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Objective:Sodium urate was used to induce acute gouty arthritis rat model, and to observe the inflammatory response of rats and the intervention effect of diclofenac sodium on the expression of Toll-like receptor-related (TLR) protein of ankle joint.Methods:Thirty males specific pathogen free (SPF) grade Wistar rats were used to develop the models. Random number table method was used to divide the rats into normal saline control group, model group, and drug group (diclofenac sodium t 1.35 mg/g body weight), 10 rats in each group. After fully grinding the sodium urate crystals, an appropriate amount of saline and Tween-80 (9∶1) was added to make a suspension, and the sodium urate crystals (25 mg/ml) were injected to the right posterior ankle of the rats in the model and drug groups. The solution was 0.2 ml, and rats in the sham group were injected with 0.2 ml of normal saline at the same location. After the model was established, drug and equal volume of purified water were administrated intragastrically once a day for 7 days. The toe volume device was used to measure the joint swelling of the rat (at 4 h, 8 h, 24 h, 48 h, 72 h) , and blood was taken from the abdominal aorta after anesthesia to determine the rat kidney function, interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) content, the rat ankle joint TLR4, myeloid differentiation factor (MyD88), NF-κBp65 protein expression were determined using Western blot and immunohistochemical methods. Multiple comparisons were carried out using single factor analysis of variance (ANOVA), comparing the two groups by using LSD- t, the comparison of different times using repetitive measure analysis of variance (repeated measures). Results:After the models were established, the rat's right ankle joint showed various degrees of redness, slow walking, and unresponsiveness. Compared with the normal saline control group, under the light microscope, the ankle synovial cells of the model group proliferated, with localized degeneration and necrosis, and many inflammatory cell infiltration. The rat serum inflammatory factors IL-1β, IL-6, TNF-α in the diclofenac sodium group [(24.6±3.3) pg/ml, (151±21) pg/ml, (61±16) pg/ml] were significantly reduce compared with model group [(28.4±4.3) pg/ml, (173±26) pg/ml, (81±5) pg/ml] ( t=2.296, P<0.01; t=2.909, P<0.01; t=2.352, P<0.01). Compared with normal saline group, variance analysis showed that the NF-κBp65, MyD88, TLR4 protein expression of ankle joint detected by Western bolt method and immunohistochemistry method was significantly increased in the model group. Compared with the model group, diclofenac sodium the ankle tissue protein expression of NF-κBp65, MyD88, and TLR4 was significantly inhibited. There were statistical significances in three groups ( P<0.05 or P<0.001). Conclusion:The level of inflammatory factors in acute gout arthritis rats model induced by sodium urate crystals is increased, and the expression of TLR4/MyD88/NF-КBp65 proteins in ankle joint tissue is increased, which affects the TLR signaling pathway. Diclofenic has inhibitory and relieving effects.
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Prosthesis for femoral head surface replacement was designed. Between January 2002 and December 2005,28 patients underwent femoral head surface replacement in Anyang People's Hospital. The diameter of the normal femoral head was determined by CT,and the prosthesis was selected according to the measured value ±1 mm. With the muscle gap approach,the center of the femoral head was determined according to the measured distance between the femoral head center and the culminated point of the round ligament fossa based on the mark of the culminated point of the round ligament pit. Additionally,the diameter of the femoral head was measured again to ensure the precise match of the prosthesis and acetabulum. X-ray films at 24-48 months show no loosening or dislocation of the prosthesis,which was well matched with acetabulum. Furthermore,the joint space did not remarkably change. The mean score was 63 before operation and 91.5 after operation respectively according to Harris sore. The successful rate is 92.86% (excellent in 23cases,good in 3,and improved in 2). The results show that femoral head surface replacement is an effective method for young patients with avascular necrosis of the femoral head at the stage of ARCO HI.
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The influence of L-arginine on endothelial nitric oxide synthase (eNOS) and cyclooxygenase 2 (COX2) was observed in experimental pulmonary thromboembolism and the action mechanism on pulmonary thromboembolism was explored. Wistar rats were randomly divided into control group, model group and treatment group. Pulmonary thromboembolism models were established by auto-blood back transfusion, and L-Arg 100 mg/kg was intraperitoneally injected after successful model preparation. The animals were sacrificed at 3 h, 1 day, 3 days and 7 days after embolism.Plasma NO, TXB2 and 6-Keto-PGF1α were detected. The expression of eNOS and COX2 protein and mRNA in pulmonary tissues was detected by immunohistochemistry and RT-PCR respectively.The results showed that pulmonary thrombosis could be seen post pulmonary embolism and inflammatory reaction was significant. Plasma NO was decreased (P＜0.01), and the levels of TXB2,6-Keto-PGF1α and T/P ratio were all elevated. The expression of eNOS protein and mRNA in the pulmonary tissue was down-regulated (P＜0.05), while that of COX2 protein and mRNA was upregulated (P＜0.01). In treatment group, the level of NO was increased, the levels of TXB2 and T/P ratio were decreased, but the level of 6-Keto-PGF1 α was increased. The expression of eNOS protein and mRNA in pulmonary tissue was upregulated (P＜0.05), while that of COX2 protein and mRNA was down-regulated (P＜0.05). In conclusion, L-arginine can educe the role of pulmonary tissue protection through up-regulating the expression of intra-pulmonary NOS and down -regulating COX2 in pulmonary thromboembolism.
