Objectives To study the effect of ubiquitin proteasome inhibitor bortezomib on apoptosis and Caspase-3 expression in human umbilical arterial vascular smooth muscle cells. Methods Human umbilical artery vascular smooth muscle cells were cultured in vitro. Different doses of bortezomib (0, 4, 10, 40nmol/L) were used to treat cell. Cell proliferation activity were detected by using CCK8 method. Western Blot assay was used to measure the expression of Pro-caspase-3 and Cleaved-PARP protein. Morphology of cells in different groups are observed by confocal microscopy and apoptosis rate were detected by flow cytometry analysis. Results Bortezomib inhibits proliferation activity of vascular smooth muscle cells. With increasing of drug concentration and treating time, cell viability are reduced (P < 0.01). Cells treated with different concentrations bortezomib for 48 h showed increased apoptosis rate (P < 0.01), decreased pro-caspase-3 protein expression and increased cleaved-PARP protein expression (P < 0.01). Laser confocal microscope found that with the increase of drug concentration, the number of apoptotic cells showed an upward trend under per high-power vision. Conclusion The ubiquitin proteasome inhibitor bortezomib may decrease the expression of pro-caspase-3 and increase Cleaved-PARP expression in human umbilical artery vascular smooth muscle cells. Additionally , it could induce apoptosis in a dose-dependent manner.

Carotid cavernous fistula is the abnormal arteriovenous communications, which can be located within the internal carotid cavernous segment itself, or between the cavernous and the internal carotid branches or external carotid meningeal branches. With the development of interventional neuroradiology, endovascular embolization has become the choice of treatment for the carotid cavernous fistula, as the technique is less invasive, simple and reliable. This paper aims to review the recent progresses in this respect in order to further improve its clinical diagnosis and treatment.

Objective To evaluate clinical value of ultrasound contrast agent the sulfur hexafluoride micro-bubble in the treatment of T2WI high signal uterine fibroids with high intensity focused ultrasound (HIFU).Methods A total of 36 patients with T2WI high signal uterine fibroids were randomly divided into experimental group and control group.Eighteen patients in experimental group received HIFU treatment with sulfur hexafluoride micro-bubble before,during and after the operation while another eighteen in control group received HIFU treatment with sulfur hexafluoride micro-bubble only after the operation.We recorded operation time (min),changing time of massive gray (s),irradiation time (s),therapeutic dose (J),incidence of complications and ablation rate (％).Results Operation time,changing time of massive gray,irradiation time,and therapeutic dose in experimental group were (101.51±43.02) min,(856.42±451.14) s,(1077.37±546.23) s,and (496581.81 ± 267192.31) J,respectively while those in control group were (261.34 ± 85.53) min,(1 833.32 ± 642.67) s,(1 890.21 ± 1 268.43) s,and (784 608.31 ± 357 621.45) J,respectively.Significant differences were found among those parameters between the two groups (P ＜0.05).Tumor ablation rate in experimental group was 83.3％ while that in control group was 55.5％,which was of significant difference between the two groups (P＜0.05).One case of muscular stiffness was found in experimental group while one case of second-degree burn and two cases of muscular stiffness were found in the control group.The incidence of complications was 1/18 (5.5％).No severe complication like bowel perforation or bladder perforation was found in the two groups.Conclusion The effect of ultrasound contrast agent sulfur hexafluoride micro-bubble can shorten operation time,improve tumor ablation rate and reduce complications and it can play a guiding role in clinical work.

Objective To evaluate the expression of vascular endothelial growth factor (VEGF)and endostatin (ES) in the serum of patients with differentiated thyroid cancer at the time of before and after operation and postoperative recurrence. Methods The serum samples were obtained from 7 patients with postoperative lung metastasis and 21 patients with local recurrence after operation.The serum samples from 30 normal subjects were obtained as control.The levels of serum VEGF and serum endostatin were analyzed by enzyme-linked immunosorbentassay (ELISA).Thelevelsofserumthyroglobulinwereanalyzedby Chemiluminescence method.Statistical analysis was performed with t-test using SPSS 13.0 software.Pearson correlation analysis was performed for the correlation between serum VEGF and serum ES and thyroglobulin.ResultsThe levels of serum VEGF and serum ES in patients with lung metastasis 3 weeks after operation were significantly lower than those before operation and at the time of recurrence [ VEGF:( 210.3 ± 30.4) ng/L vs (412.6 ±57.3) ng/L and(619.5 ± 126.4) ng/L,P ＜0.05 ;ES:(25.2 ±6.2)ng/L vs (34.3 ±7.6)ng/L and (38.6 ± 8.7) ng/L,P ＜ 0.05 ].And the levels of serum VEGF and serum ES in patients with local regional recurrence 3 weeks after operation were significantly lower than those before operation and at the time of recurrence [ VEGF:(209.3 ±36.7) ng/L vs (399.4 ±56.3) ng/L and (406.5 ±59.2) ng/L,P ＜0.05;ES:(25.7 ± 4.7 ) ng/L vs ( 35.2 ± 6.8 ) ng/L and ( 31.2 ± 7.6 ) ng/L,P ＜ 0.05 ].The expression of serum VEGF and serum ES in patients with pulmonary metastasis were significantly higher than those in patients with local regional postoperative recurrence ( P ＜ 0.05 ).There was a linear positive correlation between serum VEGF,serum ES and thyroglobulin levels in the patient with recurrent differentiated thyroid cancer( r =0.752,0.349,P ＜0.01 ).Conclusion The serum VEGF and serum ES level were significantly elevated in patients with differentiated thyroid cancer before operation and recurrence.The serum VEGF and serum ES were importantindicators to reveal the biological behaviors of differentiated thyroid cancer.

