Effect of 1-methyltryptophan on lipopolysaccharide-induced permeability and apoptosis in human umbilical vein endothelial cells / 中华急诊医学杂志

Objective:To explore the effect of 1-methyltryptophan (1-MT) on lipopolysaccharide (LPS)-induced permeability and apoptosis of human umbilical vein endothelial cells (HUVECs).Methods:HUVECs were treated with phosphate buffer saline (PBS, control group), 1 μg/mL LPS (LPS group), and LPS combined with 1 mmol/L 1-MT (1-MT group). The expression levels of the p120 concatemer (p120ctn), vascular endothelial (VE) cadherin, caspase-3, and DNA repair enzyme polyadenylate ribose polymerase-1 (PARP) after incubation at 8 h were detected using Western blot. The concentrations of kynurenine (Kyn) after incubation at 2, 4, 6, and 8 h were measured by high-performance liquid chromatography, and indoleamine2, 3-dioxygenase (IDO) activity was calculated. Comparisons among groups were performed using the LSD- t test. Results:Compared with the control group, the expression of caspase-3 [(74.01±7.91)% vs (157.14±7.63)%, P<0.01] and the concentration of Kyn were significantly up-regulated, while the expression of p120ctn [(49.12±2.15)% vs (37.61±1.80)%, P<0.01], VE-cadherin [(107.70±7.01)% vs (90.66±2.58)%, P=0.027], and PARP-1 [(67.95± 3.08)% vs (57.93±5.26)%, P=0.038] were significantly down-regulated, and IDO activity was significantly increased in the LPS group ( P<0.05). Compared with the LPS group, the expression of caspase-3 [(157.14±7.63)% vs (110.74±7.89)%, P<0.01] was significantly down-regulated, while the expression of p120ctn [(37.61±1.80)% vs (47.19±0.82)%, P<0.01], VE-cadherin [(90.66±2.58)% vs (107.27±9.89)%, P=0.029], and PARP-1 [(57.93±5.26)% vs (74.12±4.90)%, P=0.005] were significantly up-regulated, and the activity of IDO was significantly decreased over time in the 1-MT group ( P<0.05). No significant differences were observed between the PBS and 1-MT groups in the protein levels of p120ctn, VE-cadherin, and PARP-1 protein as well as Kyn concentration and IDO activity ( P>0.05), while the expression of caspase-3 was increased in 1-MT group ( P=0.001). Conclusions:LPS aggravates the permeability of HUVECs, which can be reversed by 1-MT via inhibiting IDO activity and reducing Kyn concentrations. Moreover, 1-MT can also reduce apoptosis, which may be via increasing the expression of PARP-1 and reducing the expression of caspase-3, thus protecting endothelial cells.

The study on the ratio of carotid artery/vein cross-sectional area measured by ultrasound to evaluate volume responsiveness / 中华急诊医学杂志

Objective To investigate the application of the cross-sectional area ratio of internal jugular vein and common carotid artery (IJV/CCA) in the evaluating the volume responsiveness of critically ill patients. Methods The capacity of critically ill patients were prospectively assessed. The diameter and sectional area of the IJV and CCA were measured by bedside ultrasonography. The cross-sectional area ratio of IJV/CCA was calculated and compared with the variety of cardiac output (ΔCO) after passive leg raising (PLR). Then the correlation index between the cross-sectional area ratio of IJV/CCA and ΔCO was evaluated, and the sensitivity and specificity parameters of capacity status were assessed by the cross-sectional area ratio of IJV/CCA. Results Of 55 critically ill patients in this study, 34 cases had positive volume responsiveness, and 21 case negative volume responsiveness.The general clinical data of the two groups had no statistically significant difference. The cross-sectional area ratio of IJV/CCA in the positive group was significantly less than that of the negative group (1.38±0.55 vs. 2.16±0.68, P<0.01). There was a significant correlation between the IJV/CCA cross-sectional area ratio and the ΔCO value of PLR (r=-0.67, P<0.01). When the ratio of the cross-sectional area of IJV/CCA was 1.65, the sensitivity of the assessment capacity was 86.4％ and the specificity was 78.8％. Conclusions The use of portable bedside ultrasonography is a noninvasive, convenient and reliable method to evaluate the capacity state of the critically ill patients.

