Study of glutathione protected gold nanoclusters on HeLa cytotoxicity / 国际生物医学工程杂志

Objective To investigate the effects of glutathione protected gold nanoclusters (GSH-Au NCs) on HeLa cytotoxity.Methods Fluorescence intensity were measured on GSH-Au NCs containing medium treated cells using fluorescence spectrophotometer at different time points.GSH-Au NCs uptake by HeLa cells at 1,2,6,12 and 24 h were investigated through fluorescent spectrophotometer.In vivo tumor uptake was also investigated on BALB/c tumor-bearing mice through inductively coupled plasma mass spectrometry (ICP-MS) at 24 h after intraperitoneal injection of 0.2 ml GSH-Au NCs (3 mmol/L) and distilled water (control group) respectively.The cytotoxicity of GSH-Au NCs at different doses (0.003-0.3 mmol/L) was tested at 24 and 48 h using MTT assay after interaction with HeLa cells.Results The uptake efficiency of GSH-Au NCs by HeLa cells kept increasing and reached maximum of 73.13％ at 24 h.The results of tumor-bearing mice indicated that the tumor tissue had higher uptake efficiency after 24 h (320±15) ng/g than that of control group (intraperitoneal injection of distilled water),and the difference was stastically significant (P＜0.05).HeLa cells were treated with different concentrations of GSH-Au NCs for 24 h,and GSHAu NCs had a slight effect on cell viability.With the increase of GSH-Au NCs dose,the inhibition effects on growth of HeLa cells enhanced.The cell activity of HeLa cells treated with 0.3 mmol/L GSH-Au NCs for 24 h reduced to 86％compared with that of control group (the concentration of GSH-Au NCs was 0) (P＜0.05),while there was no significant difference between the survival rate of different concentrations of GSH-Au NCs group and the control group for 48 h.Conclusions GSH-Au NCs have neglectable cytotoxity on HeLa cells even though both in vitro and in vivo uptake are high.GSH-Au NCs are suitable for biomedical application such as imaging,drug loading and targeted drug delivery.

Comparison of two mouse models of alcoholic liver disease induced by oral ethanol gavage or Lierber-DeCarli ethanol liquid diet / 中国比较医学杂志

Objective To select a simple, stable and reliable mouse model of alcoholic liver disease. Methods The mouse models of alcoholic liver disease were induced by oral gavage ethanol or Lierber?DeCarli ethanol liquid diet for 8 weeks. The food intake and body weight were recorded. Pathological changes were examined using HE staining. Liver injury was assessed by the activities of serum ALT, AST, AKP and γ?GT, and serum and hepatic TC and TG. Results After modeling, both models showed significantly increased activities of serum ALT, AST, AKP, and contents of serum and hepatic TG (P<0?05), indicating the successful development of alcoholic steatohepatitis. However, oral ethanol gavage led to body weight loss and weak mental state. Ethanol liquid diet less affected the body weight and mental state. Ethanol liquid diet enhanced liver to?body weight ratio and serum TC, but oral gavage of ethanol did not. The changes of serum ALT, AST, serum and hepatic TG, and hepatic steatosis in the ethanol liquid diet models were more severe than those in the oral gavage ethanol models, suggesting that Lierber?DeCarli ethanol liquid diet led to more serious liver injury than oral gavage ethanol. Conclusions Lierber?DeCarli ethanol liquid diet model is better than oral gavage ethanol model, and is more suitable for studies on mechanisms and evaluation of hepato?protective drugs for alcoholic liver disease.

Application of Time-resolved Fluroimmunoassay for Determination of Furaltadone Metabolite 3-Amino-5-morpholinomethyl-2-oxazolidinone / 分析化学

To detect furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone ( AMOZ ) in fish sample, an Eu3+ labeling time-resolved fluoroimmunoassay ( TRFIA ) was developed. The effects of experimental conditions including AMOZA-OVA concentration, dilution of antibody, and reaction time on the sensitivity of TRFIA were explored. The results showed that the optimized assay conditions were as follows:the AMOZA-OVA concentration was 0. 25 μg/mL; the antibody was diluted 5í104 folds, and the competitive reaction time was 50 min. Under optimal conditions, the method showed a detection limit of 0. 01 ng/mL, an IC50 of 0. 26 ng/mL and a linear range (IC20-IC80) of 0. 025-2. 83 ng/mL. The recoveries of AMOZ in fish at three spiked levels ranged from 78 . 0% to 86 . 0%, and the relative standard deviations were less than 15%. Good correlation between the ic-TRFIA and high performance liquid chromatography-tandem mass spectrometry was obtained for spiked food samples. The proposed ic-TRFIA method was suited for the determination of AMOZ residue in food samples.

