Human sTNFR1 (soluble tumor necrosis factor receptor 1) gene was amplified by RT-PCR from Hela cells. A recombinant expression vector of sTNFR1-MBP was constructed in pMAL-c2x, and transformed into E. Coli JM109.It was sequenced and confirmed to be identifical to the sTNFR1 gene in data bank. Recombinant protein sTNFR1-MBP was induced by IPTG and purified by Amylose resin Affinity Chromatography. sTNFR1-MBP was binded to sTNFR1's antibody in Western-blotting. From MTT assays, the results showed that sTNFR1-MBP could effectively block the cytotoxicity mediated by TNF?on QSG7701 cells. Annexin V-FITC staining and flowcytometry were used to observe the recombinant protein's anti-apoptosis capacity and the recombinant protein has marked anti-apoptosis effect in vitro.sTNFR1-MBP had good biological activity and it will be employed in further study.

OBJECTIVE: To investigate the effect of diazepam and modafinil on acute hepatic failure in mice. METHODS: Acute liver failure was induced in male Kunming strain mice by enterocoelia injecting the mice with D-GalN and LPS . The mice in the treatment groups were given corresponding drug 2 h before the administration of D-GalN and LPS, and the mice in the control group were given the same dose of distilled water. The 24-hour survival rate, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were compared. Serum levels of TNF-alpha and IL-1 and the levels of SOD, MDA, GR, GSH, NO and NOS in the liver were determined. RESULTS: Treatment with diazepam increased the survival rate and improved liver histological feature. Diazepam inhibited the serum levels of ALT, AST, TNF-alpha and IL-1, and reduced levels of MDA, NO and NOS and increased levels of GR and SOD in the liver. Modafinil decreased liver histological feature, increased the serum levels of ALT, AST, TNF-alpha and IL-1, increased level of MDA, and inhabited levels of SOD and GR in the liver. CONCLUSION Treatment with diazepam may suppress the D-GalN/LPS-induced acute hepatic failure and modafinil may facilitate the acute hepatic failure.
Animals ; Benzhydryl Compounds ; adverse effects ; therapeutic use ; Diazepam ; adverse effects ; therapeutic use ; Galactosamine ; Lipopolysaccharides ; Liver ; pathology ; Liver Failure, Acute ; chemically induced ; drug therapy ; pathology ; Male ; Mice ; Modafinil ; Random Allocation

BACKGROUND: Platelet-rich plasma (PRP) and naringin can both promote proliferation and induce osteogenic differentiation of mesenchymal stem cells. However, their combined use is rarely reported. OBJECTIVE: To observe the effect of PRP combined with naringin on the osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)in vitro. METHODS: BMSCs at passage 3 were divided into four groups: (1) blank control group, cells were cultured in α-MEM; (2) PRP group, cells were cultured in α-MEM containing PRP; (3) naringin group, cells were cultured in α-MEM containing naringin; and (4) combined group, cells were cultured in α-MEM containing PRP and naringin. The contents of used PRP and naringin were 12.5% and 50 μg/L respectively. Cell proliferation was detected by MTT assay. Expression of related genes in hBMSCs was detected by RT-PCR. Alkaline phosphatase staining, collagen type I immunohistochemical staining, and alizarin red staining were used to analyze the osteogenic differentiation of hBMSCs. RESULTS AND CONCLUSION: The proliferation of hBMSCs was increased in each group, especially in the combined group. Cells in all the groups except the blank control group were positive for alkaline phosphatase staining, collagen type I immunohistochemical staining, and alizarin red staining, and the positive effect was more obvious in the combined group. However, negative or weakly positive response was found in the blank control group. At 7 and 14 days, the expression of alkaline phosphatase and collagen type I was significantly higher in the PRP, naringin and combined groups than the blank control group (P < 0.05); at 14 days, the expression of alkaline phosphatase and collagen type I was significantly higher in the combined group than the PRP and naringin groups (P < 0.05). To conclude, PRP combined with naringin can promote the proliferation of hBMSCs and induce the osteogenic differentiation of hBMSCs. Moreover, there is a synergistic effect between PRP and naringin.