Functional effect of the long non-coding RNAs on HIV-1 infection in monocytes / 安徽医科大学学报

Objective To identify the expression of long non-coding RNAs(lncRNAs) and their putatively modulated cytokines in human immunodeficiency virus(HIV)-1 infected monocytes and to explore the role of lncRNAs in immunomodulation of HIV-affected host cells.Methods RNAs arrays were used to screen the lncRNAs differentially expressed in HIV-infected monocytes and subsequently confirmed by quantitative real time PCR (qRT-PCR).A siRNA was designed and transfected to the human monocytes, followed by interleukin(IL)-1β, IL-10 and tumor necrosis factor(TNF)-α detection with ELISA.Results The results of the screening assays and qRT-PCR showed that the expression of lncRNA-n266623 was up-regulated in monocytes after HIV-1 infection;the secretions of IL-1β,IL-10 and TNF-α in human monocytes were significantly enhanced following transfection of siRNA targeting lncRNA-n266623 in the monocytes.Conclusion The HIV-1 infection can promote the expression of lncRNA-n266623 which may inhibit the expressions of IL-1β, IL-10 and TNF-α in monocytes and negatively regulate the immune response to HIV-1 infection.

Study of IFN-inducible double-stranded RNA dependent protein kinase on antiviral activity of HBV in vitro / 中国药理学通报

Aim To construct and express the eukary-otic expression vector of double-stranded RNA-depend-ent protein kinase (PKR)fusion green fluorescent and analyse its antiviral activity of HBV in vitro.Methods The PKR gene was cloned into an empty expression vector pEGFP-N1 using molecular clone technology. After being confirmed by restriction enzyme digestion and sequencing methods,the recombinant plasmid was named as pEGFP-PKR that was subsequently transfect-ed into HepG2.2.15 cells using LipofectamineTM2000. The expression level of PKR in HepG2.2.15 cells was confirmed by using fluorescent microscopy. Mean-while,HBV DNA and HBsAg/HBeAg were detected by real-time PCR and electrochemiluminescence meth-od,respectively.Results Both restriction enyme di-gestion and sequencing assays showed that the recombi-nant vector pEGFP-PKR was successfully constructed in our study.Fluorescent microscopy observation indi-cated that the fusion protein pEGFP-PKR expressed ef-ficiently in HepG2.2.15 cells.Moreover,compared with the empty vector group,the expression of HBV antigen in supernatants was significantly decreased (P<0.05 ).However,the extracellular HBV DNA ex-pression was not inhibited significantly.Conclusion In vitro,PKR proteion has certain antiviral activity of HBV.

Recombinant expression of Schistosoma japonicum fructose-1,6-bisphos-phate aldolase and its expression in different developmental stages of S. ja-ponicum / 中国血吸虫病防治杂志

Objective To clone express and purify Schistosoma japonicum fructose?1 6?bisphosphate aldolase SjFBPA in E. coli and observe its expression in different developmental stages of S. japonicum. Methods FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid and inducibly expressed with IPTG in E. coli BL21. SDS?PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA rSjFBPA . Then rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS?PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore SjFBPA mRNA was ana?lyzed in different developmental stages of S. japonicum by RT?PCR. Results SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro?tein could specifically reactive to the anti?His?tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT?PCR showed that SjFBPA mRNA was expressed in cercaria schistosomulum adult worm and egg of S. japonicum. Conclusion SjFBPA is successfully recombined and expressed in a prokaryotic system and SjFBPA mRNA is expressed in cercaria schistosomulum adult worm and egg of S. japonicum.