Effect of Wnt pathway on bone damage and bone marrow adipogenesis induced by radiochemotherapy / 国际生物医学工程杂志

Cancer has become the second largest life-threatening disease nowadays.Radiotherapy and chemotherapy are still important treatments for cancer.However, they tend to produce a lot of serious adverse effects including bone damage and bone marrow fat, etc.Based on recent research, the research progress on canonical Wnt pathway and its impact on stromal stem cells differentiation into osteoblasts and adipocytes are reviewed.Radiochemotherapy-induced bone damage and bone marrow fat is closely related to canonical Wnt pathway.In experimental assay and clinical application, Wnt pathway antagonists, such as Dickkopf-1 (DKK-1), sclerostin, and secreted frizzled-related protein 1 (sFRP-1) are used to relieve bone damage.Wnt pathway is expected to become a potential target for the therapy of bone damage and bone marrow fat induced by raidochemotherapy.

Indirect Competitive Chemiluminescent Enzyme Immunoassay Method for Determination of Dimethyl Phthalate in Soy Sauce and Liquor / 分析化学

4-Amino dimethyl phthalate as the hapten was coupled to carrier protein and then used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to dimethyl phthalate ( DMP) was thus obtained, and on the basis of this, an indirect competitive chemiluminescent enzyme-linked immunoassay ( icCLEIA ) was developed. The experimental parameters of icCLEIA were optimized as follows: the concentration of coating antigen was 50 μg/L, the primary antibody concentration was 92. 5 μg/L, the secondary antibody concentration was 1μg/mL, distilled water (pH 6. 0) was used as diluent solution and the competitive reaction time was 40 min. Under the optimal conditions, the icCLEIA exhibited a linear working range from 0. 74μg/L to 30. 32μg/L with the limit of detection of 0. 29μg/L. The cross-reactivity of thirteen structural analogues was lower than 5%. The recovery of DMP from spiked liquor and soy sauce samples ranged from 80 . 2% to 116 . 0% and the average RSD was less than 3 . 6%. The detection results of the spiked liquor and soy sauce samples were consistent with those by standard gas chromatography-mass spectrometry method. The developed icCLEIA method exhibited a practical potential for detecting DMP residue in food samples.

Direct Competitive Enzyme-Linked Immunosorbent Assay for Detection of Acrylamide in Food Samples / 分析化学

Due to the low molecular weight and simple structure, the production of specific antibodies against acrylamide is unavailable. In this study, a novel hapten was synthesized through the derivatization of acrylamide and 4-mercaptophenylacetic acid. The hapten was then coupled to carrier protein and used to immunize New Zealand rabbits. Polyclonal antibody which showed specific binding to the acrylamide derivative ( hapten) was obtained. The antibody was labeled with horseradish peroxidase ( HRP) and used to develop a direct competitive enzyme-linked immunosorbent assay ( dc-ELISA) . The dc-ELISA was used to determine the content of acrylamide derivative, and then transferred to the content of acrylamide. The assay showed an IC50 value of 45. 49 μg/L, a limit of detection of 3. 0 μg/L and the linear range of 9. 2-195 μg/L for acrylamide. The recovery of acrylamide from spiked food sample was determined ranging from 83 . 6% to 112 . 7%. Good correlations between the results of dc-ELISA and standard HPLC-MS/MS were obtained. The proposed dc-ELISA is suitable for the determination of acrylamide in food samples.

