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OBJECTIVE： To study improvement effects of different proportions of total glucosides of ginseng （TGG）， total glucosides of moutan cortex （TGM） and paeonol containing serum on the injury of human umbilical vein endothelial cells （HUVEC） injury induced by hydrogen peroxide （H2O2）， screen the optimal proportion and investigate its mechanism. METHODS： The rats were randomly divided into blank group （distilled water）， TGG group （TGG， 2.025 g/kg）， TGM group （TGM， 4.05 g/kg） and paeonol group （paeonol， 1.08 g/kg）， with 12 rats in each group. They were given relevant medicine twice a day for consecutive 7 days. 1 h after last medication， the blood samples were collected via abdominal aorta to prepare drug containing serum. Using survival rate of HUVEC as evaluation indexes， different proportions of TGG， TGM and paeonol containing serum as factors， L9（34） orthogonal test was designed to optimize the optimal proportion of 3 kinds of drug containing serum. HUVEC were divided into blank group， model group， TGG group， TGM group， paeonol group and optimal proportion group. Except that blank group were treated with relevant medium， other group were treated with 1.2 mmol/L H2O2 to induce HUVEC injury， and then TGG group （volume fraction of drug containing serum was 0.000 5％）， TGM group （volume fraction of drug containing serum was 0.000 5％）， paeonol group （volume fraction of drug containing serum was 1％） and optimal proportion group were intervened with drug containing serum. The levels of LDH， NO and ET-1 in cells were detected by microplate method and ELISA. RESULTS： The optimal proportion of drug containing serum were TGG 0.000 5％， TGM 0.000 5％ and paeonol 1％. Compared with blank group， the levels of LDH and ET-1 were higher in model group （P＜0.01）， while NO level was lower （P＜0.05）. Compared with model group， the levels of NO were higher in TGG group， TGM group and optimal proportion group （P＜0.01）， while the levels of LDH and ET-1 were lower （P＜0.05 or P＜0.01）. Compared with TGG group， TGM group and paeonol group， the level of LDH was lower in optimal proportion group （P＜0.05 or P＜0.01）， while the level of NO was higher （P＜0.05 or P＜0.01）. CONCLUSIONS： TGG and TGM combined with paeonol can significantly improve HUVEC injury induced by H2O2， and the mechanism of which may be associated with the decrease of LDH and ET-1 and the increase of NO.
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Objective To establish a rapid method for the detection of SMCY antigen. Methods To Use the technology of colloidal gold immunochromatography with double antibody sandwich assay, the gold labeled pad was coated with colloidal gold labeled SMCY rabbit polyclonal antibody, colloidal gold strip was made for detection of serum, which include 12 serum samples of pig, cattle, dog, chicken and mice and 50 serum samples of human. Results The colloidal gold test strips showed obvious specificity in human and common animal sera and could distinguish between male and female sera. Conclusion This method can be used to identify the serum of women and men, and has certain species specificity, which provides a direction for forensic science to rapidly identify gender of human samples.
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Objective with the advantages of rapidity in detection protein, We selected the gender-specific amino acid sequence based on human SMCY and SMCX, cloned and expressed SMCY gender-specific fusion antigens. The rabbits were immunized with purified antigens to obtain the polyclonal antibodies. A new method was established for rapidly sex identification of forensic evidence samples by detecting SMCY antigens with the corresponding polyclonal antibodies. Methods We found three differential fragments by analyzing the sequence of human SMCY and SMCX. Then we cloned this three fragments and ligated as a new recombinant.This SMCY gender-specific fusion antigen gene was sub-cloned into pET-28a and expressed in Escherichia coli. The fusion antigen was purified by Ni-NTA column. The rabbits were immunized with purified antigen to produce the specific polyclonal antibodies.The reactivity of the polyclonal antibody was evaluated by ELISA and Western blotting. We developed a colloidal gold test strip for detecting the gender of human samples. Results We successfully selected gender-specific amino acid sequence, the SMCY gender-specific fusion antigen was expressed by prokaryotic expression and the polyclonal antibody was prepared by immunizing rabbit. The results of colloidal gold strip tests showed that there is a significant difference between male and female serums. Conclusion The results showed that the SMCY gender-specific fusion antigen could be recognized by the polyclonal antibody.The colloidal gold strip tests made by SMCY gender-specific fusion antigens and the corresponding polyclonal antibodies could be used for rapidly determining the gender of forensic evidence samples.
