Objective To construct DNA vaccine expressing Mycobacterium tuberculosis(Mtb) immunodominant antigen Ag85A and analyze its anti-tuberculosis T cell responses in BCG primed-mice after DNA vaccination boosting.Methods The coding gene of Ag85A mature fragment was amplified by PCR with H37Rv genomic DNA as template,and then cloned into the eukaryotic expression vector pVAX1 to construct Ag85A DNA vaccine.After purification,Ag85A DNA vaccine was injected intramuscularly twice in BCG primed-mice with BCG vaccination and DNA vaccination alone as control.Eight weeks post-vaccination,spleen lymphocytes were separated and were then used to analyze Mtb antigen specific effector T cell response and polyfuntional IFN-γ/TNF-α/IL-2 secreting CD4+ T cell frequencies and intensities,and CD8+T cell responses by IFN-γ ELISPOT assay and intracellular staining,respectively.Results Compared to BCG vaccinated-and DNA vaccinated-mice,Ag85A DNA boosting not only enhanced significantly BCG primed-mice IFN-γ+TNF-α+IL-2+,IFN-γ+ IL-2+,TNF-α+IL-2+ and IL-2+ CD4+ T cell frequencies and IL-2 secretion,but also improved significantly IFN-γ-secreting and IL-2-secreting CD8+ T cell frequencies.Condusion Ag85A DNA vaccine was constructed successfully and was demonstrated to enhance significantly BCG primed-mice Mtb antigen specific CD4+ and CD8+ T cell responses when boosting,which is beneficial to improve BCG immunogenicity and its waning immune protection against Mtb.

AIM: To investigate the ethanolic and aqueous extracts from Sancao prescription (Spica prunellae, Oldenlandia diffuse (willd) Roxb, Herba agrimoniae) on the proliferation of human lung adenocarcinoma cell line (A549). METHODS: 95% ,60% and 30% ethanolic extract and aqueous extract were prepared from Sancao pre-scription. The MTT assay was used to determine the inhibitory action against the proliferation of A549. RESULTS: IC_(50) of 60% ethanolic extract over A549 was one of the lowest in extracts. Combination of 60% and 90% ethanolic extract showed the synergistic antitumour activity. CONCLUSION: Ethanolic extract of Sancao prescription has and effect on human hung adenocarcinoma(A549).

Objective To investigate the effects of aquaporin 3 (AQP3) and phospholipase D2 (PLD2) on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431.Methods Three small interfering RNAs (siRNAs) were constructed targeting the AQP3 and PLD2 genes separately,and transfected into A431 cells using liposomes.Then,fluorescence quantitative PCR was performed to find the most efficient siRNAs.Western blot was conducted to detect the protein expression levels of AQP3 and PLD2 in A431 cells after transfection with the selected AQP3-siRNA and PLD2-siRNA.Some A431 cells were divided into five groups:normal control group without any treatment,transfection reagent group treated with the oligofectamine reagent only,negative control group transfected with the negative control siRNA,AQP3-siRNA group transfected with the selected AQP3-siRNA,PLD2-siRNA group transfected with the selected PLD2-siRNA.After additional culture,cell counting kit-8 assay was performed to evaluate the proliferation of A431 cells,flow cytometry to detect the apoptosis of A431 cells after annexin V-fluorescein isocyanate/propidium iodide double-staining.Statistical analysis was carried out by the paired t test.Results The transfection with AQP3-siRNA and PLD2-siRNA induced a significant decrease in the mRNA and protein expressions of AQP3 and PLD2 respectively in A431 cells when compared with the untransfected cells.Compared with the negative control group,the proliferation of A431 cells was significantly decelerated at 24,48 and 72 hours after transfection in the AQP3-siRNA group (t =24.10,11.00,9.54,respectively,all P ＜ 0.01) and PLD2-siRNA group (t =30.47,7.02,8.73,respectively,all P ＜ 0.01).A significant increase was observed in the apoptosis of A431 cells at 48 and 72 hours after transfection with AQP3-siRNA (t =11.36,20.91,respectively,both P ＜ 0.01),and at 72 hours after transfection with PLD2-siRNA (t =4.86,P ＜ 0.05) compared with the negative control group.Conclusion The down-regulation of AQP3 and PLD2 expressions by siRNA can inhibit the proliferation,but induce the apoptosis,of A431 cells.

