Objective To assess the effect of intracoronary attenuation,slice thickness,and window setting on CT attenuation measurement of non -calcified coronary plaque in ex vivo specimen.Methods Twenty adult cadaver heart specimens were used.Five solutions of contrast medium diluted with saline were prepared as follows:1/200,1/50,1/40,1/30,and 1/20.CT scans were performed after intracoronary iniection of each solution.The solution of 1/200 was used to measure baseline values of plaques.and the other four solutions were used to imitate the different intracoronary attenuation.The plaque attenuations were measured with region of interest under four intracoronary attenuations,two slice thickness (0.625 mm and 1.250 mm),and two window settings(WW 1000 HU,WL 150 HU and wW 500 HU,WL 200 HU),respectively.The result was compared with factor analysis and partial correlation analysis.Results Twenty four plaques met the study criteria.The baseline CT attenuation of plaques under two window settings were(23.90±6.87)HU(setting 1)and(29.00±7.26)HU(setting 2)respectively.Intracoronary attenuation and window settings had significant effect on CT attenuation measurement of non-calcified plaques(F=45.674 and 79.094,P＜0.01),whereas slice thickness of 0.625 mm and 1.250 mm showed no significant effect on CT attenuation measurement(F=1.033,P＞0.05).Comparing with baseline vslues.plaque attenuations on 1/40 and 1/30 solutions that can imitate lumen attenuation in clinical practice were increased.Conclusions Intraeoronary attenuation and window settings had significant effeet on CT attenuation measurement of non-calcified plaques,while slice thickness of 0.625 mm and 1.250 mm had no significant effect.Plaque attenuation would increase with the increase of intracoronary attenuation.

Objective To construct a recombinant adenovirus vector ( Ad-hIL-17F) expressing human interleukin 17F (hIL-17F) and to investigate the effects of expressed hIL-17F on angiogenesis. Methods The hIL-17F fragments was amplified by PCR using pUCm-T/hIL-17F plasmids as templates and then cloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-hIL-17F.The pAdTrack-CMV-hIL-17F transfer vector was linearized with PmeI digestion and then transformed into competent BJ 5183 with pAdEasy-1 backbone vector for homologous recombination .Then it was linearized with PacI digestion and transfected into human embryonic kidney 293 (QBI-293A) cells to construct Ad-hIL-17F.RT-PCR analysis and indirect immunofluorescent assay (IFA) were performed to determine the expressions of hIL-17F.MTT assay was used to detect the inhibitory effects on cell growth of ECV 304 .The expressions of VEGF and Ang-1 in 293 A and ECV304 cells were measured by ELISA .The effects of Ad-hIL-17 F on the expressions of VEGF in 293A cells were analyzed by Real-Time PCR.Results The sequencing result verified that hIL-17F gene fragment was correctly inserted in the vector .The expressions of hIL-17F gene at mRNA and pro-tein levels were confirmed by RT-PCR and IFA.Ad-hIL-17F could significantly inhibit the growth of ECV304 cells and down-regulate the expressions of VEGF and Ang-1 in 293A and ECV304 cells.Conclu-sion Ad-hIL17F expressing hIL-17F was successfully constructed .The expressed hIL-17F could inhibit the angiogenesis through down-regulating the expressions of VEGF and Ang-1.

Objective:To study the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the MG-63 osteosarcoma xenografts in nude mice.Methods:Ad-ING4 was transfected into QBI-293 cells and harvested.15 nude mice of the subcutaneous tumor models were established with MG-63 osteosarcoma cells and were randomly divided into PBS,Ad-GFP and Ad-ING4 groups.Then PBS(100 μl),Ad-GFP(100 μl,10~9pfu/ml) and Ad-ING4 (100 μl,10~9pfu/ml) for each one were given respectively QOD for 5 times,with intratumor injections.Tumor volume changes were monitored;and the 15 mice were sacrificed 2 weeks after treatment,the tumors were removed,weighed and ratios of tumor-suppression were calculated.The morphological changes of apoptotic tumor cells were observed under microscope.Bcl-2,Bax,Caspase-3,VEGF,CD34 expression was tested by immumohistochemistry.Results:High titer(10~9pfu/ml)adenoviral vector of ING4 gene were obtained.In nude mice bearing MG-63 osteosarcoma xenografts,the growth of MG-63 tumors treated by intratumoral injecting of Ad-ING4 was significantly suppressed,compared with PBS group and Ad-GFP group.The ratios of tumor weight-suppression of Ad-ING4 group was 59.3%(P＜0.05).Immumohistochemistry displayed that the expression of Bax,Caspase-3 was up-regulated and the expression of Bcl-2,VEGF,CD34 was down-regulated by Ad-ING4.Conclusion:Ad-ING4 can inhibit the growth of MG-63 osteosarcoma xenografts in nude mice,which may be via activating the apoptosis pathway and inhibiting tumor angiogenesis.

