Molecular Characteristics of gp90 Gene of 14 Reticuloendotheliosis Viruses Isolated in China

[Objective] The paper was to study molecular characteristics of gp90 gene of 14 Reticuloendotheliosis viruses isolated in China.[Method] The surface envelop gene gp90 of 14 REV strains isolated from different commercial layer farms in China were amplified,and their nucleotide sequences were determined.[Result] Sequence analysis showes that 14 REV strains are more identical to the subtype 3 isolates than to the early Chinese REV isolates.In addition,14 REV strains have a high identity with some REV strains in US and Taiwan.[Conclusion] The study provided necessary information for further understanding the evolution of REV.

Effect of Echinacea Polysaccharide on Secretion of TNF-α mRNA by IEC-6 Injured by LPS

Objective] This study was conducted to investigate Echinacea polysaccha-ride （EPS） on expression of tumor necrosis factor （TNF） under injury of intestinal epithelial cel s （lEC-6） by lipopolysaccharide （LPS）, so as to discuss the action mechanism of EPS to injured cel s. [Method] Total DNA was extracted with TRlzon reagent, TNF-α mRNA was amplified, and the amplification products were subjected to agarose gel electrophoresis and imaging. [Result] 50 μg/ml EPS could partial y in-hibited the production of TNF-α mRNA by lEC-6 under the stimulation of LPS, while the inhibition of 200 and 500 μg/ml EPS on the level of TNF-α mRNA gradual y in-creased with the concentration increasing; and lEC-6 cel s pretreated with 50, 100, 200 and 500 μg/ml EPS for 24 h and then stimulated by 10 μg/ml LPS for 1 and 4 h were analyzed by RT-PCR method, and it was found that the expression of TNF-α mRNA induced by LPS could be effectively inhibited by EPS, and the inhibition rate at 4 h was higher than that at 1 h. [Conclusion] EPS could play its role of protecting intestinal mucosa by inhibiting the secretion of TNF-α mRNA by cel s un-der the stimulation of LPS, and such inhibition effects of EPS had concentration dependency and time dependency.

VEGI-armed oncolytic adenovirus inhibits tumor neovascularization and directly induces mitochondria-mediated cancer cell apoptosis

Vascular endothelial cell growth inhibitor （VEGI） is a member of the tumor necrosis factor superfamily and plays an important role in vascular homeostasis. In this study, to investigate the anticancer therapeutic potential of this gene, a secreted isoform of VEGI （VEGI-251） was inserted into a selectively replicating adenovirus with E1B 55 kDa gene deletion （ZD55） to construct ZD55-VEGI-251. We report here that secreted VEGI-251 produced from ZD55- VEGI-251-infected cancer cells potently inhibits endothelial cell proliferation, tube formation in vitro and angiogen- esis of chick chorioallantoic membrane in vivo. Additionally, ZD55-VEGI-251 infection leads to a much more severe cytopathic effect than control viruses on several human cancer cell lines, including cervical cancer cell line HeLa, hepatoma cell line SMMC-7721 and colorectal cancer cell line SW620. Further study reveals that the increased cytotoxicity is a result of VEGI-251 autocrine-dependent, mitochondria-mediated apoptosis accompanied by caspase-9 activation, enhanced caspase-3 activation and PARP cleavage. Moreover, ZD55-VEGI-251-treatment of athymic nude mice bearing human cervical and colorectal tumor xenografts markedly suppressed tumor growth. Our findings indicate that the combined effect of antiangiogenesis and apoptosis-induction activity makes the VEGI-251-armed oncolytic adenovirus a promising therapeutic agent for cancer.

Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript Ⅱ KDR-TK plasmid by enzymatic digestion with Xho I and Sal I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble, pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP) expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to theprodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively. CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs. Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

Increased Activity of the Human Telomerase Catalytic Subunit Promoter by the VEGF Enhancer in Human Cancer Cells

We attempted to improve the activity of hTERT promoter by fusing the vascular endothelial growth factor （VEGF） enhancer. To determine the potential as cancer specific promoters, we measured the reporter gene transfection assay driven by the hTERT promoter and the VEGF enhancer in human cancer cells. We found that the hTERT promoter containing VEGF enhancer conferred strong expression of the reporter gene only in different cancer cell lines but not in normal human cells. Retrovirus vector expressing HSV-TK controlled by the hTERT promoter and the VEGF enhancer was constructed. A549 cells infected with LN-enh-hT-TK was significantly suppressed and induced to apoptosis more than those infected with LN-hT-TK. The apoptosis ratio ofA549 cell infected with two kinds of retrovirus cell with GCV in lower concentration is 20.94% and 50.7%. It suggested that there is significant differentiation between the assay groups. Our results demonstrated the possible application of hTERT promoter and the VEGF enhancer in targeted cancer gene therapy.

