Objective To investigate the protective effect of Lycium barbanun glycopeptide(LbGP)on human keratinocyte HaCaT cells against radiation-induced cytotoxicity and its mechanism. Methods HaCaT cells were exposed to radiation with linear accelerator(rate 6 Gy/min)at doses of 4,8,12,16,20,24 and 28 Gy respectively,and the cell activity was detected by CCK-8 assay. HaCaT cells were treated with LbGP(0,0. 05,0. 1,0. 5,0. 8,1. 0,1. 5 and 3 mg/mL)for 4 h,exposed to radiation of 12 Gy,and detected for the cell viability by CCK-8 assay. HaCaT cells were divided into control group(without LbGP and radiation),radiation group(radiation of 12 Gy)and LbGP + radiation group(0. 8 mg/mL LbGP for 24 h,radiation of 12 Gy). After irradiation for 1 h,the reactive oxygen species(ROS)production was measured by flow cytometry,the superoxide dismutase(SOD)activity was determined by WST-8 assay and the expression of nuclear factor-E2 related factor 2(Nrf2),p-Nrf2,NADPH quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)were detected by Western blot;The mRNA transcription levels of Nrf2,HO-1 and NQO1 were detected by qRT-PCR at 1,3 and 5 h after irradiation. Results Radiation of12 Gy induced about 50% cell death,and 0. 8 mg/mL LbGP showed the best protective effect on the activity of irradiated cells. After irradiation for 1 h,compared with the control group,the content of ROS in HaCaT cells increased significantly in radiation group(F = 2. 55,P < 0. 001),the activity of SOD decreased significantly(F = 1. 23,P < 0. 01),the contents of NQO1 and Nrf2 protein had no significant difference(F = 1. 78 and 1. 00,respectively,P > 0. 05),the content of HO-1protein increased significantly(F = 1. 37,P < 0. 05),and the content of p-Nrf2 protein decreased significantly(F = 2. 75,P < 0. 01);Compared with the radiation group,the content of ROS in HaCaT cells of LbGP + radiation group decreased significantly(F = 3. 61,P < 0. 001),the activity of SOD increased significantly(F = 1. 23,P < 0. 05),and the contents of Nrf2,p-Nrf2,HO-1 and NQO1 protein increased significantly(F = 4. 00,2. 25,6. 25 and 1. 27,respectively,P < 0. 05). In addition,the mRNA levels of Nrf2,HO-1 and NQO1 genes in LbGP + radiation group were significantly higher than those in radiation group at 1,3 and 5 h after irradiation(F = 0. 20～36. 00,P < 0. 05). Conclusion LbGP can mitigate oxidative stress damage of HaCaT cells induced by radiation and protect cell activity,which may play a role by activating Nrf2 and increasing the levels of its downstream antioxidant enzymes SOD,HO-1 and NQO1.

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.
Animals ; Antibodies, Bispecific ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Escherichia coli ; Fibronectins ; chemistry ; immunology ; Humans ; Mice ; Single-Chain Antibodies ; biosynthesis ; immunology ; Tumor Necrosis Factor-alpha ; immunology

Traditional T vector cloning method requires onerous procedures for identifying recombinant, and directional cloning was impossible. In order to overcome these problems, we have devised a directional T vector pETG based on pET-23a(+). For gene cloning, 7 bp partial LacO sequence was introduced into DNA fragment to reconstitute a full length LacO with Bfu I digested T vector. After transformation, blue colonies were selected on LB plate supplemented with X-gal. Restriction enzyme digestion and PCR identification showed that all blue colonies contained the directionally inserted recombinants and the recombinant efficiency was nearly 100%. We have successfully cloned 103 genes from human liver cDNA; in the study complicated procedures for screening of recombinant were not required. Eight pETG clones were picked for protein expression, and all the clones successfully produced corresponding proteins. We demonstrated that the directional T vector was successfully constructed, and it was very suitable for gene cloning and expression.
Cloning, Molecular ; methods ; DNA, Complementary ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; genetics ; Genetic Vectors ; genetics ; Humans ; Liver ; chemistry ; Polymerase Chain Reaction ; methods ; Recombinant Proteins ; biosynthesis ; genetics

Objective: To construct a novel carrier with core-shell structure-inner core of pFGF2-EGFP-loaded TACS coated by hydroxybutyl chitosan (HBC), and to explore its effects on granular fat graft survival. Methods: The core-structured particles (TACS-pFGF2-EGFP) and core-shell-structured particles (HBC@ TACS-pFGF2-EGFP) were prepared to explore the release pattern of pFGF2-EGFP of these particles. The expression of FGF2 protein was detected by Western-Blot in 293T cells transfected with the sustained - release microspheres in vitro. Cell proliferation assay demonstrated that 10μg/ml pFGF2 plasmid could promote 293T cells growth. Eighteen New Zealand white rabbits were used for adipose tissue transplantation experiment. Rabbit left ear was treated as experimental group, 2 ml fat granules and HBC@TACS-pFGF2-EGFP were implanted; rabbit right ear was used as control group, 2 ml fat granules and HBC@TACS-empty plasmids were transplanted. The specimens were harvested at 4, 8, 12 weeks separately after fat transplantation. Gross view, HE staining, and immunohistochemical staining were performed to observe graft survival, biological characteristics, and neovascular density. Results: HBC@TACS-pFGF2-EGFP particles could sustained release Pfgf2 gene in vitro, and successfully express FGF2 protein after transfecting 293T cells. At different time points after transplantation, the volume of adipose tissues was gradually reduced with time. The fat volume and survival rate of adipose tissues in the experimental group were significantly higher than that in the control group (P<0.05). HE staining showed that the arrangement of new adipocytes in the experimental group was more regular than that in the control group. Immunohistochemistry staining showed that the micro vessel density and FGF2 protein expression level in the adipose tissue of the experimental group were higher than those in the control group (P< 0.05). Conclusions HBC@TACS-pFGF2-EGFP particles with sustained release of FGF2 can improve the survival of granule fat transplantation.