Clinic study of serum product advanced oxidation protein changes of patients with chronic obstructive pulmonary disease / 中国综合临床

Objective To investigate the serum levels of advanced oxidation protein products (AOPP) in patients with chronic obstructive pulmonary disease (COPD) as well as explore the relevance of its clinical significance AOPP and COPD.Methods Fifty-four patients with mild/moderate COPD (COPD group) were enrolled in this study,who were treated in the No.100th Hospital of the Chinese People's Liberation Army from Apr.2011 to Nov.2013.Thirty healthy volunteers (control group) at same period were selected as control group and the general condition of two groups were matched.AOPP,superoxide dismutase(SOD) and malondialdehyde (MDA) levels were measured.Results The serum AOPP,MDA and SOD in control group were (45.78 ± 12.54) μmol/L,(2.96 ± 0.55) μmol/L and (78.40 ± 8.37) kU/L respectively.And its were (68.93 ± 10.62) μmol/L,(6.07 ± 2.44) μmol/L and (53.66 ± 5.99) kU/L respectively in COPD group.The differences were statistically significant (t =-8.57,-9.14,14.38 ; All P values were less than 0.01).The serum AOPP,MDA and SOD in mild COPD group were (65.56 ±9.65) μmol/L,(4.21 ± 1.83) μmol/L and (62.97 ± 6.28) kU/L respectively,and (71.79 ± 11.37) μmol/L,(7.43 ± 3.12) μmol/L and (41.25 ± 5.89) kU/L respectively in moderate CODP group.The differences were statistically significant (t =-2.17,-4.80,13.00; P ＜ 0.05 or P ＜ 0.01).Conclusion The increased levels of serum AOPP is important pathological changes in patients with COPD,which may be involved in the occurrence and development of COPD,and is the reaction of oxidative stress injury in patients with COPD early sensitive indicator.

The effect of salvianolate combined Qumei trimetazidine on chronic heart failure / 中国综合临床

Objective To explore the effect of salvianolate combined with Qumei trimetazidine on cardiac function in patients with chronic heart failure.Methods Seventy-four patients with chronic heart failure were randomly divided into treatment group and control group (37 cases per group).Patients in control group were treated with the regular treatment scheme including digitalis,diuretics,vasodilators,angiotensin converting enzyme inhibitor(ACEI),angiotensin receptor blockers (ARB) or β blocker therapy for 24 weeks treatment.Patients in treatment group were given the regular treatment scheme plus salvianolic acid and Qumei trimetazidine treatment,of which,the dose of salvianolic was 0.2 g into 5％ glucose injection 250 ml or 0.9％ sodium chloride injection 250 ml by intravenous injection,1 times/day,and Qumei trimetazidine for 20 mg,3 times/day,for 24 weeks.Cardiac function was observed in patients of two groups before and after treatment.The level of brain natriuretic peptide (BNP) was measured.Results Heart function were improved,the total effective rate in treatment group was 91.9％ (34/37),higher than that of control group (70.3％ (26/37),x2 =5.638,P ＜ 0.05).In treatment group,left ventricular ejection fraction (LVEF),stroke volume (SV),cardiac output (CO) of patients after treatment were (52 ± 7) ％,(65.10 ± 12.87) ml,(5.65 ± 1.18) L/min respectively,significant different from that before treatment ((39 ±5)％,(46.53 ± 12.14) ml,(4.79 ± 1.02) L/min,and the differences were statistic significant (t =9.192,6.384,3.352,P ＜ 0.05).Meanwhile,in treatment group,systolic pressure,diastolic pressure,heart rate,left ventricular end diastolic diameter (Dd),left ventricular diastolic posterior wall thickness(PWT),interventricular septal thickness (IVST),left ventricular mass (LVMW),plasma brain natriuretic peptide of patients after treatment were (105 ± 8) mmHg,(75 ± 9) mmHg,(76±8) time/min,(48.7 ±3.7) mm,(9.1 ±1.4) mm,(8.7 ±1.2) mm,(170±59) g,(104.1 ±19.5) ng/L respectively,significant different from that of before treatment((134 ± 12) mmHg,(84 ±8) mmHg,(118 ±11) time/min,(55.2 ±7.8) mm,(11.7 ±2.3) mm,(10.5 ±2.4) mm,(228 ± 111) g,(568.7±179.5) ng/L t=-12.231,-4.546,-18.782,-4.579,-5.874,-4.080,-2.806,15.652,P ＜ 0.01).The same trend was seen in control group in terms of LVEF,SV,systolic blood pressure,heart rate,PWT,plasma BNP before and after treatment(LVEF:(38 ±6)％ vs.(43 ± 8)％ ;:(46.76 ± 11.80) ml vs.(58.69 ± 11.58) ml; systolic blood pressure:(132 ± 10) mmHg vs.(116 ± 11) mmHg; heart rate:(116 ± 10) time/min vs.(77 ±9) time/min;PWT:(11.5 ±2.6) mm vs.(10.4 ±2.0) mm;plasma BNP:(570.2 ± 177.3) ng/L vs.(211.6 ± 21.2) ng/L;t =3.041,4.389;-6.546,-17.632,-2.039,12.21 ;P ＜ 0.05 or P ＜ 0.01).Moreover,after treatment,systolic pressure,diastolic pressure,LVEF,SV,CO,Dd,PWT,IVST,LVMW,plasma brain natriureticpeptide in treatment group were significantly better than that of control grouo (t =-4.919,-2.867,5.510,2.252,2.581,-2.319,-3.238,-3.628,-2.231,-22.701,P ＜0.01 or P ＜ 0.05).Conclusion The effect of salvianolate combined Qumei trimetazidine on treating chronic heart failure is significant,and there is a reverse effect on the left ventricle.