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The influence of L-arginine on endothelial nitric oxide synthase (eNOS) and cyclooxygenase 2 (COX2) was observed in experimental pulmonary thromboembolism and the action mechanism on pulmonary thromboembolism was explored. Wistar rats were randomly divided into control group, model group and treatment group. Pulmonary thromboembolism models were established by auto-blood back transfusion, and L-Arg 100 mg/kg was intraperitoneally injected after successful model preparation. The animals were sacrificed at 3 h, 1 day, 3 days and 7 days after embolism. Plasma NO, TXB2 and 6-Keto-PGFla were detected. The expression of eNOS and COX2 protein and mRNA in pulmonary tissues was detected by immunohistochemistry and RT-PCR respectively. The results showed that pulmonary thrombosis could be seen post pulmonary embolism and inflammatory reaction was significant. Plasma NO was decreased (P<0.01), and the levels of TXB2, 6-Keto-PGF1alpha and T/P ratio were all elevated. The expression of eNOS protein and mRNA in the pulmonary tissue was down-regulated (P<0.05), while that of COX2 protein and mRNA was upregulated (P<0.01). In treatment group, the level of NO was increased, the levels of TXB2 and T/P ratio were decreased, but the level of 6-Keto-PGF1alpha was increased. The expression of eNOS protein and mRNA in pulmonary tissue was upregulated (P<0.05), while that of COX2 protein and mRNA was down-regulated (P<0.05). In conclusion, L-arginine can educe the role of pulmonary tissue protection through up-regulating the expression of intra-pulmonary NOS and down -regulating COX2 in pulmonary thromboembolism.
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Objectives To construct a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) and clone differentially expressed genes related to DPCs in anagen. Methods Total RNA was isolated from DPC of anagen and telogen follicles. Then ds cDNAs were synthesized in turn using SMART cDNA synthesis technique. After cDNAs from anagen and telogen follicle DPCs were hybridized with each other twice and underwent two rounds of nested PCR, PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library. Selected clones were verified by reverse Nothern blot and DNA sequencing, and the acquired sequences were analyzed for homology based on Genbank nucleotide database. Results cDNA subtractive library of DPCs in anagen follicle was set up successfully with high subtractive efficiency. Thirty-five genes were identified with 22 known functional genes and 13 unknown functional genes. Conclusions These results demonstrate the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression might be useful for elucidating the genetic events in hair follicle growth regulation.
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Objective To identify the expression of gene HSPC011(HSPC,hematopoietic stem/pro-genitor cell)in the dermal papilla cells of hair follicle and clone its cDNA.Methods The expression of gene HSPC011was confirmed by intracellular mRNA hybridization in situ;the objective cDNA was amplified by RT-PCR(reverse transcription PCR).Results By in situ hybridization,it was shown that gene HSPC011expressed in the coagulated dermal papilla cells,but not in the non-coagulated dermal papilla cells and the dermal fibroblast;The objective cDNA with a fragment of430bp was amplified by RT-PCR,and a recombi-nant eukaryotic expressing plasmid pCI-neo/HSPC011was constructed.By enzyme cutting and sequencing analysis,the objective cDNA was completely identical with gene HSPC011.Conclusion Gene HSPC011was clearly shown to express in the dermal papilla cells,and the expression of HSPC011was possibly related with the differentiation and functional status of the dermal papilla cells.
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Objective To screen and analyze genes differentially expressed within dermal papillae cells(DPC)with aggregative behavior.Methods Total RNA was extracted from DPC with and without ag-gregative behavior,and double-stranded cDNA were synthesized by using SMART cDNA synthesis.The cD-NA fragments of differentially expressed genes in dermal papillae cells with aggregative behavior were isolat-ed by suppression subtractive hybridization,sequencing,and then subtracted library was set up.Positive clones were screened by PCR method and verified by cDNA dot blot and then analyzed through homologous retrieving.Results A subtractive cDNA library of DPC with aggregative behavior was successfully construct-ed.The results of screening and cloning of the library showed that DPC with aggregative behavior could ex-press genes related to homologous aggregation,regnlation of growth,differentiation and development,and sig-nal transduction proliferation and cycle control,which included known genes(capping protein,paladin,vas-cular endothelial growth factor),hematopoietic stem/progenitor cells(HSPC)related genes(HSPC011and HSPC016)and a new gene.Conclusions The construction of subtractive library of DPC lays solid founda-tion for screening and cloning new and specific genes related to aggregative behavior of DPC.Several genes may cooperatively involve in homologous aggregation,and regnlation of growth of DPC.Among these genes,capping protein and palladin may be closely related to aggregative behavior of DPC,and VEGF and HSPC re-lated clones may be responsible for the status of higher proliferation of DPC.
