prolong the mean survival time of mice with graft-versus-host disease.Conclusions These results demonstrate that CD40/CD40L interactions play an important role in the pathogenesis of graft-versus-host disease and suggest clinical potential for CD40-Ig in the prevention and treatment of human graft-versus-host disease.

OBJECTIVEObserving the dynamic change characteristics of serum liver function indexes in occupational dermatitis medicamentosa-like of trichloroethylene patients with liver damage, we can underlie for guiding therapy, prognosis and mechanism of dermatitis medicamentosa-like of trichloroethylene patients with liver damage.

METHODSWe collected serum of 10 cases of occupational dermatitis medicamentosa-like of trichloro-ethylene patients with liver damage from different time points since they were hospitalized, using automatic biochemistry analyzer to detect total protein (TP), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), albumin/globulin ratio etc 11 liver function biochemical indicators. We used Excel to establish database, professional drawing software gnuplot to draw dynamic variation diagram of each index.

RESULTSThe variation range of 11 liver function indexes of 10 cases was TP 43.2-74.2 g/L, ALB 24.6-44.6 g/L, A/G 0.77-2.10, TBIL 3.7-268.2 umol/L, DBIL 1.0-166.0 umol/L, IBIL 2.4 -167.5 umol/L, ALT 11-5985 U/L, AST 14-5586 U/L, GGT 15-1500 U/L, ALP 35-309 U/L, S/L 0.07-1.94, respectively. TBIL, DBIL, ALT, AST, GGT, ALP concentration significantly increased, especially ALT, AST, GGT, ALT topped 5985 U/L, AST topped 5586 U/L, GGT topped 1500 U/L. But TP, ALB and S/L significantly decreased, TP lowest to 43.2 g/L, S/L lowest to 0.07. A/G basically remained unchanged, but IBIL didn't change regularly.

CONCLUSIONThe early liver damage in dermatitis medicamentosa-like of trichloroethylene patients was serious, and repeatedly attacked, so we should lead to enough attention to the clinical work and prevention. This also provided the basis for studying the mechanism of trichloroethylene poisoning.

Adolescent ; Adult ; Bilirubin ; blood ; Dermatitis, Occupational ; blood ; physiopathology ; Female ; Humans ; Liver ; enzymology ; physiopathology ; Liver Function Tests ; Male ; Trichloroethylene ; Young Adult

By using the cell density, cell viability, size distribution of cell aggregates,specific consumption rate of glucose (q_ glc ), specific production rate of lactate (q_ lac ), lactate transform rate (Y_ lac/glc ) and amino acids utilization as the evaluation indexes, the growth and metabolism of HEK293 cells under carrier-free immobilization culture mode were examined and compared with those of HEK293 cells cultured in static tissue flasks. It was found that HEK293 cells grown as suspended cell aggregates in spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells in stationary anchored culture, and HEK293 cells under carrier-free immobilization culture mode as suspended aggregates in stirred bioreactor facilitate perfusion performance and increase unit productivity. Cultivation of HEK293 cells in carrier-free immobilization culture mode has potential for further improving mammalian cells culture technique.

Objective To study the apoptosis of facial motor neurons and the expression of apoptosis-related genes, Bcl-2 and Bax, in the animal model of viral facial paralysis. Methods Total of 84Balb/c mice were divided into viral inoculation group and nerve transaction group. The animals were executed 1, 3, 7, 10, 15, 20 and 30 days after being operated respectively. The histopathological features of facial neurons in brain stem were observed by HE and Nissl stain. The changes of facial neuronal apoptosis were observed by TUNEL The changes of expression of Bcl-2 and Bax genes in facial neurons were observed by immunohistochemistry staining. Results After nerve transection, increased apoptotic cells were found in homolateral facial motor neucleus and the peak appeared at 10 and 15 days. The level of Bcl-2 expression inneurons declined while the expression of Bax increased gradually. Correspondingly, the ratio of Bcl-2/Bax declined. In the viral inoculation group, no visible change of apoptosis and Bax expression, but the level of Bcl-2 and the ratio of Bcl-2/Bax increased gradually. Conclusions Comparing to axotomy, facial motor neucleus in HSV-1 infective animal model are free of apoptosis. Both the mild form of lesion and the ability to block apoptosis of HSV-1 are likely to be involved into the phenomenon. Bcl-2 and Bax might interfere with the apoptotic response.

CD40/CD40L, besides B7/CD28, is an alternative important costimulation signal transduction pathway. It plays a pivotal role in T cell activation. Moreover, it may play a critical role at many levels of sensitization and effector phases of allograft rejection. In order to get the fusion protein of human CD40 extracelluar region and IgG 1 Fc fragment, and investigate the potential role of blocking CD40/CD40L costimulation pathway in immunotherapy, total RNA was extracted from human lymphoma cell line Daudi, and CD40 gene extracelluar region was amplified by RT-PCR. The PCR products were inserted into pGEM T Easy vector, and the cloning vector pGE40 was obtained. The DNA sequence was analyzed by automatic DNA sequencer. After sequencing, the transient expressing vector was constructed by inserting correct fragment into pIG vector, which contains the genomic human IgG1 Fc (hinge, CH2 and CH3) gene. Hence the recombinant fusion expression vector was constructed successfully, and named after pIG/40 Ig. Then, COS-7 cells were transfected through DEAE-Dextran/chloroquine method. The CD40-Ig fusion protein expressed in COS-7 cell culture supernatant was identified by sandwich ELISA and Western blot. Result showed that the CD40-Ig fusion protein can be detected by sandwich ELISA in the cell culture supernatant. Western blot analysis also showed that it could react with McAbs of mouse anti-human CD40 G28-5 and mouse anti-human Ig gamma chain. There is only one obvious band at the position of relative molecular weight 50 kD, and it is equivalent to the expected value. Above all, the recombinant fusion expression vector pIG/40 Ig was constructed, and CD40-Ig fusion protein gene was expressed in COS-7 cells successfully. It could be laid a foundation to investigate the potential role of CD40/CD40L pathway as the target of GVHD prevention and therapy.