The clinical efficacy of salvianolate on patients with chronic heart failure and plasma brain natriuretic peptide levels / 中国综合临床

Objective To investigate the effect of salvianolate on chronic heart failure in patients with cardiac function and plasma brain natriuretic peptide effect.Methods Sixty-eight cases with chronic heart failure patients were randomly divided into treatment group and control group (34 cases for each group).Patients in control group were given the conventional treatment,in treatment groups were given conventional treatment plan plus salvianolic acid at dose of 0.2 g added 5％ glucose injection 250 ml (or 0.9％ sodium chloride injection 250 ml),1 times a day for 12 weeks.The cardiac function was recorded and brain natriuretic peptide level was measured before and after treatment.Results After 12 weeks of treatment,the total efficiency in treatment group was 91.2％ (31/34)) higher than that in control group(70.6％ (24/34)),and the difference was statistically significant (x2 =9.399,P ＜ 0.01).Before treatment,the left ventricular ejection fraction (LVEF),stroke volume(SV),cardiac output(CO) in treatment group were (38 ±6)％,(44.64 ± 11.03) ml,(4.81 ± 1.03) L/min respectively,differed from that after treatment ((51 ± 8) ％,(63.21 ± 11.94) ml,(5.67 ± 1.17) L/min),and there were significant differences between before and after treatment (t =-7.580,-8.975,-3.233 respectively; P ＜ 0.01).The levels of systolic blood pressure,diastolic blood pressure,heart rate,left ventricular end-diastolic internal diameter (Dd),the left ventricular diastolic wall thickness (PWT),diastolic interventricular septal thickness (IVST),left ventricular mass (LVMW),brain natriuretic peptide in treatment group before treatment were (131 ± 11) mmHg,(85 ± 7) mmHg,(116 ± 9) times/min,(55.1 ± 7.9) mm,(11.8 ± 2.4) mm,(11.4 ± 2.3) mm,(231 ± 112) g,(572.9 ± 183.6) ng/L respectively,significant differed from those of after treatment((104 ± 7) nmHg,(76 ± 8) mmHg,(75 ± 7) times/min,(48.8 ± 3.9) mm,(9.2±1.3) mm,(8.9± 1.1) mm) (172 ±57) g,(101.8 ± 18.5) ng/L respectively),and the differences were significant (t =12.075,4.937,20.961,4.169,5.556,5.721,2.738,14.886 ; P ＜ 0.01).The levels of LVEF,SV in control group before treatment were (37 ±7)％ and (44.87 ± 10.82) ml,differed from those of after treatment((42 ± 9)％ and (56.70 ± 10.60) ml;t =-2.556,-4.554;P ＜ 0.01).The systolic blood pressure,heart rate,Dd,IVST,plasma brain natriuretic peptide in control group before treatment were (130 ±12) mmHg,(114 ± 10) times/min,(54.8 ± 8.7) rmm,(11.3 ± 2.6) mm,(574.1 ± 181.4) ng/L respectively,significantly differed from those of after treatment ((115 ± 9) mmHg,(76 ± 8) times/min,(50.6 ±8.3) mm)(9.9±1.3) mm,(215.7 ±23.2) ng/L;t=5.830,17.304,2.037,2.806,11.427;P＜0.01 or P ＜ 0.05).The levels of systolic blood pressure,diastolic blood pressure,LVEF,SV,CO,PWT,IVST,plasma brain natriuretic peptide in treatment group were better than that in control group (t =-4.601,-3.093,4.358,3.253,2.802,-3.066,-3.425,-27.985,P＜0.01).Conclusion Salvianolate is proved to be better drug on treating chronic heart failure curative with left ventricular reverse effect and less adverse reaction.