BACKGROUND:Previous studies have demonstrated that regenerated silk fibroin film can promote pcDNA3.0-vascular endothelial growth factor 165(rEGF165) transfected L929 cells to express VEGF.OBJECTIVE:To investigate the effects of regenerated silk fibroin film on expression of cytokines related to angiogenesis in the fibroblasts transfected by adenovirus mediated-VEGF165(Ad-VEGF165).DESIGN,TIME AND SETTING:ontrolled observational cell gene engineering experiment performed by analysis of variance at the Laboratory of Cellular and Molecular Biology,Soochow University between November 2007 and ApriJ 2008.MATERIALS:Regenerated silk fibroin film was provided by Professor Li Ming-zhong,who was from Department of Material Science and Engineering,Soochow University.METHODS:The QBI-293A and WI-38 fibroblasts cultured on the regenerated silk fibroin film.polyvinyl chloride film and (Ad-GFP)and treated by phosphate buffered saline(PBS)for controls.MAlN OUTCOME MEASURES:VEGF mRNA was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR);the expression levels of VEGF,angiogenin 1(Ang 1),fibroblast growth factor 2(FGF2),and platelet-derived growth factor(PDGF)were detected by enzyme-labeled immunosorbent assay(ELISA).RESULTS:The VEGF mRNA expression in the fibroblasts cultured on the regenerated silk fibroin film was increased but that in the fibroblasts cultured on the polyvinyl chloride film was signifcantly decreased(P<0.05).ELISA results demonstrated that not only VEGF gene expression in 293A and WI-38 cells transfected bv Ad-VEGF165 cultured on regenerated silk fibroin film was high,but also Ang 1 expression increased significantly(P<0.05).Meanwhile,the expression levels of FGF2 and PDGF were normalin the fibroblasts cultured on the regenerated silk fibroin film.CONCLUSION:Adenovirus vector can be effciently transfected into fibroblasts cultured on the regenerated silk fibroin film and can express VEGF and Ang 1 protein with highly biological activity,which accelerates angiogenesis.Regenerated silk fibroin film also can maintain the normal expression levels of FGF2 and PDGF,which ale related to wound healing.

Objective To construct a recombinant adenoviral vector carrying and co-expressing human inhibitor of growth 4(ING4) and human interleukin-24(IL-24) mediated by poly( A)-Promoter[Ad-ING4-poly(A)-Promoter-IL-24, referred to as Ad-ING4-IL-24] and explore its effect on the growth of HepG-2 human hepatocellular carcinoma cellsin vitro. Methods The poly(A)-Promoter(hEFl-elF4g) (Sal Ⅰ and Not Ⅰ ), ING4 ( Bgl Ⅱ and Sal Ⅰ ), and IL-24 ( Xho Ⅰ and Xba Ⅰ ) fragments were amplified by PCRusing pORF-mbcl-2α, pcDNA3.0-IL-24, and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-poly (A)-Promoter-lL-24, respectively. The pAdTrack-CMV-ING4-poly (A)-Promoter-IL-24 transfer vector linearized with Pme Ⅰ digestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-l-pAdTrack-CMV-ING4-poly ( A )-Promoter-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰ digestion and transfected into the human embryonic kidney 293 (QBI-293A) cells by liposome, leading to formation of the recombinant adenoviruses Ad-ING4-IL-24co-expressing ING4 and IL-24. The Ad-ING4-IL-24 were amplified in QBI-293A cells and its titer was up to 3.5 × 109 PFU/ml. Adenovirus-mediated ING4 and IL-24 genes expression in HepG-2 cells was examined by RT-PCR and Western blot. The growth-suppressing and apoptosis-inducingg effect of Ad-ING4-IL-24 coexpressing ING4 and IL-24 on HepG-2 human hepatocellular carcinoma cells was assessed by MTT assay and FCM, respectively. Results DNA sequencing showed that the ING4, poly (A)-Promoter, and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in HepG-2 cells. Adenovirusmediated ING4 and IL-24 co-expression significantly suppressed HepG-2 hepatocellular carcinoma cell growth and induced cell apoptosis, and the effect of Ad-ING4-IL-24 group was more significant than AdING4 and Ad-IL-24 group. Conclusion The adenoviral vector co-expressing ING4 and IL-24 mediated by poly(A)-Promoter(Ad-ING4-IL-24) was successfully constructed. Ad-ING4-IL-24 had marked anti-tumor effect in suppressing HepG-2 human hepatocellular carcinoma cell growth and inducing cell apoptosis in vitro. Compared with Ad-ING4 and Ad- IL-24, Ad-ING4-IL-24 enhanced anti-tumor effect.

