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Objective:To investigate the production of cytokines from macrophages induced by Treponema pallidum membrane proteins Tp0971.Methods: The Tp0971 was amplified by PCR from a preparation of T.pallidum genomic DNA and then sub-cloned into the prokaryotic expression vector pET28a(+)to construct the recombinant plasmid pET28a(+)/Tp0971.The recombinant plasmids were transfected into E.coli Rosseta strain to express recombinant protein Tp0971 by IPTG induction.The expression products were purified by Ni-NTA affinity chromatography,and the concentration was determinated by BCA method.Detoxi-Gel was used to remove endotoxin contamination in during the protein preparation.After induced by PMA,cells were incubated with various concentrations of recombinant proteins Tp0971.The expression levels of IL-8,IL-1β and IL-6 were detected by ELISA.Cells were pretreated with anti-TLR2 antibody or TLR2 siRNA,or pyrrolidine dithiocarbamate,an inhibitor of NF-κB,for evaluation of the role of TLR2 and NF-κB in the production of cytokines by ELISA.Results: Tp0971 gene were amplified successfully by PCR,and the recombinant plasmids were confirmed by enzyme digestion and sequencing.SDS-PAGE results showed three recombinant proteins were expressed as the soluble with a relative molecular weight of 29 kD.0.5-10 μg/ml of Tp0971 could stimulate macrophages to produce IL-8,IL-1β and IL-6 dose-dependently.After cells were pretreated with siRNA or neutralizing antibody targeting TLR2 or the PDTC,the Tp0971 induced protein expression levels of proinflammatory cytokines were significantly reduced in macrophages.Conclusion: Tp0971 induces macrophages to produce proinflamatory cytokines via TLR2/NF-κB pathway.
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ObJective To observe the efficay and adverse reactions of olmesartan combined with hydrochlorothiazide on patients with primary moderate hypertension,compared with the therapy of benazapril associated with amlodipine.Methods 116 primary moderate hypertension patients were randomly divided into the treatment group and control group.Patients in treatment group were treated with olmesartan combined with hydrochlorothiazide oral administration,and control group were given benazapril combined with amlodipine oral administration,which lasted for 3 weeks.Every subject was measured blood pressure before and after the drug administration and observed adverse reaction.Cardiac hypertrophy was detected by UCG.A comparison was made in effectiveness of the drug and cardiac hypertrophy between two groups.Results There was significant difference in effectiveness of two anti-hypertension therapies,and cardiac hypertrophy in the treatment group was lighter than that in control group.Conclusion The therapy of olmesartan combined with hydrochlorothiazide was more effective than that of benazapril combined with amlodipine,also more depressed cardiac hypertrophy.
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Objective To investigate the immune response to and protective effect of a bivalent DNA vaccine expressing interleukin-2(IL-2)and Gpd proteins in New Zealand rabbits.Methods Seventy-two male New Zealand white rabbits were equally and randomly divided into 4 groups to be immunized with recombinant plasmids pcDNA3.1(+)/Gpd-IL-2(pcD/Gpd-IL-2),pcDNA3.1(+)/Gpd(pcD/Gpd),empty plasmid pcDNA3.1(+)(pcD)and phosphate buffered saline(PBS),respectively.Immunization was carried out by intramuscular injection at multiple sites with a 2-week interval for 3 times.On week 10 after the initial immunization,the rabbits were challenged intradermally with T.pallidum(Nichols strain).Enzyme-linked immunosorbent assay(ELISA)was used to quantify the serum level of anti-Gpd antibodies in the rabbits and the level of IL-2 and interferon(IFN-γ)in the supernatant of Gpd protein-stimulated spleen cells from the rabbits at different time pionts.MTT assay was conducted to detect the proliferation response of spleen cells collected from the rabbits on day 0,14,28,140 and 168 after the challenge.Results Compared with pcD and PBS,both the vaccines pcD/Gpd and pcD/Gpd-IL-2 elicited significantly higher levels of anti-Gpd IgG antibodies in rabbits at different time points during the vaccination and infection period,with the titers peaking at 1 ∶ 1024 and 1∶4096,respectively(both P ＜ 0.01).There were also significant differences in the serum levels of anti-Gpd IgG antibodies between the pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits at different time points(all P ＜0.01).The levels of IL-2 in the supematant of spleen cells from pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits on week 8 after the immunization were 110 ± 12.6 and 167 ± 15.7 μg/L respectively,and those of IFN-γwere 225 ± 17.6 and 447 ± 22.4 μg/L respectively,significantly higher than those in that from the other two groups of rabbits(all P ＜ 0.01).Furthermore,an apparent proliferation response was observed in spleen cells from pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits with a higher stimulation index compared with pcD-and PBS-immunized rabbits(all P ＜ 0.01).Dark-field microscopic examination of early-stage infected lesions revealed that pcD/Gpd-IL-2-immunized rabbits had a lower detection rate(17.5％)of Tp from lesions,occurrence of ulcerative lesions(15％)and shorter curing time compared with pcD/Gpd-immunized rabbits.Conclusion The recombinant plasmid pcDNA3.1(+)/Gpd-IL-2 could induce protective humoral and cellular immune response more efficiently in rabbits.