Timing effect of paeoniflorin on liver fibrosis in mice infected with Schistosoma japonicum / 中国人兽共患病学报

To probe the effect of paeoniflorin on periovular granuloma and liver fibrosis in mice infected with Schistosoma japonicum in different times of infection and the treatment with praziquantel (PZQ). The models of hepatic fibrosis induced by S.japonicum were established by exposure of BALB/c mice percutaneously through the tail to cercariae of S.japonicum. and mice with treatment were randomly divided into 3 groups: i.e. groups of pre-treatment (I), group of simultaneous treatment (Ⅱ) and group of post-treatment (III). All groups, except the normal control group, were orally introduced with PZQ. And mice in the paeoniflorin-treated group and control group were separately introduced with paeoniflorin and 0.5% sodium carboxymethycellulose respectively. The treatments in group I, II and III were started 30 days before PZQ usage, simultaneously with PZQ or 30days-after PZQ usage respectively. Mice in these groups were sacrificed on the 102, 132 or 162 days after infection. Then the serum levels of hyaluronic acid (HA), amino-terminal peptide of type III procollagen (PIIIP) and liver hydroxyproline (Hyp) were detected. The histopathology was examined by HE and Masson staining; the degree of hepatic fibrosis and the area of egg granuloma were analyzed. The expression of collagen I was examined by immunohistochemical method. It was found that the area of granuloma and degree of hepatic fibrosis in the paeoniflorin-treated groups in group I and III were significantly lower than those in the model control groups. Also, paeoniflorin could induce decreas expression of collagen I. Meanwhile the levels of serum HA, PIIIP and liver Hyp were all reduced in comparison with those in the control group (P<0.05 or P<0.01). However, in group Ⅱ, no significant difference was noted between the treated and the control group in most data. Paeoniflorin also showed the effects to reduce the size of periovular granuloma and to reduce the expression of type I collagen, thereby to resist the development of hepatic fibrosis caused by S. japonicum.-It is evident that PAE shows an efficaciously therapeutic effect on the development of liver fibrosis of shistosomiasis, whenever it is administered before or after the usage of schistosomicides.

Inhibition of IL-13-induced collagen I by sIL-13Rα2 in NIH-3T3 fibroblast cells and the profile of sIL-13Rα2/IL-13 in mice with schistosomiasis / 中国人兽共患病学报

To determine the inhibition of IL-13 by recombinant sIL-13Rα2 in NIH-3T3 fibroblast cells for its potential therapeutic value in hepatic fibrosis caused by Schistosoma japanicum in mice . IL-13 and sIL-13Rα2 from liver of BALB/c mice infected with S.japonicum at different infection time (weeks 0,6,8,10 and 12) were analyzed by ELISA and RT-PCR. The recombinant sIL-13Rα2 expression plasmidwas constructed, followed by transfection into NIH-3T3 fibroblast cells. TypeⅠcollagen produced by NIH-3T3 cells were examined by RT-PCR and Western blotting. It was demonstrated that the expression of IL-13 increased gradually after infection, reached peak density (16.1586 pg/mL)at week 8 and then reduced but was still higher than the level of control mice(3.4146 pg/mL;P =0.017 ). The secretion of sIL-13R α2 reached to its peak 10 weeks after infection(4827.426 pg/mL)and then reduced slowly but still higher than normal(4057.112 pg/mL; P=0.021). Meanwhile, the changes in mRNA level of IL-13 and sIL-13R α2 were coincided with that examined by ELISA. Both IL-13 and sIL-13Rα2 reached their peak density (P=0.033) at week 8 and 10 (P=0.025) respectively, and they were followed by a slower degree of decrease. The sIL-13Rα2 could significantly inhibit the effect of IL-13 on NIH-3T3 fibroblast cells, showing decreased mRNA level(P =0.012)and protein level of typeⅠcollagen compared with normal groups(P =0.031). It is concluded that the sIL-13Rα2 can inhibit the effect of IL-13 on NIH-3T3 fibroblast cells which leads to a reduced production of typeⅠcollagen, demonstrating its potential therapeutic value in hepatic fibrosis of schistosomiasis.