Objective To construct the recombinant plasmid of fusion protein containing respiratory syncytial virus (RSV) cytotoxicity T lymphocyte (CTL) epitope M2:81-95 and heat shock protein (HSP) 70L1, and to investigate its immunogenicity after prokaryotic expression. Methods HSP70L1 gone was cloned from SMMC7721 cells. The M2:81-95 gene fragment was amplified by polymerase chain reaction (PCR) method. Plasmid pET-HSP70L1-M2 : 81-95 (pET-HSP70L1-M2) was constructed, identified and transferred into E. coli BL21 (DE3). Expression of HSP70L1-M2 : 81-95(HSP70L1-M2) was induced by isopropy-β-D-thiogalaetosidc (IPTG). The expressed protein was purified by affinity chromatography and renatured by gradient dialysis. The BALB/c mice were immunized with this fusion protein. IgG antibodies and the subtypes were detected by enzyme-linked immunosorbent assay (ELISA), and CTL responses were determined by methyl thiazolyl tetrazolium (MTT). Results The recombinant plasmid pET-HSP70L1-M2 was successfully constructed. The fusion protein HSP70L1-M2 was expressed in E. coll. The purified protein induced strong RSV-and CTL epitope-specific CTL responses and high titer of protein specific lgG antibody 4.87±0.35. The subtypes were IgG1 (5.53±0.28) and lgG2a (4.40±0.21) and IgG1/ IgG2a ratio was balanced. The titers of lgG, IgG1 and IgG2a in PBS control group were 0.33±0.17, 0.51±0.21 and 0, respectively, which werc significantly lower than those in immunized group (t = 3.512, 3.681, 5.856; P<0.05). Conclusions The recombinant plasmid pET-HSP70L1-M2 is successfully constructed and the fusion protein is expressed and purified. HSP70L1-M2 induced strong RSV-and CTL epitope-specific CTL responses and mixed T helper cell (Th)1/Th2 response in BALB/c mice.

Objective: To investigate the regulation of respiratory syncytial virus CTL epitope fused with G protein antigen fragment G1 to the specific immunoresponses. Methods: The recombinant plasmid pET-DsbA-G1 or pET-DsbA-G1F/M2 was transferred into E.coli BL21(DE3) and the fusion protein DsbA-G1F/M2 or DsbA-G1 was expressed.The expressing product was induced and purified by affinity chromatography. The two proteins were used to immunized BALB/c mice i.p, respectively. Serum and spleen cells were collected regularly. RSV-specific CTL responses were measured by MTT, IgG and IgG1 and IgG2a antibodies by ELISA, neutralizing antibodies by plaque reduction assay. Results: The recombinant proteins were expressed successfully and the purity was over 90% after purified by affinity chromatography. The protein DsbA-G1F/M2 induced significant RSV-specific CTL responses, while DsbA-G1 without CTL epitope did not induce detctable CTL responses. Strong IgG antibody responses were elicited,indicated by both. IgG1 and IgG2a titers induced by DsbA-G1F/M2, while only IgG1 was induced by DsbA-G1.The ratio of IgG1/IgG2a was downregulated significantly. Both antigens induced high level of neutralizing antibodies, but the latter was a little lower. Conclusion: DsbA-G1F/M2, the fusion protein of a CTL epitope and G protein fragment G1 can induce both cellular immunity and humoral immunity. The activation of CTLs downregulates the ratio of IgG1/IgG2a.The more balanced immunoresponse is advantageous for improving safety of the candidate vaccine.