Objective To establish Ad-hLIF transgenic feeder cells for the expansion of umbilical cord blood CD34+ HSPC in vitro and study the SCID mice model of hematopoietic stem/progenitor cell (HSPC) transplantation. Methods The expression of objective gene in Ad-hLIF transgenic feeder cells was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting(MACS) was detected by the flow cytometry. After expanded with various combinant of cytokines and transgenic feeder layer cells for 28 d, the quantity of mono-nuclear cell (MNC) and CD34+ cells rate was detected in different time. MNC after expansion stained by CFDA SE was injected to the sublethally irradiated SCID mice. Humanize gene Alu was detected by RT-PCR and fluorescence microscope. Results The green fluorescence was observed in the transgenic cells infected with 50MOI( multiplicity of infection) Ad-hLIF, and the objective gene was confirmed by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by MACS could reach 95.60% ±2.58%, Ad-hLIF transgenic feeder cells and various cytokines system increased MNC by 356.95±0.87 fold, and maximal expansion of CD34+ cells was observed during 0-14 d of culture, then down-expansion gradually. Four weeks after transplanted in SCID mice, fluorescently-labeled humanize cells still can be observed. The existence of the humanized gene Alu was confirmed by RT-PCR. Conclusion Ad-hLIF transgenic feeder cells can effectively proliferate umbilical cord blood CD34+ HSPC in vitro and delay it differentiate, what's more, it has high transplant efficacy and haematogenesis activity.

To investigate the inhibitory effect and anti-cancer mechanism of adenovirus mediated IL-24 gene expression on the human U251 glioma cell. U251 glioma cells were infected with Ad-IL-24 at various multiplicity of infection (MOIs). Cell proliferation was determined by MTT assay. Cell apoptosis was detected by flow cytometry and Hochest staining. The transcription of apoptosis-related genes was analyzed by reverse transcription-PCR (RT-PCR), and the expression of Cleaved Caspase-3 was analyzed by Western blotting. The result showed that the growth of U251 glioma cells was significantly inhibited by Ad-IL-24 at the MOI of 100. The apoptotic rate of U251 glioma cells was 42% 72 h after infection with Ad-IL-24. Four days after infection, the growth of the U251 glioma cells was inhibited to 50%. RT-PCR showed that Ad-IL-24 not only up-regulated expression of bax/bcl-2, ICE, C-myc, p53 and down-regulated the expression of HIF-1alpha, but also enhanced Caspase-3 activation, eventually resulting apoptosis. Taken together, these results suggest that infection of U251 glioma cells with Ad-IL-24 can inhibit growth and induce apoptosis significantly by the regulation of apoptosis-related genes.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; Brain Neoplasms ; genetics ; pathology ; Cell Proliferation ; drug effects ; Genetic Therapy ; Glioma ; genetics ; pathology ; Humans ; Interleukins ; genetics ; metabolism ; Recombination, Genetic ; Tumor Cells, Cultured

Objective To investigate the image quality and radiation dose of the wider detector array CT scanner with low dose scanning mode in young children with congenital heart disease.Methods Totally 100 consecutive pediatric patients younger than 3 years with congenital heart disease were enrolled.They were divided into two groups.The low dose group with fifty patients underwent axial CT scanning with ECG gating,and the control group with fifty patients were scanned with volume helical shuttle (VHS) technique.CT number and noise of two groups images at the level of ascending aorta,main pulmonary artery,left ventricle,descending aorta and adjacent muscle were measured,and the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated.The double blind method was used to evaluate subjective image quality of the level of intra-cardiac,extra-cardiac and coronary artery.Effective dose was also calculated for both groups.Results No significant difference was found in the CT number,image noise,SNR,and CNR between the two groups in the same anatomic regions (P ＞ 0.05).No significant difference was found in subjective image quality between the two groups for the intra-cardiac and extra-cardiac structure(P ＞0.05).The subjective image quality of coronary artery was significant higher in low dose group than the control group(4.10 ± 0.90 vs.2.88 ± 0.82,Z =-5.818,P ＜ 0.05).Effective dose was (0.57 ± 0.30)mSv in group A and (2.39 ± 1.15)mSv in group B with dose savings of 76％ (t =-11.642,P ＜ 0.05).Conclusions The wider detector array CT scanner with low dose scanning mode can improve image quality with lower radiation dose.