Effects of pulsed electromagnetic fields on homing genes of mouse osteoblasts / 国际生物医学工程杂志

Objective To investigate the effects of pulsed electromagnetic fields (PEMF) on homing and proliferation-related genes of mouse osteoblasts.Methods 9 week-old C57BL/6 mice were treated with PEMF (70 Hz,1 mT) for 4～5 weeks,while mice in control group didn't not receive PEMF.Bone marrow cells of femurs and tibias were flushed out,and the bones were minced and incubated at 37 ℃ with a type Ⅰ collagenase.Bone associated mononuclear cells (MNCs) were isolated via density centrifugation with Lymphoprep.Magnetic cell sorting was used before flow-cytometric sorting,and the ALCAM+Sca-1-cells were collected.The homing and proliferation-related genes expressed in ALCAM+Sca-1-cells were detected with high throughput microarray and RT-PCR.Results The expression of Jag1 and Ang-1 in mouse osteoblasts increased under the effects of PEMF.Conclusions PEMF may have regulation effects on HSC (hematopoietic stem cell) survival through modulating the homing and proliferationrelated genes in ALCAM+Sca-1-osteoblasts.

Effect of ionizing radiation on the expressions of RANKL and OPG mRNA and proteins in osteoblasts / 中华放射医学与防护杂志

Objective To investigate the effect of radiation on the expressions of RANKL and OPG in osteoblasts in order to disclose the molecular mechanism of bone injury induced by ionizing radiation.Methods The osteoblasts were differentiated from MC3T3-E1 cells.After 2 or 4 Gy137 Cs γ-irradiation,the mRNA and protein expression levels of RANKL and OPG of osteoblast precursor and osteoblast were detected by real-time PCR and Western blot.Results The expressions of RANKL mRNA (t=5.41,P＜0.05)and protein(t=68.37,P＜0.01)were up-regulated after 4 Gy irradiation,while the expressions of OPG mRNA(t=5.20,7.02,P＜0.05)and protein(t=7.78,9.45,P＜0.05)were down-regulated after 2 and 4 Gy irradiation.Conclusions 2 and 4 Gy ionizing radiation alters RANKL/RANK/OPG pathway in osteoblasts,which may promote the osteoclast differentiation and maturation and hence promote bone resorption of osteoclasts.

Effect of dibutyl phthalate and di-(2-ethylhexyl) phthalate on urine SOD activity and MDA content in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of dibutyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) on urine superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in rats.</p><p><b>METHODS</b>According to 2×2 factorial analysis, 60 adult male SD rats were randomized into 10 groups (n=6), including a control group (fed with sesame oil), 3 DBP groups (fed with DBP at the doses of 30, 100 and 300 mg/kg), 3 DEHP groups (with DEHP at 50, 150, and 450 mg/kg), and 3 DBP+DEHP groups (with 30 mg/kg DBP+50 mg/kg DEHP, 100 mg/kg DBP+150 mg/kg DEHP, and 300 mg/kg DBP +450 mg/kg DEHP). The agents were administered in a single dose through gavage in a volume of 2 ml. After the treatments, the 24, 48, 72, and 96 h urine samples were collected to determine the SOD activity and MDA content.</p><p><b>RESULTS</b>DBP and DEHP, either alone or in combination, significantly decreased SOD activity and increased MDA content in the urine collected at 24 h but not at the other time points. Such changes were gradually reversed with time.</p><p><b>CONCLUSION</b>DBP or DEHP treatment alone can result in significant oxidative damage in the kidney of rats, and the toxic effect of the combined exposure is even more obvious.</p>

Expression and molecular evolution of recombinant acetylcholinesterase for detection of pesticide residues: a review / 生物工程学报

Acetylcholinesterase (AChE) plays a key role in the pesticide determination. However, the extraction of AChE from natural materials has the disadvantages of low yield, complex purification and poor stability. Therefore, the preparation of recombinant AChE with high performance becomes the hot topic of researchers in recent years. In this article we summarize the progress in the expression of recombinant AChE and the improvement of its analytical characteristic. Finally, we point out that the directed evolution strategy combined with surface display technology is the future trend on improving recombinant AChE activity.