Objecfive To observe the ability of dengue virus type 1-4 envelope domain Ⅲ fusion protein to inhibit virus infection and analyze the neutralizing ability of polyclonal antibodies against rEⅢ.Methods After being connected by linker peptide.EⅢ protein of Dengue virus serotypes 1-4 were expressed in E coli BL21(DE3) then purified.Fusion proteins were verified by Western Blot and ELISA.Rabbits were immunized with fusion proteins to produce anti-rE Ⅲ serum.The activity of anti-rEⅢ serum were detected through indirect immunofluorescence assay test.Inhibition of dengue virus type 1 to 4 infection in BHK-21 cells by rEⅢ fusion protein were tested.Neutralizing activity of anti-rEⅢ serum was analyzed.Results Dengue virus type 1 to 4 envelope domain Ⅲ recombinant fusion protein was expressed in Ecoli BL21 and purified successfully.Then rEⅢ fusion protein and anti-rEⅢ serum were analyzed respectively and rEⅢ fusion protein can effectively inhibit dengue virus type 1 to 4 from infecting BHK cells.The anti-rE Ⅲ serums can neutralize dengue virus type 1 to 4 but with different neutralizing titer.Conclusion Dengue virus type 1-4 envelope domain Ⅲ fusion protein can directly inhibit DV infeetion.Antibodies induced by rE Ⅲ fusion proteins can neutralize dengue virus type 1-4.

OBJECTIVEA proteomics approach was applied for finding multi-drug resistance (MDR) related proteins in hepatic cancer cell lines.

METHODSTwo-dimensional electrophoresis (2-DE) was used to separate the total proteins of vincristine-resistant hepatic cancer cell line HepG2/VCR and its counterpart HepG2. The differential expression proteins between the two cell lines were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Heat-shock protein 27 (HSP27), one of the differential expression proteins in the development of MDR of HepG2/VCR, was analyzed by HSP27 antisense oligonucleotides (ASOs).

RESULTSAs a high expression protein in HepG2/VCR, HSP27 was identified, and the suppression of HSP27 expression by HSP27 ASO enhanced the vincristine chemosensitivity of HepG2/VCR (P less than 0.05).

CONCLUSIONHSP27 is linked to MDR in human hepatic cancer cell line HepG2/VCR.

Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; HSP27 Heat-Shock Proteins ; metabolism ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism

OBJECTIVESTo study the relationship between quasispecies of hepatitis B virus and the clinical manifestations of their infection, and to find the answer of why different quasispecies of HBV with the same genotype can induce different clinical situations.

METHODSSixty serum samples, in all of which HBVs of genotype B exist, taken from 32 chronic asymptomatic carriers and 28 severe chronic hepatitis patients, were collected to detect quasispecies of HBV DNA by melt curve approach. Then the relationship between quasispecies of HBV of the same genotype and the clinical situation of their infection was studied by comparing the wave crests of the two sample groups.

RESULTSThe data of the 60 serum samples of HBV of genotype B detected by melt curve showed that HBV DNA in severe chronic hepatitis patients had more wave crests than that in chronic asymptomatic carriers (P < 0.05), suggesting that HBV in severe chronic hepatitis patients had more quasispecies than in the chronic asymptomatic carriers.

CONCLUSIONThe numbers of quasispecies of HBV correlate with the clinical situations of their infection. In the patients infected by HBV of the same genotype, those who have more HBV quasispecies would have more severe clinical manifestations.

Adolescent ; Adult ; Child ; Female ; Genotype ; Hepatitis B virus ; classification ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Middle Aged

Out of the 20 channels in the channel and collateral system, only 5 enter the brain, with unclear circulation pathway in the brain. Electrophysiologic and imaging studies indicate that the signal induced by acupuncture at acupoints can enter the brain no matter whether the channel connecting the acupoint enters the brain. Therefore, the authors put forward the hypothesis of "all the 12 channels enter the brain", i.e., the hypothesis of "channels and collaterals in brain". In the theory system of channels, less channels enter the brain with unclear circulation pathway. This possibly is related with that sensation is main way for descovery of channels. In future, we should adopt modern scientific and technical ways and strengthen the study on circulation of channels in the brain, so as to perfect the channel theory.
Acupuncture Therapy ; Brain ; physiology ; Humans ; Medicine, Chinese Traditional ; Meridians