Objective To investigate the value of parametric imaging of CEUS in uterine fibroids with different signal intensity on T2WI by high intensity focused ultrasound (HIFU),and dynamic vascular pattern (DVP).Methods Totally 16 patients with single uterine fibroids were divided into high intensity group (n=6),isointensity group (n=4) and low intensity group (n=6) according the signal intensity on T2WI before HIFU treatment.CEUS were performed on each patient.SonoLive CAP software was used to analyze the CEUS images and DVP parametric images were reconstructed.Results The maximum intensity,rising time,time to peak and mean transit time were (235.40± 35.46)％,(22.80± 3.16)s,(25.09±2.44)s,(125.78 ± 27.63)s in high intensity group,(71.97± 2.43) ％,(24.85±3.22)s,(32.81±3.92)s,(66.52±3.48)s in isointensity group and (16.17±2.83) ％,(25.42±2.66)s,(32.82±3.76)s,(64.27±3.33)s in low intensity group.There were statistically significant differences among three groups (all P＜0.05).DVP curve was divided into two types:non-washout in high intensity group and cystic type in low intensity group.Conclusion CEUS and DVP imaging can directly quantitively display the difference of blood perfusion among the uterine fibroids with different signal intensity on T2WI which can provide important information for HIFU treatment in uterine fibroids.

Objective To evaluate the expression of serum vascular endothelial growth factor(sVEGF) in differentiated thyroid carcinoma(DTC)patients before and after thyroidectomy and when recurrence occured.To analyze the relationship between serum VEGF and serum thyroglobulin(Tg)level.Methods sVEGF levels of 30 cases of DTC (7 patients with pulmonary metastasis,23 patients with locoregional recurrence)were determined by enzyme-linked immunosorbent assay(ELISA) before and after thyroidectomy and when recurrence occured.The levels of serum Tg and thyroglobulin antibodies (TgA) were detected by chemiluminescence method.The results were compared with those of the 30 cases of normal controls.Results sVEGF levels had no significant difference between pulmonary metastasis group and locoregional recurrence group before operation (t =0.373,P ＞ 0.05),but they were significantly higher than those in the normal control group(t =8.597 and 14.14 respectively,all P ＜ 0.05).sVEGF levels in patients with pulmonary metastasis or locoregional recurrence were significantly reduced at the 3rd week after operation and the difference had statistical significance with those before operation(t =5.977,11.50,P ＜ 0.05).The difference of postoperative level of sVEGF in patients with pulmonary metastasis,locoregional recurrence and in patients of normal control group had no statistical significance (t =1.593,1.525,P ＞ 0.05).sVEGF levels in patients with pulmonary metastasis or locoregional recurrence were significantly higher after recurrence than those before recurrence (t =7.387,12.16,P ＜ 0.05).After recurrence sVEGF levels in patients with pulmonary metastasis were significantly higher than those in patients with locoregional recurrence (t =4.167,P ＜ 0.05) It showed a positive linear correlation between serum Tg level and sVEGF level in DTC patients with recurrence(r =0.786,P ＜ 0.01).Conclusion sVEGF is significantly elevated in DTC patients before operation and with postoperative recurrence,making it an important indicator to reveal the biological behaviors of DTC.

Objective:To investigate whether His-tag to change the immunogenicity of recombinant protein G1F/M2 of respiratory syncytial virus.Methods:The G1 and F/M2 gene fragments were amplified by PCR method and then ligated into the expressing vector pET-His or pET-DsbA-His.Each recombinant plasmid was transferred into E.coli BL21(DE3) and the expression was induced by IPTG.The expressed His-G1F/M2 or DsbA-His-G1F/M2 was purified by affinity chromatography.The latter was digested with thrombase and G1F/M2 was purified by affinity chromatography.His-G1F/M2 or G1F/M2 was used to immunize BALB/c mice.Anti-RSV antibody was measured by ELISA and RSV-specific CTL responses by MTT.Results:No significant difference was observed between the level of anti-RSV antibody or RSV-specific CTL response induced by G1F/M2 and that by His-G1F/M2.Conclusion:His-tag does not change the immunogenicity of recombinant protein G1F/M2 of respiratory syncytial virus.

Objective To investigate immune responses and protective effect induced by two recombinant proteins of hepatitis C virus(HCV)in BALB/c mice.Methods BALB/c mice were immunized with recombinant proteins HCV-T and(or)HCV-F4HVR1 three times.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Five mice were sacrificed after 14 days of the last immunization.Splenic cells were isolated and levels of interferon(IFN)-γ,interleukin(IL)-4 and cytotoxic T lymphocyte(CTL)cytotoxicity assay were measured in vitro.The remaining mice were subcutaneously injected with 1.0×106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results Compared with phosphate bufter saline(PBS)group,combined immunization with HCV-T and HCV-F4HVR1 induced higher levels of specific IgG against HCV-F4HVR1(t=3.815,3.762,P＜0.05),HCV-NS3-specific CTL response(t=3.971,P＜0.05)and IL-4(t=3.512,3.417,P＜0.05)and IFN-γ(t=3.813,3.426,3.671,P＜0.05)secretions.Conclusion High levels of specific humoral immunity and cellular immunity are induced in vivo after combined immunization with HCV-T and HCV-F4 HVR1,which could effectively prevent from the attack of SP2/0-NS3 cells.