Lipopolysaccharide upregulates the expression of Toll-like receptor 4 in human vascular endothelial cells

OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA was extracted from ECV304 cells and isolated human umbilical vein endothelial cells (HUVECs) exposed to LPS, respectively. The quantification of TLR2 and TLR4 mRNA in HUVECs and EVC304 cells was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ECV304 cells and HUVECs were able to express TLR2 and TLR4 mRNA, but the expression levels of TLR4 appeared to be stronger than those of TLR2. LPS could upregulate the expression levels of TLR4 obviously, whereas it had no effect on the expression level of TLR2. CONCLUSIONS: Our data indicate that TLR4 may be the LPS signal transducer in endothelial cells and plays important roles in the cell activation of LPS. The ECV304 cell line is a better experimental model than isolated HUVECs in the research of endothelial cells.

Anti-tumor effect of human endostatin mediated by retroviral gene transfer in nude mice

OBJECTIVE: To explore the inhibitory effect of human endostatin gene mediated by retroviral vector on the growth of human liver carcinoma. METHODS: A recombinant retroviral plasmid containing human endostatin gene and signal peptide was engineered and transferred into PA317 cells to produce retrovirus. Human liver carcinoma cells (SMMC7721) were infected with the above retrovirus to build a stable endostatin-transfected liver carcinoma cell line (SMMC-endo). The control liver carcinoma cell line (SMMC-pLncx) was developed in a similar way except that the plasmid was replaced by an empty retroviral vector. Immunohistochemistry and Western blot were used to test the expression and secretion of human endostatin. The biological activity of the expressed human endostatin was assessed by endothelial cell proliferation assay. The growth rates of SMMC-endo and control SMMC-pLncx cells in vivo and in vitro were also observed. RESULTS: The expression and secretion of human endostatin by endostatin-transfected SMMC-endo cells were confirmed by immunohistochemistry and Western blot. Compared with the control group, concentrated supernatant of SMMC-endo cells remarkably inhibited the proliferation of human umbilical vein endothelial cells by 48%, significantly higher than the inhibition by the control (10.2%; P

Extracellular domain of kinase domain region mediated by adeno-associated virus inhibits growth and angiogenesis of bladder cancer in Balb-c mice

OBJECTIVE: To verify whether the extracellular domain of kinase domain region (KDR) has anti-angiogenesis activity in vivo. METHODS: cDNA was cloned into adeno-associated virus (AAV) vector pSNAV and transfected to baby hamster kidney (BHK) cells. Recombinant AAV was obtained from the cell culture supernatant after adding helper virus. Recombinant AAV-infected human bladder cancer EJ cell line (EJ cells) were injected subcutaneously into Balb-c nude mice. Tumor specimens were removed from the mice, paraffin-embedded and sliced, then stained by immunohistochemistry. Microvessel density (MVD) was determined under a microscope. RESULTS: The tumor volume developed by EJ cells transfected with the extracellular domain of KDR was significantly smaller (1.70 +/- 0.18 cm(3)) compared with that in the control (5.62 +/- 0.67 cm(3)) (P

Preparation of enzymatically cross-linked sulfated chitosan hydrogel and its potential application in thick tissue engineering

For the requirement of preliminary vascularization, the scaffolds for thick tissue engineering should have not only good cell affinity, but also anticoagulant ability. In this paper, enzymatically cross-linked hydrogel scaffolds based on sulfated chitosan （SCTS） were prepared. Firstly, sulfated chitosan-hydroxyphenylpionic acid （SCTS-HPA） conjugate was synthesized, and the structure of SCTS-HPA was identified by FITR and ~H NMR. And then the enzymatically cross-linked hydrogels were pre- pared in presence of horseradish peroxidase （HRP） and hydrogen peroxide （H202）. The gelation time, mechanical property, morphology and cytotoxicity to human umbilical vein endothelial cells （HUVECs） of the hydrogel were evaluated in vitro, the tissue compatibility of SCTS-HPA scaffold was studied in vivo. The results showed that the gelation time, mechanical property, morphology of the dehydrated hydrogel could be controlled by the the concentration of HRP and H202. The cytotoxicity test showed that the hydrogel extracts have no cytotoxicity to HUVECs. The in vivo assay indicated that SCTS-HPA scaffold have good tissue compatibility with no thrombus formation. All these results indicated that the SCTS-HPA scaffold could be used as a thick tissue engineering scaffold.