Sag5b:a novel gene for differentiation of strain virulence of Toxoplasma gondii / 中国人兽共患病学报

A promising genetic marker, sag5b, was cloned and expressed and the difference of the genes between highly virulent strain (RH) and less virulent strain(Prugniaud) of Toxoplasma gondii was compared. The PCR-generated product of sag5b was subcloned into T easy vector and plasmid pET28a consecutively. The fusion expression was induced by IPTG and identified by SDS-PAGE and Western blotting. The immunoreactivity of recombinant SAG5B was identical to that of native SAG5B on the membrane of tachyzoites of RH strain. The brains of mice infected with Prugniaud strain of T. gondii were homogenated. Sag1 was successully cloned by PCR from both RH strain tachyzoites and the homogenized brain tissues of mice infected with low virulent strain of Prugniaud,whereas sag5b was only detected in RH strain but not in Prugniaud strain, indicating that sag5b could be used as a genetic marker for differentiation of strain virulence. Expression and vaccination of the virulence-associated gene into mice failed to induce obvious protective immunity against the challenge of RH strain.

Expression of 14-3-3 sigma gene in patients with breast cancer and its clinical significance / 临床检验杂志

Objective To investigate expression of 14-3-3 sigma gene in patients with breast cancer and its clinical significance.Methods Expression of 14-3-3 sigma gene was semi-quantitated by using RT-PCR and western-blot in 40 specimens of breast cancer and 18 specimens benign breast disease tissue.Results Of 40 cases of breast cancer 35(87.5%)were negative for 14-3-3 sigma gene in RT-PCR,32(80%)were negative in Western-blot,and 31(77.5%)were negative in both RT-PCR and western-blot.Besides,the expression in 2 cases was down-regulation in both the 2 method.In 18 specimens with benign breast disease tissue the expression of 14-3-3 sigma gene was detectable,which was demonstrated by RT-PCR or western-blot.Conclusion Inactivation and down-regulation of 14-3-3 sigma gene is a frequent event in breast cancer,and it may contribute to diagnosis of breast cancer.

AMPLIFICATION, CLONING AND PROKARYOTIC EXPRESSION OF GENE FOR MAJOR SURFACE ANTIGEN OF TOXOPLASMA GONDII / 中国人兽共患病学报

Aim To amplify P30 gene and express P30 fusion with GST Methods P30 gene was smplified from T. gondii chromosomal DNA and ligated to pGEM-T and pGEX-4T-1. Screening-positive recombinants were induced for expression, which was subsequently detected by WB Results P30 gene was amplified and GST-fusion was confirmed by rabbit antiT. gondii serum. Conclusions The construction of pGEM-T-P30 and pGEX-4T-1-P30, together with the recombinant protein would lay a base for further investigation of P30 at a molecule-level and application to diagnosis and vaccination

Immunolocalization of the Signaling Protein l4-3-3 of Schistosoma japonicum / 中国寄生虫学与寄生虫病杂志

Objective To study the localization of the signaling protein 14-3-3 of Schistosoma japonicum (Sj14-3-3)in the parasite. Methods Cercariae were collected from the infected Oncomelania hupensis for the infection of rabbits. Fifteen-day-old schistosomula and adult worms obtained from infected rabbits 15 and 42 days post-infection were used for frozen sections and indirect immunofluorescence staining with monoclonal antibody to rSj14-3-3. Results The results showed that the Sj14-3-3 distributed mainly in the tegument, subtegument, muscle, and parenchyma of both adult worms and 15-day-old schistosomula. Conclusion The wide distribution and large sites of Sj14-3-3 in the parasite were clearly demonstrated, which established a significant clue for further studies of biologic actions and application of 14-3-3 protein.