Objective To explore the value of transthoracic echocardiographic suprasternal view in　the diagnosis of the subtype of the patent ductus arteriosus(PDA).Methods Sixty-five cases with PDA were examined by transthoracic echocardiographic suprasternal view and parasternal great artery short axis view respectively before closure therapy.The diameters of both the aotic and pulmonary side were detected,and subtype diagnoses were made.The results were compared with those from digital subtraction angiography (DSA).Results The demonstrated rates of PDA were 100％ on the parasternal great artery short axis and suprasternal views.Of the 65 cases,12 cases,19 cases and 19 cases were the funnel type of PDA checked on the parasternal great artery short axis view,suprasternal view and DSA respectively.The demonstrated rate of the parasternal great artery short axis view was lower than that of the suprasternal view (x2 =5.14,P ＜0.025 ).The diameter of the aotic side of PDA was (8.31 ± 2.76)mm,(10.87 ± 3.26) mm and (11.15±3.29)mm and the diameter of the pulmonary side of PDA was (5.69± 2.82)mm,(5.75 ± 2.63)mm and (6.09 ± 2.78) mm respectively on the above two views and DSA.Conclusions The diameter of the aotic side of PDA can be accurately detected by using superasternal view,which would be helpful for the diagnosis of PDA subtype.

Objective To evaluate the application of three dimensional time-of-flight (3D-TOF) MRA in screening intracranial aneurysms in the population of Wenling community. Methods A total of 2 124 patients with suspicious intracranial aneurysm in Wenling community, who received 3D-TOF MRA and three dimensional digital subtraction angiography(3D-DSA) during the period from September 2011 to August 2012, were enrolled in this study. The epidemic data of intracranial aneurysm in Wenling community were analyzed, the effectiveness of 3D-TOF MRA in detecting intracranial aneurysm was assessed, and the consistency between 3D-TOF MRA and 3D-DSA (regarded as the golden standard) in detecting intracranial aneurysm was statistically analyzed. Results The results of 3D-TOF MRA showed that the morbidity of intracranial aneurysm in the population of Wenling community was 6.87% (146/2 124), among which the morbidities in males and females were 48.63% (n=71) and 51.37% (n=75) respectively; the mean age of patients was (41.2±11.6) years old. The accompanying diseases included hypertension, diabetes mellitus, arteriosclerosis and cerebrovascular lesions. 3D-TOF MRA examination revealed 149 intracranial aneurysms, among which misdiagnosis was made in 5 patients and missed diagnosis in 2 patients. The sensitivity, specificity and accuracy of 3D-TOF MRA in diagnosing intracranial aneurysm were 98.63% (144/146), 99.72%(1 773/1 778) and 99.67%(2 117/2 124) respectively. No statistically significant difference in measuring the longitudinal diameter and neck width of intracranial aneurysms existed between 3D-TOF MRA and 3D-DSA examinations (P>0.05). Conclusion In detecting intracranial aneurysm, 3D-TOF MRA carries higher sensitivity, specificity and accuracy, and its non-invasive advantage is more suitable for the screening of intracranial aneurysms.