Objective:To study the expression of MMP1 and TIMP1 in simple and radiation-combined wound healing and their effects on the healing process and tissue remodeling. Methods: A rat model of radiation-combined wound healing was used. Immunohistochemistry and in situ hybridization were performed which enabled the detection of MMP1 and TIMP1 expression in the healing process. Ultrastructural changes were observed with transmission EM. Results: The wound healing process was impaired and delayed. In rats receiving 25 Gy of gamma ray locally the irradiated wounds healed 6 days later than non-irradiated controls. The following changes in MMP1 and TIMP1 expression were found: (1) In the early inflammatory phase and in the period of granulation tissue formation, MMP1 expression in the newly-formed epidermis of irradiated wounds approximated that in the controls. Later, the epidermal expression of MMP1 in radiation wounds was comparatively increased with the delay of the healing process.On days 3 to 14 after wounding, TIMP1 was weakly positive in the proliferating keratinocytes of control wounds and became negative after epidermal covering, whereas no or only slight epidermal expression was detected in radiation wounds before epidermal covering.(2)MMP1 and TIMP　1 expression in radiation wounds was markedlydecreased in fibroblasts　, endotheliocytes and macrophages as compared with the controls. The expression phase was prolonged due to the delay of the healing process.Conclusions:The reduced expression of MMP1 and TIMP1 in granulation tissue retards such important processes as cell migration, angiogenesis and tissue remodeling, thus retarding the healing process. The expression of MMP1 in the newly-formed epidermis may help the process of reepithelialization,but in the late healing period, overexpression of MMP1 and decreased expression of TIMP1 in the epidermis may hinder the establishment of basal membrane and the formation of granulation tissue, and thus affect the matrix remodeling process.

Carboxypeptidase B is a metalloenzyme, which is widely used for commercial and research purposes. Commercially available CPB purified from porcine or bovine pancreas is very expensive, and is not totally free from other proteases. In order to express the rat proCPB in Pichia pastoris, total RNA extracted from SD rat pancreas cells was reversely transcripted to synthesize cDNA, and the proCPB ORF was synthesized by PCR. After digestion with Xho I and EcoR I , the fragment was inserted into pPIC9, and the recombinant plasmid was named as pPIC9-proCPB. By digestion with Sac I , the lined pPIC9-proCPB was transformed into Pichia pastoris strains GS115 with PEG1000 and integrated into their genomes. In the inducement of methanol, recombinant proCPB was successfully expressed in Pichia pastoris, and could be secreted into the supernatant in the culture. After optimizing the fermentation conditions, a higher production could be obtained when GS115-proCPB was induced in BMGY (pH6.0) at 28CC, with addition of 0.5% casein. The yield of recombinant protein reached 500mg/L, achieving over 94% of total protein in the culture supernatant. The purity of recombinant CPB can reach 96% after two step phenyl sepharose F F purification, and 38% of total protein can obtained after optimizing the pufication method. Comparing to the specific activity 180u/mg of CPB purchased from Sigma, the specific activity of recombinant CPB is 110u/mg. Mass spectrometry analyses showed the mass of the recombinant CPB was 35.1 kD, which is very close to the theory value 35.2 kD. Amino acid sequencing of N-terminal of recombinant CPB further indicated proCPB was expressed successfully and modificated correctly after translation.
Animals ; Carboxypeptidase B ; genetics ; isolation & purification ; metabolism ; Catalysis ; Cattle ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Enzymologic ; Kinetics ; Mass Spectrometry ; Molecular Weight ; Pichia ; genetics ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; chemistry ; isolation & purification ; metabolism ; Sequence Analysis, Protein ; Substrate Specificity ; Swine

Apoptosis ; drug effects ; HeLa Cells ; Hepatocytes ; cytology ; drug effects ; Humans ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Transfection ; Tumor Necrosis Factor-alpha ; pharmacology