Protective Immunity Induced by Recombinant Signaling Protein 14-3-3 Vaccine of Schistosoma japonicum / 中国寄生虫学与寄生虫病杂志

Objective To evaluate the immunoprotective effect of Schistosoma japonicum (Chinese strain) recombinant signaling protein 14-3-3(rSj14-3-3), and to observe the synergism of rSj14-3-3 and rSjGST proteins as candidate vaccine and the effect of ??-T cells activated by Mtb against Schistosoma japonicum. Methods BALB/c mice immunized with rSj14-3-3 and rSjGST purified through SDS-PAGE, electroelution and dialysis were challenged by cercaria infection. Six weeks after challenging infection, the mice were killed and the worm and egg reduction rates were calculated. Results Worm reduction rate was found to be 32.20% in rSj14-3-3+Freund adjuvant group, 31.10% in rSj14-3-3+rSjGST+Freund adjuvant group, 27.96% in rSj14-3-3+Mtb group, 26.00% in rSj14-3-3+rSjGST+Mtb group, and 27.10 % in rSjGST+Mtb group, respectively, number of eggs in liver tissue was reduced by 50.40%, 53.30%, 51.10%, 58.60% and 51.30%, respectively. Conclusion rSj14-3-3 could induce partial immunity against Schistosoma japonicum in BALB/c mice, and might serve as a candidate vaccine; ??-T cell activated by Mtb played a role in anti-Schistosoma japonicum similar to the immune reactions induced by Freund adjuvant, but no synergistic effect combined with rSjGST was observed.

Cloning and Expression of the Signaling Protein 14-3-3 of Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To clone and express the cell signaling protein 14-3-3 gene from Toxoplasma gondii RH strain. Methods Toxoplasma RH strain tachyzoites, which maintained by mouse passage, were harvested from ascites of mice and genomic DNA was prepared. A pair of primers were designed and synthesized based on the sequence of Toxo 14-3-3 cDNA. A specific fragment of Toxo 14-3-3 gene was obtained by RT-PCR amplification from Toxoplasma genomic DNA. The PCR products were ligated to pGEM-T. The EcoRI / Xho I restricted fragments, confirmed by PCR and EcoRI / XhoI digestion, were cloned into expression vector pET28a and the recombinants were transformd into E.coli BL21. Fusion expression was induced by isopropyl-beta-D-thiogalactoside (IPTG) and confirmed by Western blotting with rabbit anti-Toxoplasma sera. Results The molecular size of Toxo 14-3-3 was 803 bp, which is highly homologous to the previous report cloned from the parasites of intestinal epithelial stage in cat. High expression was obtained in pET28a/ Toxo 14-3-3/E.coli BL21 when confirmed by Western blotting. Conclusion The recombinant construction of Toxo 14-3-3 was generated and expression was induced.

The Role of TGF?1 and IL-13 in Cellular Signal Transduction of Hepatic Fibrosis of Schistosomiasis / 中国寄生虫学与寄生虫病杂志

Liver fibrosis is characterized by an abnormal hepatic accumulation of extracellular matrix (ECM) that results from both increased deposition and reduced degradation of collagen fibres. Some studies show that transforming growth factor ?1(TGF-?1), alternatively activated macrophage (aaM) and interleukin 13(IL-13) play a key role in the evolution of fibrosis, of which TGF-?1 and IL-13 become research hotspots. TGF-?1 mainly activates hepatic stellate cells (HSC) through TGF-?1/Smad signal pathway, while IL-13 seems to play a rather crucial role through JAK-STAT6 signal pathway. aaM is an important source of TGF-?1 and activated with Il-13. This paper reviews the role of those signaling molecules in cellular signal transduction of hepatic fibrosis of schistosomiasis japonica, and provides some targets for future drug development.