AIM:To investigate the effects of resveratrol ( Res) on the proliferation of ARPE-19 cells and to ex-plore the possible mechanisms.METHODS:After ARPE-19 cells were treated with Res at concentrations of 0, 50, 100, 150, 200 and 300 μmol/L for 24 h, 48 h and 72 h, the effects of Res on the proliferation of the cells were tested by CCK-8 assay.The ARPE-19 cells were treated with Res at concentrations of 0, 100, 150 and 200 μmol/L for 48 h.The effects of Res on the cell cycle and apoptosis were detected by flow cytometry with Annexin V-FITC/PI staining.The protein expression of proliferating cell nuclear antigen (PCNA) was detected by immunofluorescent assay.The mRNA expression of PCNA, P21 and P27 was determined by real-time PCR.RESULTS:The results of CCK-8 assay showed that Res inhibited the prolifera-tion of ARPE-19 cells in a time-and dose-dependent manner.The treatment with Res for 48 h resulted in an arrest of cell cycle at S phase without increasing cell apoptosis.Res inhibited the protein expression of PCNA in ARPE-19 cells.The re-sults of real-time PCR showed that Res increased the mRNA expression of P21 and P27, and decreased the mRNA expres-sion of PCNA.CONCLUSION: Res inhibits the proliferation of ARPE-19 cells and induces the cell cycle arrest at S phase.The mechanism may be related to up-regulation of P21 and P27, and down-regulation of PCNA.

Objective To explore changes of left ventricular filling during the strain phase of Valsalva maneuver (VM) and its mechanism. Methods Thirty healthy volunteers were recruited to perform VM with a load of 40 mmHg. Left ventricular filling parameters (E, A, E/A ratio, e and E/e ratio) were determined by echocardiography at baseline,at the first beat and at the second beat during the strain phase of VM,respectively. Results Compared to those at baseline, E, E/A ratio and E/e ratio increased ( P ＜0.05) while A and e did not change ( P＞0.05) at the first beat during the strain phase. Compared to those at the first beat during the strain phase,E, E/A ratio and E/e ratio decreased ( P ＜0.05) while A and e did not change ( P ＞0. 05) at the second beat during the strain phase. Conclusions Left ventricular filling decreased at the second beat during the strain phase of VM, which is different from the present knowledge that left ventricular filling would begin to decrease 4-5 beats later during the strain phase of VM. Positive intrathoracic pressure decreases left-side heart and pulmonary vessel' transmural pressure while increases the blood resistance, which may be the reason that E, E/A ratio and E/e ratio decreased at the second beat during the strain phase of VM.

[ ABSTRACT] AIM:To investigate the effects of transplantation of bone marrow mesenchymal stem cells ( BMSCs) modified by bcl-2 gene on myocardial cell apoptosis, angiogenesis and cardiac function in the rabbit after acute myocardial in-farction ( MI) .METHODS:The rabbit BMSCs were isolated, cultured and purified in vitro.The BMSCs were transfected with adenovirus or adenovirus-Bcl-2.The rabbit model of MI was established by ligation of left anterior descending branch. The rabbits were injected with Ad-Bcl-2-BMSCs ( MI+Bcl-2-BMSCs group) , Ad-BMSCs ( MI+BMSCs group) and DMEM ( MI group) in infarction marginal zone 2 weeks after ligation.The cardiac function was evaluated by echocardiography.The apoptosis of myocardial cells was measured by TUNEL.The mRNA expression of VEGF was detected by real-time PCR.The expression of CD31 was examined by immunohistochemical staining, and new blood capillaries were counted at 4 weeks after BMSCs transplantation.The correlation of the above values with cardiac function was analyzed.RESULTS: The cardiac function was better, the apoptotic rate was lower, the mRNA expression of VEGF and the capillary density were higher in both MI+Bcl-2-BMSCs group and the MI+BMSCs group than those in MI group, and those in MI+Bcl-2-BMSCs group in-creased more obviously .The left ventricular ejection fraction ( LVEF) had a negative correlation with the myocardial cell ap-optosis rate.A positive correlation with the mRNA expression level of VEGF and the capillary density was also observed. CONCLUSION:The transplantation of BMSCs modified by bcl-2 gene significantly reduces the myocardial cell apoptosis, promotes angiogenesis, improves heart function of the rabbits with MI.