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Objective To clone, express Tp0319 gene from Treponemapallidum (T. pallidum), and to assess the immunocompetence of recombinant protein. Methods The immuno-dominant region of Tp0319gene was chosen by computer analysis, amplified from T. pallidum complete genome by PCR, subcloned into the expression vector pQE32 to construct a recombinant plasmid, pQE32/Tp0319, which was then expressed in E. coli M15. The recombinant protein was purified with Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot. New Zealand rabbits were immunized with the recombinant protein, and the titer of anti-Tp0319 antibodies in sera from immunized rabbits were measured with indirect ELISA. Also, indirect ELISA with the recombinant Tp0319 as coating antigen was performed to detect the anti-Tp0319 antibody in sera from 200 normal human controls and 200 patients with syphilis. Results The prokaryotic expression vector pQE32/Tp0319 was constructed successfully, and the recombinant protein Tp0319 with a molecular weight of about 30 000 was attained. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the specific antibody titer was more than 1: 10 240 after immunization for 3 times. Western blot proved that the recombinant protein could specifically react with anti-T. pallidum IgG antibody-positive sera. Indirect ELISA was successfully developed with the recombinant Tp0319, and detected antibodies to T. pallidum in control sera with a sensitivity and specificity of 100% (40/40), respectively. Compared with T. pallidum particle agglutination (TPPA) assay, the sensitivity and specificity of the indirect ELISA were 92.6% and 100%, respectively, in the detection of T. pallidum in sera from patients and controls, and the concordance between the indirect ELISA and TPPA was 96%. Conclusions The prepared recombinant protein shows a satisfactory immunocompetence, which may lay a foundation for its further application in the serodiagnosis of syphilis.
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Objective To construct a recombinant plasmid encoding Tp0821,a membrane lipoprotein of T. pallidum,express and purify this protein,and to evaluate its immunocompetence.Methods The recombinant plasmid pQE32/Tp0821 was constructed and induced to express the corresponding protein.Then,New Zealand rabbits were immunized with purified recombinant protein to prepare polycional antibodies,and the titer of polyclonal antibody was determinated.Indirect ELISA was developed with the recombinant protein of T. pallidum as coating antigen to detect 80 control sera and 150 FTA-ABS-positive sera.Results The recombinant plasmid pQE32/Tp0821 was constructed and a fusion protein with expected molecular weight was expressed.Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the antibody titer reached 1:6400.Compared with FTA-ABS test,the indirect ELISA showed a sensitivity and specificity of 92.6%and 98.6%,respectively,in the detection of control and clinical sera.Conclusion The recombinant protein Tp0821 shows excellent immunocompetence,which can be applied to the serological diagnosis of syphilis.
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Deregulation of cell cycle is one of important mechanisms leading to breast cancer. It has been revealed that cyclin E and p27 are the most important regulatory factor for cell cycle control and a better marker for evaluating prognosis of breast cancer compared to other biological markers.
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Objective To detect the expression of Cytokeratin 19 (CK19) and human mammaglobin (hMAM) mRNA in the peripheral blood of breast cancer patients dynamically and to evaluate its clinical significance.Methods The expression of CK19 and hMAM mRNA was evaluated by real-time fluorescent quantitative reverse transcriptase pelymerase chain reaction in the peripheral blood of 40 breast cancer patients and 10 healthy volunteers.Results The expression of CK19 and hMAM in the peripheral blood of 40 breast cancer patients were higher than healthy donors.The positive expression rate of CK19 and hMAM was descending tendency during treatment.The different staging expression rate had no differences between before treatment and during treatment,but after treatment the expression rate had remarkably differences (CK19:P=0.044;hMAM:P=0.005).If the expression of CK19 and hMAM was continual positive,the rate of recurrence and metastasis would be higher (P＜0.001).Coneluslons Real-time fluorescent quantitative RT-PCR may detect the expression of CK19 and hMAM in the peripheral blood of breast cancer patients,and it is a sensitive and specific technique.The resuits suggest a possible use of this approach for evaluating prognosis,monitoring recurrence.
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Objective To examine the advanced oxidation protein products (AOPP) in patients with acute coronary syndrome(ACS) and discuss the relationship between oxidative stress with the development of atherosclero-sis(AS). Methods Plasma were collected in 59 acute myocardial infarction (AMI) patients including 35 patients underwent selective PCI,24 patients underwent emergency PCI,43 unstable angina pectoris(UA) patients and 10 non-coronary artery disease (non-CAD) patients. All cases underwent coronary angiography (CAG). Plasma was collected immediately,post-24 hours and post-48 hours after admission. AOPP was determined by measurements of absorbance (A) at 340 nm under acidic conditions via spectrophotometry. Results AOPP was (236.42±30.41) ( n = 35 ), ( 207.84±29.50 ) mmol/L ( n = 35 ), ( 227.79 ± 35.18 ) mmol/L ( n = 31 ) respectively immediately, post-24 hours and post-48 hours after admission in AMI ( selective PCI ), ( 239.95 ±39.94 ) mmol/L ( n = 43 ), (175.92 ±29.46) mmol/L(n =38) ,and (156.54 ±28.29) mmol/L(n =35) in UA group and (57.41 ± 13.60) mmol/L( n = 9 ), (56.11 + 11.90) mmol/L ( n = 10 ) and ( 61.75 ± 12.28 ) mmol/L ( n = 8 ) in non-CAD group. Compared with normal group ( without CAD ) , significantly higher plasma AOPP was detected in AMI ( selective PCI) and UA patients ( P < 0.05 ). AOPP level was significantly increased in AMI selective PCI patients as compared with that of emergency PCI group immediately and post-24 hours after admission( P <0.01 ) ,and post-48 hours after admission( P < 0.05 ), but there was no statistical significance between emergency PCI and UA group( P > 0.05 ). Conclusions Oxidative stress is an important step in the development of atherosclerosis, and the higher levels of AOPP in ACS patients show that AOPP may be as good markers in these patients.