Objective: To investigate the effect of adding glutathion(GSH) to tumescent solution on autologous fat graft survival. Methods: 14 male and female New Zealand rabbits were divided into experimental group and control group randomly, 7 in each group. Experimental group: The donor areas of the rabbits were injected with 3 ml of tumescent solution with GSH. Control group: The donor areas of the rabbits were injected with 3 ml regular tumescent solution. DCFH-DA probe was used for fluorescent staining of harvested fat cells. Then stained fat cells were measured for the intracellular reactive oxygen species(ROS)content by fluorescence microplate. The grafts were harvested at 3 months after transplantation and assessed by general observation, volume measurement, wet weight measurement, HE staining for the number of fat cells, and CD34 immunohistochemical staining for the measurement of micro-vascular density. T test was performed by using SPSS 24.0. Results: The intracellular ROS content of harvested fat cells in experimental group was lower than that in control group, and the difference was statistically significant (P<0.05). At 3 months after transplantation, the wet weight of fat grafts in experimental group was (1133.21±87.97) mg and that in control group was (718.79±79.27) mg. The volume of fat grafts in experimental group was (1.00±0.04) ml and that in control group was (0.59±0.03) ml. The number of fat cells in experimental group was (746.6±15.7)/10 high magnification vision and that in control group was (350.1±32.4)/10 high magnification vision. The density in group experimental was (8.1±2.0)/high magnification vision and that in control group was (6.7±2.4)/high magnification vision. The grafts′ volume, wet weight, and the number of fat cells in experimental group were higher than those in control group, and the differences were statistically significant (P<0.05). The difference of micro-vascular density between experimental group and control group had no statistical significance (P>0.05). Conclusions The addition of GSH to tumescent solution optimizes the process of autologous fat harvest, thereby improving the survival of fat graft.

Objective To investigate the value of T2WI histogram analysis in differential diagnosis of glioblastoma multiform (GBM) from solitary metastasis.Methods Data of 103 patients with pathologically confirmed GBM (GBM group,n=57) and solitary brain metastasis (solitary brain metastasis group,n =46) were retrospectively reviewed.All patients underwent conventional MR scanning,including axial T1WI,T2WI,FLAIR and contrast-enhanced T1WI before surgery.The histogram metrics,including mean,standard deviation (SD),median,kurtosis and skewness were calculated from ROI,which were manually placed on the maximal section of the solid part of tumors on T2WI by using Image J software.ROCs were generated to evaluate differential diagnostic performance of the histogram metrics with significant difference between both groups.Results The values of mean,SD and median were significantly higher in GMB group than those in solitary brain metastasis group (P＜0.05).The areas under ROC curve of mean,SD and median was 0.772 (95％ CI [0.681,0.862],P＜0.001),0.719 (95％ CI [0.616,0.822],P＜0.001) and 0.767 (95％ CI [0.674,0.860],P＜0.001),respectively;and the diagnosis cutoff value of mean,SD and median was 509.575,58.844 and 550.500,respectively.The sensitivity of the three parameters was 0.719,0.702 and 0.719,and the specificity was 0.783,0.652,and 0.826,respectively.Conclusion The value of mean,SD and median of T2WI histogram analysis can be helpful to differentiating GBM and solitary brain metastasis,of which the mean value is the best for differential diagnosis.

Objective: To investigate the clinical features of children with infectious mononucleosis (IM), to compare the difference of therapeutic effects between general treatment to antiviral therapy for IM. Methods: This prospective study analyzed the clinical data and laboratory test results of 201 cases with IM in our hospital from January 1, 2016 to December 31, 2016. The follow-up period was 6-12 months. The patients were divided into two groups according to the order of admission. The clinical symptoms and laboratory test results of the two groups were observed after hospitalization. Results: Of the total of 201 patients, male to female is 1.72∶1; Age: 8 months to 13 years 6 months (average 4.8±2.8 years), The disease frequently occurred in summer and autumn, accounted for 64.18%.The major clinical manifestations was fever (97.51%), angina (79.10%), enlarged of lymph node(68.66%), eyelid swelling (67.16%), hepatomegaly (53.73%) and splenomegaly (46.77%). There was no statistical difference in duration of fever, improved angina time, lymph nodes(liver, spleen) enlargement recovery time, heterotypic lymphocytes normalization time, lymphocyte function normalization time.There was significant difference in reducing the serum/plasma or total blood EBV-DNA in the short term between antiviral group and general treatment group (P<0.01), but for the long-term reduction of EBV-DNA, there was no significant difference (P>0.05). The low affinity EBV-CA-IgG was converted to high affinity EBV-CA-IgG in 89.41% of patients after 3 months and in 98.68% of patients after 6 months. With the recovery of the disease, the positive rate of EBV-CA-IgM and EBV-EA-IgG decreased. But the positive rate of EBV-NA-IgG increased. Serum/plasma EBV-DNA was completely overcast after 3 months, but after 12 months, the patient’s total blood EBV-DNA positive rate was still 71.11%. Conclusions In general IM patients, there was no significant difference between antiviral therapy and general treatment, so antiviral therapy is not generally recommended. EBV DNA in the whole blood of patients with IM will continue to be positive more than 6-12 months, indicating that to observe changes of IM should not use whole blood samples for EBV DNA testing.

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.
Acetylglucosamine ; Acetylglucosaminidase ; Bacillus coagulans ; Chitin ; Chitinases ; Hydrogen-Ion Concentration ; Recombinant Proteins/genetics*