To study the inhibitory effect and anti-cancer mechanisms of interleukin 24 (IL-24) on human osteosarcoma cell MG-63, we delivered IL-24 into MG-63 cells in vitro and in vivo by adenovirus. The expression level of IL-24 was detected by RT-PCR and fluorescence microscope; the growth inhibition, apoptosis rate and apoptosis body were measured by MTT, Flow cytometry and Hoechst staining respectively. Furthermore, we analyzed the expression of bcl-2, bax, caspase3 genes by RT-PCR after overexpression of IL-24. For in vivo study, we first established the MG-63 tumor model by grafting MG-63 cells in athymic nude mice; and then injected Ad-IL-24 into the tumors. Two weeks after injection, we sacrificed the mice, removed the tumors, weighed and calculated the ratios of tumor-suppression. We also detected the expressions of Bcl-2, Bax, Caspase-3 and CD34 with immumohistochemistry. Our in vitro results indicated that Ad-IL-24 was transcribed and translated in MG-63 osteosarcoma cells. More interestingly, IL-24 inhibited the growth of MG-63 cells and induced apoptosis by up-regulation of bax, caspase-3 and down-regulation of bcl-2. The in vivo data showed that IL-24 suppressed the tumor growth conspicuously through down-regulating the expression of bcl-2, and up-regulating the expression of bax, caspase-3. This study would provide evidence for the gene therapy of IL-24 on osteosarcoma.
Adenoviridae ; genetics ; metabolism ; Animals ; Apoptosis ; genetics ; Bone Neoplasms ; pathology ; therapy ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Genetic Therapy ; Humans ; Interleukins ; biosynthesis ; genetics ; Mice ; Mice, Nude ; Osteosarcoma ; pathology ; therapy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; metabolism

Objective To investigate the accuracy,image quality and effective dose (ED)of the whole-heart CT scanner in infants with congenital heart disease.Methods Totally 86 consecutive pediatric patients younger than 2 years old with congenital heart disease were enrolled.They were divided into 2 groups:whole-heart CT scanner with low dose group,43 patients(28 males,15 females,aged 12 d -19 months)underwent CT acquisition by using the whole-heart CT,and the other 43 patients(23 males,20 females,aged 19 d-16 months)examined with volume helical shuttle (VHS)of high definition CT were assigned as VHS group.With surgical results as the standard,the sensitivity, specificity,positive predictive value (PPV),negative predictive value (NPV)and the diagnostic accuracy of the 2 groups for cardiovascular abnormalities were evaluated.Attenuation and noise of 2 groups of ascending aorta,main pul-monary artery,and muscle were measured,and the signal -to -noise ratio (SNR)and contrast -to - noise ratio (CNR)were calculated.The double blind method was used to evaluate the subjective image quality of the level of intra-cardiac,extra-cardiac and coronary artery.Both the volumetric CT dose index (CTDIvol)and dose-length product(DLP)of each child were recorded,and the ED was also estimated in both groups.Results By using surgical findings as the reference standard,a total of 124 and 113 separate cardiovascular anomalies were confirmed by the whole-heart CT scanner in the low dose group and the VHS group.The diagnostic accuracy in 2 groups was 99.2%and 98.8%,respectively,without significant difference(χ2＝0.035,P＝0.852).The sensitivity,specificity,PPV and NPV in 2 groups were 97.8%,99.7%,98.4%,99.6% and 95.2%,99.3%,98.2%,98.7%,respectively.No signifi-cant difference was found in the attenuation,image noise,SNR,and CNR between 2 groups in the same anatomic regions (all P＞0. 05).No significant difference was found in subjective image quality between 2 groups on the intra-cardiac and extra-cardiac structure(all P＞0.05).But the subjective image quality of coronary artery was significantly higher in whole-heart CT scanner in the low dose group than that of the VHS group(4.02 ± 0.91 vs.2.79 ± 0.74),and the differ-ence was significant(Z＝ -5.562,P＝0.000).ED was (0.59 ± 0.31)mSv in whole-heart CT scanner in the low dose group and (2.28 ± 1.07)mSv in the VHS group,reflecting dose savings of 74% by using the whole-heart CT scanner with high temporal resolution,and the difference was significant(t＝ -11.285,P＝0.000).Conclusions The whole-heart CT scanner with low dose can improve image quality with lower ED,especially for the image quality of coronary artery,which is an effective examination method for the diagnosis of congenital heart disease of children, especially for complex congenital heart disease.