Construction and verification of lentivirus vector expressing mouse notch intracellular domain / 国际生物医学工程杂志

ObjectiveNotch signaling is highly conservative in evolution and plays an important role in cell's proliferation and differentiation.Construction of tentiviral vector containing Notch intracellular domain (NICD) would lay the foundation for the study of Notch signaling.MethodsTotal RNA was extracted from the myeloid tissue of C57BL/6 mice.cDNA was composed via reverse transcription.NICD sequence was obtained by PCR and recombined into lentiviral vector.Lentiviral vector with NICD was infected into target HEK293T cell.Real-time PCR and Western blot were used to examine NICD expression in HEK293T cell.ResultsNICD expression increased in HEK293T cells.ConclusionThe successful construction of lentiviral vector involving mice NICD expression provides the foundation for the future study.

Effect of irradiation on RANKL/OPG mRNA levels in mouse osteoblast in vitro / 中华放射医学与防护杂志

Objective To study the influence of irradiation on the osteoblast function by the gene expression changes of RANKL and OPG.Methods Bone marrow stromal cells were induced to develop into early and mature osteoblasts in vitro.The characterization of osteoblasts was indentified by ALP staining.The RANKL and OPG mRNA levels in early and mature osteoblasts, which exposed to 0 -4 Gy radiation were determined by RT-PCR.Results Bone marrow stromal cells had been induced to early and mature osteoblasts by osteoblast differentiation medium in vitro.In early stage of osteoblast, RANKL mRNA expression levels treated with 1Gy irradiation was 2.83-fold higher than those other irradiation dosage groups.The RANKL mRNA expression levels of each group in early stage of osteoblasts were significantly higher than those in the mature counterpart ( t = 8.34 - 103.57, P ＜ 0.05 ).The ratio of RANKL/OPG mRNA was obviously greater in early osteoblast compared with the mature cells ( t = 2.84 - 20.99, P ＜0.05 ), and it was the highest in 1Gy irradiation treated early osteoblast.Conclusions Radiation exposure of the early osteoblasts promotes osteoclasts function and results in the bone loss.

Effect of radiation on collagen type I mRNA expressions in mouse osteoblast in vitro / 国际生物医学工程杂志

Objective Cancer radio-therapy may induce bone damage of the patients.collagen type I gene expressions in osteoblast after radiation indicates the influence of radiation on the function of early and late osteoblast.Methods Bone marrow stromal cells were differentiated into osteoblasts in vitro.and the characteristics was indentified.The collagen type I expressions in early and late stage osteoblasts exposed to 1～4Gy radiation were examined by RT-PCR.Results Compared to control group,collagen type I gene expressions increased in early osteoblast after 1～3 Gy radiation (P＜0.05),while the gene expressions in late osteoblast that cultured 10 days decreased.Collagen type I gene expression in late stage ostoblast after 4 Gy irradiation was greatly higher than that in early stage osteoblast (P＜0.01).Conclusion After 1～3 Gy irradiation,the collagen type I expression in early osteoblast was enhanced,indicating the increased ability of bone formation.The exposure to 1～3 Gy decreased collagen type I expression in late osteoblast and weakened the ability of bone formation.The result of high expression of collagen type I in late osteoblast after 4 Gy irradiation may be the manifestation of compensatory function.

Effects of pulsed electromagnetic fields on the precursor cells of osteoclasts and osteoblasts / 国际生物医学工程杂志

Objective To study the in vitro and in vivo effects of pulsed electromagnetic fields (PEMFs) on osteoclast and osteoblast precursor cells.MethodsTo observe the in vitro effect of PEMFs,femur bone marrow cells of 8 week old female SD rats were collected.According to different treating doses,rats were divided into four treatment groups and one control group.After the treatment,the clones of granuloeyte/maerophage colony forming unit(CFU-GM) and fibroblast colony forming units(CFU-F) were measured respectively.To observe the combined in vitro and in vivo effect of PEMFs,8 week old female SD rats were randomly divided into three groups: 2-70 group,ovariectomization (OVX) group and SHAM group.Rats in the 2-70 group and OVX group were bilateral ovariectomized,while rats in the SHAM group were sham-ovariectomized.12 weeks after ovariectomization,the 2-70 group was exposed to PEMFs while the other groups were left untreated.Then,femur bone marrow cells of the rats were collected.According to the way whether the groups were treated with PEMFs,the cells were divided into six groups: 2-70 with/without treatment,OVX with/without treatment,SHAM with/without treatment.After the treatments,the clones of CFU-GM and CFU-F were measured respectively.Resultsin vitro effect of PEMFs: Compared with the control group,the CFU-GMin the treated groups reduced while the CFU-F increased.PEMFs effect in vitro and in vivo: The CFU-F in treated groupsincreased,whileno.significantdifferencesofCFU-GMwerefoundamongthegroups.Conclusion PEMFs has inhibitory effect on osteoelast precursor cells and enhances the proliferation of osteoblast precursor cells when simply applied in vitro.When PEMFs was applied in combined manner of in vitro and in vivo,it shows that PEMFs enhance the proliferation of osteoblast precursor cells but has no inhibitory effects on osteoelast precursor cells.