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OBJECTIVE To investigate the effect of tranilast on cardiac fibroblasts proliferation and phenotype transformation incubated with a high concentration of glucose and the possible mechanism. METHODS The cardiac fibroblasts were divided into seven groups in accordance with different nutrient solutions:normal control group(5.5 mmol · L-1 glucose),hypertonic group(5.5 mmol · L-1 glucose+mannitol 25 mmol · L- 1),high glucose group (25 mmol · L- 1 glucose),tranilast intervention groups (25 mmol·L-1 glucose+tranilast 50,100 and 200μmol·L-1),and activin receptor-like kinase 7(ALK7) inhibitor group(25 mmol · L-1 glucose+10μmol · L-1 SB431542). The cell proliferation was detected by MTT method. The transformation of cardiac fibroblasts was determined by immunfluorescence staining. The expression of fibroblast specific protein 1 (FSP-1),α-smooth muscle actin (α-SMA),transforming growth factor-β1(TGF-β1)and ALK7 was assessed by Western blotting. RESULTS Compared with nor?mal control group,A492 nm of cells in high glucose group was significantly increased(P<0.01),while the expression of α-SMA,TGF-β1 and ALK7 protein in high glucose group was significantly increased(P<0.01),but FSP-1 protein was significantly decreased(P<0.01). There was no difference between normal control group and hypertonic group. Compared with high glucose group,A492 nm of cells in tranilast or SB431542 intervention groups were decreased(P<0.05),and the expression of α-SMA,TGF-β1 and ALK7 protein was significantly decreased(P<0.05),but the expression of FSP-1 protein was increased (P<0.05)in tranilast or SB431542 intervention groups. CONCLUSION Tranilast can inhibit the proliferation and phenotype transformation of cardiac fibroblasts induced by high glucose,which may be related to down-regulation of the expression of ALK7.
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TcdA and B toxins secreted by Clostridium difficile( CD) are two important causes of diseases in organisms. The expression of tcdA and tcdB genes is regulated by a few factors located in the pathogenicity locus ( PaLoc) .Studies have indicated that the tcdC gene is likely to act as a negative regulator of toxin gene expression.So far, it has been debatable whether tcdC gene is regarded as a negative regulator.The mechanism of tcdC gene in pathogenesis remains unclear.In this paper, the structure and function of the tcdC gene are summarized, which will help study the mechanism of tcdC gene and obtain optimal drug targets.
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Objective To construct prokaryotic expression vectors for glutamate dehydrogenase(GDH)of Clostridium difficile(CD),and express recombinant GDH in Escherichia coli,and identify its antigenicityed.Methods The entire gene of GDH was cloned from ATCC43255 genome DNA.The recombinant antigens were expressed in E.coli with IPTG induction and purified by Ni-NATBeads.The antigenicity was detected using CD Qick Chek Complete dual-antigen EIA. Results Prokaryotic expression vectors of CD GDH were constructed successfully.The antigen could be identified by specific anti-GDH antibodies.Conclusion The GDH antigen can be used to prepare corresponding antibodies,which facilitate the development of immunoassay for CD GDH.
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Objective To develop a method to determine Pb and Cd in Qingkailing Injection by graphite furnace atomic absorption spectrometry, and search their source. Methods The samples, digested via microwave, were determined the contents of Pb and Cd by graphite furnace atomic absorption spectrometry in raw material, intermediate and finished product of Qingkailing Injection. Results The standard curve of Pb was Y=0.007 3X+0.011 6, and Cd was Y=0.056 7X+0.060 4. The regression equation of Pb and Cd was 97.0% and 95.6%, respectively. Content determination of Pb and Cd in Qingkailing Injection revealed that Pb and Cd in finished product came from raw materials. Conclusion The method is rapid, high sensitive and accurate, and can be applied to the inspection of Pb and Cd in Qingkailing Injection.