Transformation of Schistosomulae by Electroporation and Transient Expression of the Enhanced Green Fluorescent Protein (EGFP) Gene / 中国寄生虫学与寄生虫病杂志

Objective To explore the possibility of heterogenous gene to express in juvenile Schistosoma japonicum and the application of electroporation in transformation of schistosomulae. Methods The plasmids of pEGFP-C1 were introduced into mechanically transformed schsitosomula with electroporation. The presence, transcription and translation of the transgene in electroporated schistosomula were confirmed by PCR, RT-PCR and Western blotting analysis respectively using the genomic DNA, total RNA and protein extracted and isolated from schistosomula cultured in vitro for 48 hours. Meanwhile, localization of EGFP within electroporated schistosomula was performed with confocal laser scanning microscope. Results 760 bp and 276 bp amplified products by PCR and RT-PCR were found coincident with the expected size and expression of EGFP gene in elctroporated schistosomula was confirmed by Western blotting. Fluorescence of EGFP was localized in tegument and subtegument of the electroporated schistosomula with confocal microscopy, especially in the anterior part of the worm. Conclusion The heterogenous gene of EGFP has been successfully introduced into juvenile S. japonicum by electroporation and the expression of transgene was confirmed with molecular and microscopical methods.

Effect of astragalosides on schistosomal hepatic fibrosis in vitro / 中国药理学通报

Aim To observe the effect of astragalosides on proliferation and collagen synthesis of HSC-T6 cells driven using schistosoma japonicum soluble egg antigen-activated macrophage conditioned medium(SEA-MCM).Methods SEA-MCM was prepared by injection of Schistosoma japonicum SEA via mice peritoneal.The proliferation and collagen synthesis of HSC-T6 cells stimulated with SEA-MCM were measured using MTT colorimetric assay and()~3H-proline incorporation respectively.Results The proliferation and collagen production of HSC-T6 cells were significantly promoted by SEA-MCM.They were significantly suppressed after being treated with AST(32.5、65、130 mg?L~(-1)) showing concentration-dependent effect.Conclusion:Astragalosides may inhibit HSC-T6 cells proliferation and collagen production that was driven by SEA activated MCM in vitro.

Role of p38 and phosphatidylinositol 3 kinase(PI3K) in the signal transduction of apoptosis of activated human ??T cells induced by restimulating with Mycobacterium Tuberculosis antigen / 中国免疫学杂志

Objective:To establish the model of apoptosis of activated human ??T cells induced by restimulating with Mycobacterium tuberculosis antigen(Mtb-Ag). To investigate the roles of p38 and phosphatidylinositol 3 kinase(PI3K) pathways in the apoptosis of activated human ??T cells induced by restimualting wih Mtb-Ag.Methods:Mtb-Ag activated human T cells(MtbAT) were cultured for 15 days to 25 days and restimulated with three concentrations of Mtb-Ag for 24 hours, and the apoptosis of ??T cells were measured by flowcytometry(FCM) using Annexin-V-FITC/PI staining. Mtb-AT were restimulating with Mtb-Ag(10 ?g/ml) for 3, 6, 12 and 24 hours, the apoptosis of ??T cells were detected. Mtb-AT cells were pretreated with SB203580(an inhibitor for p38 pathway), or LY294002(an inhibitor for PI3K pathway) for 60 minutes, and restimulating with Mtb-Ag for 3 hours, the apoptosis of ??T cells were detected.Results:Both 10.0 and 20.0 ?g/ml Mtb-Ag significantly induced the apoptosis of ??T cells(P0.05). Compared with control, the apoptosis of ??T cells could be significantly induced by restimulating MtbAT with Mtb-Ag(10.0 ?g/ml) for 3, 6, 12 and 24 hours(P0.05) in the percentages of apoptosis of ??T cells restimulated by Mtb-Ag(10.0 ?g/ml) between for 3 hours and for 24 hours, the percentages of apoptosis of the latter is higher than the former about 7.55%. The apoptosis of ??T cells induced by restimualting wih Mtb-Ag could be inhibited by SB203580(80.0 ?mol/L) or LY294002(10.0 ?mol/L), the inhibition rate of apoptosis was 91.6% and 43.1%, respectively.Conclusion:We established the model of apoptosis of activated human ??T cells by means of using Mtb-Ag(10.0 ?g/ml) to restimulate activated ??T cells for 3 hours. The test of inhibitors of signalling molecule suggested the signalling pathways including p38 and PI3K, participated in the apoptosis of activated human ??T cells restimulated by Mtb-Ag.