Production and Identification of Monoclonal Antibody Against Flumequine and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay / 分析化学

The hapten of Flumequine(FLU) with four carbon atoms spacer arm(FLUABA) was synthesized and coupled to bovine serum albumin(BSA) as immunogen using activated ester method. Balb/c mice were immunized by the artificial immunogen and the splenocytes of immunized mice were fused with Sp2/0 cells to obtain the monoclonal antibody(McAbs). A hybridoma cell line(DB6-E7) secreting anti-flumequine McAbs was obtained by limited dilution method and screened by indirect enzyme-linked immunosorbent assay(ELISA) using heterogenous coating antigen. The results showed that the subtype of the McAb was IgG_1, and the affinity was 8.19×10~8 L/mol. The haptens of FLU, FLUABA and FLUACA, with different space arm, were separately linked to ovalbumin(OVA) for heterologous or homologous coating antigen. The results of indirect ELISA and indirect competitive ELISA(icELISA) indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. By heterologous coating antigen(FLU-OVA), the icELISA showed an IC_(50) value of 26.33 μg/L, LOD of 4.0 μg/L, and the workable range of 8-114 μg/L (IC_(20)-IC_(80)). Cross-reactivity studies showed that the McAbs were quiet specific for FLU, no cross-reactivity(<0.1%) was detected between the obtained McAbs and the quinolones compounds or other structural similarity compounds. The developed icELISA for FLU can satisfy the detection criteria of flumequine in animal food-products.

Construction and expression of anti-clenbuterol single chain Fv recombinant vector / 生物工程学报

To construct the recombinant vector pBV220-scFv and express anti-clenbuterol (CBL) scFv antibody in Escherichia coli, we amplified the scFv gene using plasmid pCANTABSE-CBL as a template, recombined it with pPICZalphaA, then amplified the scFv-His-tag gene from plasmid pPICZalphaA-scFv and linked it with expression plasmid pBV220. We identified the recombinant plasmid by restrictive enzyme digestion, PCR amplification and sequence analysis. Finally, we transformed the recombinant vector into E. coli DH5alpha that was temperature-induced and expressed recombinant protein. We identified the recombinant protein by SDS-PAGE, Western blotting and indirect competitive ELISA. The results show that recombinant plasmid pBV220-scFv contained the inserted fragment with highest homology about 99.8%. The expression of scFv induced by temperature show 37 kD Mw and anti-His-tag mAb recognized-activity by SDS-PAGE and Western blotting respectively, and could competitively combine with CBL, the IC50 is 4.55 ng/mL. The recombinant plasmid pBV220-scFv is constructed and expresses the scFv gene of CBL in E. coli successfully. This study suggests the corresponding immunoassay methods could be set up by the recombinant scFv.

Determination of hupzine A in Huperzia serrata by HPLC / 中草药

Object To compare the hupzine A (Hup A) in Huperzia serrata (Thunb.) Trev. obtained by different extracting methods and investigate the amount of alkaloids and the content of Hup A from different parts of the plants and from different places. Methods Using HPLC for the determination of Hup A. Results The content of Hup A in the stem and leaf is richer than that in the root. The content of Hup A from Guizhou, Guangdong and Anhui Provinces is 0.018%, 0.021% and 0.020% repectively; The difference of extract method of Hup A is no prominence. Conclusion The content of Hup A in the ground is richer than that of underground, and there are some difference in the content of Hup A obtained from different places.