[ ABSTRACT] AIM:To investigate the different dose of perindopril on cardiac function in the rabbits with ische-mic cardiac dysfunction .METHODS:Male rabbits weighing 2.5~3.0 kg ( n=30) were randomly divided into 3 groups (n=10):high dose perindopril group (HD group), low dose perindopril group (LD group) and cardiac dysfunction group (CD group).The Left anterior descending coronary artery of the rabbits was ligatured for model preparation .In HD group, the rabbits were treated with perindopril split normal saline solution (1 g/L)2 mL· kg-1 · d-1 .In LD group, the rabbits were treated with perindopril split normal saline solution (0.33 g/L)2 mL· kg -1 · d-1.In CD group, the rabbits were treated with normal saline solution 2 mL· kg-1 · d-1 .Four weeks after treatment , the cardiac function was measured via echocardiography , the mRNA expression of angiotensin-converting enzyme 2 ( ACE2 ) and angiotensin type 2 receptor (AT2R) was analyzed by real-time PCR, serum angiotensin (Ang)-(1-9) and Ang-(1-7) levels were detected by ELISA. RESULTS:Compared with CD group , the cardiac function of the 2 groups treated with perindopril was significantly im-proved (P<0.01), and more improvement in HD group was observed than LD group (P<0.05).The serum angiotensin ( Ang)-(1-9) and Ang-(1-7) level and the mRNA expression of ACE 2 and AT2R in the 2 groups treated with perindopril were significantly improved (P<0.01).Compared with LD group, the mRNA expression of ACE2 and AT2R and the ser-um levels of Ang-(1-9) in HD group were significant improved (P<0.05), while no difference of serum Ang-(1-7) level was observed.Correlation analysis revealed that the improvement of the cardiac function was associated with serum Ang -(1-9) level, mRNA expression of ACE2 and AT2R (P<0.01), but has no significant correlation with serum Ang-(1-7) lev-el.CONCLUSION:High dose of perindopril may improve more cardiac function in ischemic cardiac dysfunction model in rabbits.The mechanism may relate to increasing serum Ang-(1-7) level to activate AT2R.

Objective: To observe the impact of cardiac contractility modulation (CCM) on myocardial remodeling in rabbit model of chronic heart failure (CHF) with its possible mechanism. Methods: Rabbit HF model was established by ascending aortic root ligation; the animals were divided into 3 groups: Sham group, the animals received thoracotomy without aortic ligation, HF group and HF+CCM group, the HF animals received CCM treatment for 4 weeks. n=10 in each group. Cardiac function was measured by echocardiography at 12 and 16 weeks in each group respectively; myocardial tissue fibrosis and pathological changes were examined by Masson staining; plasma BNP level was assessed by ELISA; protein expressions of collagen I, collagen II, MMP2,MMP9, TIMP1 and galectin-3 in myocardial tissue were determined by Western blot analysis. Results: ① By echocardiography: with 12 weeks treatment, compared with Sham group, HF group and HF+CCM group had increased LVESD, LVEDD and decreased LVFS, LVEF, all P<0.05; with 16 weeks treatment, compared with HF group, HF+CCM group had improved LVESD, LVEDD, LVEF and LVFS, all P<0.05. ② Pathological changes:compared with Sham group, HF group showed increased collagen content in myocardial tissue, P<0.05; CCM treatment could partially decrease collagen accumulation, P<0.05. ③ After 12 weeks treatment, compared with Sham group, HF group and HF+CCM group presented elevated plasma BNP level, P<0.05; after 16 weeks treatment, compared with HF group, HF+CCM group presented reduced plasma BNP, while it was still higher than that in Sham group, P<0.05. ④ By Western blot analysis: compared with Sham group, HF group demonstrated increased protein expressions of collagen I, collagen II, MMP2, MMP9, TIMP1 and galectin-3 in myocardial tissue; the above indexes were much lower in HF+CCM group while still higher than those in Sham group, all P<0.05. Conclusion: CCM could improve myocardial remodeling in rabbit model of CHF which might be related to down-regulated protein expressions of collagen I, collagen III, MMP2, MMP9, TIMP1 and galectin3 in myocardial tissue.