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Objective: To observe the efficacy of azithromycin in treating the bacterial infectious diseases. Methods: Intravenous administration of azithromycin was carried out in 40 cases. Results: The clinical cure rate and eradication rate were 85.0% and 84.8%, respectively. Drug sensitivity tests showed that more isolates were sensitive to azithromycin (87.9%) than to erythromycin (42.4%). The MIC of azithromycin was lower than that of erythromycin. In addition, the adverse effects occurred with low frequency and were usually mild. Conclusion: Azithromycin is effective and safe in treating bacterial infectious diseases.
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Objective To establish a new method for detecting pemphigus antibody (PAb). Methods PI 1 anti idiotype monoclonal antibodies against pemphigus and purified PAb from the serum of a patient with active pemphigus were used to establish an ELISA system for detecting the PAb. Results The result showed the purified PAb was IgG4 subclass, the sensitivity and specificity for the detection of PAb were high in the ELISA system, the sensitivity and specificity were not significantly different among the IIF, indirect ELISA and ABC ELISA. The standard curve for detecting the concentration of the purified PAb was primarily obtained in the study. Conclusion The ELISA system for the detection of PAb in the sera of patients is a good qualitative method, it might be of value in the clinical diagnosis of pemphigus. It is expected to quantitatively detect PAb of pemphigus patients in the future with the ELISA system established, which is directed to the IgG4 subclass of PAb, so it may be of value in the study of IgG4 subclass in the pathogenesis of pemphigus.
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Objective To introduce the amino acid substitution for HP V16E749-57(HLA-A2-restricted CTL epitope) and to identify the novel epitopes.Me thods Quantitative method was used to evaluate the affinity of the substituted peptides.To determine the peptide candidates to be synthesized and identified,the molecular models of the HLA-A2-peptide complex and CTL epitope candidates b ound to the HLA-A2 molecule were established by computer molecular modeling.Pep tides were synthesized and purified with standard Fmoc assay,lactate dehydrogen ase (LDH) release assay was used to determine their abilities of inducing the ge neration of specific CTLs.Results Modified peptides met the requirements of H LA-A2-restricted CTL epitopes.Peptide RLHYNIVTF had the abilitiy of inducing th e generation of specific CTLs.Conclusions Compared with HPV17E749-57 the mod ified peptide RLHYNIVTF has a higher antigenicity and affinity to HLA-A2.So,pe ptide RLHYNIVTF may be used as one of the HLA-A2-restricted candidate epitopes,instead of HPV17E749-57,for peptide vaccine in the treatment of HPV infection.
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Objective To identify the relationship between expression of hematopoeitic stem/progenitor cell(HSPC)-related gene HSPC016 and aggregative behavior of the dermal papilla cells of hair follicles. Methods The aggregated human dermal papilla cells, non-aggregated human dermal papilla cells and the human dermal fibroblasts were used in this study. Expression of HSPC016 mRNA was investigated in the three cell groups by intracellular mRNA hybridization in situ and RT-PCR. Results By in situ hybridization, it was shown that gene HSPC016 specifically expressed in the aggregated dermal papilla cells, but not in the non-aggregated human dermal papilla cells and human dermal fibroblasts. A 200bp fragment of target cDNA was amplified from RNAs of the aggregated human dermal papilla cells by RT-PCR, but could not from RNAs of other two cell lines. Conclusions Gene HSPC016 was specifically expressed in the aggregated dermal papilla cells, and expression of HSPC016 might be related to the differentiation and functions of dermal papilla cells.
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Objective To clone, express and purificate human MRPS17 cDNA in Escherichia coli (E. coli). Methods MRPS17 cDNA was obtained from total RNA isolated from primary cultured human hair papillary cells by RT-PCR and sequencing method, and then it was inserted into prokaryotic expression vector pET28a for the IPTG-induced expression in E. coli BL21 (DE3). The expression product fused with 6?His at C-terminal was analyzed by Western blotting, and purified by using Ni 2+-NTA ion exchange resin. The purity of MRPS17 protein was analyzed by SDS-PAGE. Results Human MRPS17 cDNA was obtained, and the expression plasmid pET28a-MRPS17 was constructed successfully. Western blot analysis showed that human MRPS17 with 13kD molecular weight was expressed in E. coli at 20℃. The purity of the recombinant MRPS17 was more than 90% after purification using Ni 2+-2NTA ion exchange resin. Conclusion The sequence of MRPS17 cDNA was consistent with the known sequence from the Genbank. MRPS17 is successfully induced and expressed in E. coli.