Direct evidence of the existence of specific LPS binding sites on vascular endot helial cells

Objective:To investigate the binding characteri stics of endothelial cell (EC) with LPS free from the participation of serum fac tors. Methods:Laser confocal microscope was employed in the observat ion of the binding of EC with FITC-LPS. The KD and the binding sites of each EC were calculated by radioligand binding assay of receptors (RBA) using [ 3H] -LPS. Results:The binding of EC with LPS was saturable, time and con centration dependent and it could be competed with overdosed LPS of the same typ e. The fluorescence mainly distributed in cytoplasm, especially near the nucleus , which could also be stained. Conclusions:There might be some specific LPS binding sites exi sting on ECs and LPS could function intracellularily.

Proteomic analysis of human umbilical vein endothelial cells exposed to PM_(2.5)

Exposure to fine ambient particulate matter(PM_(2.5)) is known to be associated with cardiovascular disease. To uncover the molecular mechanisms involved in cardiovascular toxicity of PM_(2.5), we investigated alterations in the protein profile of human umbilical vein endothelial cells(HUVECs) treated with PM_(2.5) using two-dimensional electrophoresis in conjunction with mass spectrometry(MS). A total of 31 protein spots were selected as differentially expressed proteins and identified by matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) MS. The results demonstrated that DNA damage and cell apoptosis are important factors contributing to PM_(2.5)-mediated toxicity in HUVECs. It is further proposed that PM_(2.5) can inhibit superoxide dismutase(SOD) activity and increase reactive oxygen species(ROS) and malonaldehyde(MDA) production in a concentration-dependent manner. Induction of apoptosis and DNA damage through oxidative stress pathways may be one of the key toxicological events occurring in HUVECs under PM_(2.5) stress. These results indicated that the toxic mechanisms of PM_(2.5) on cardiovascular disease are related to endothelial dysfunction.

Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor on survival of random flaps in rats

Objective: To evaluate the effect of local application of vascular endothelial growth factor ( VEGF ) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.Methods: Thirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm × 2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1 012 pfu replication-deficient recombinant adenovirus carrying VEGF ( AdCMV-VEGF group, n = 10 ), 1012 pfu recombinant β-galactosidase adenovirus ( AdCMV-Gal group, n = 10) and 1 ml saline (saline group, n = 10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation.Results: The survival rate of the flaps in the AdCMV-VEGF group increased significantly as compared with those of the AdCMV-Gal group (P < 0.01) and the saline group ( P < 0.01). Immunohistochemical staining showed that VEGF was expressed in the survival flaps injected with AdCMV-VEGF. Histological analysis showed that more granulation tissues and angiogenesis were observed in the AdCMV-VEGF group than those in the AdCMV-Gal and the saline groups.Conclusions: Local application of adenovims-mediated VEGF165 cDNA may efficiently improve the survival of ischemic skin flaps.

In vivo antiplasmodial activity of Bombax buonopozense root bark aqueous extract in mice infected by Plasmodium berghei

OBJECTIVE: To evaluate the in vivo antiplasmodial activity and the oral acute toxicity of the Bombax buonopozense root bark aqueous extract.METHODS: The in vivo antiplasmodial activity of the root bark aqueous extract of Bombax buonopozense against early and established rodent malaria infections in chloroquine sensitive Plasmodium berghei strain in mice was investigated, and oral acute toxicity of the aqueous root bark extract of Bombax buonopozense was also evaluated in mice.RESULTS: The findings of this study revealed significant(P < 0.05) and dose dependent decrease in parasitaemia in the parasitized groups treated with varying doses of the extract(50-200 mg/kg p.o.) in both suppressive and curative tests. There was also significant decrease in parasitaemia density in the chloroquine treated group. The aqueous extract was found no toxicity in mice and the oral LD50 was determined to be greater than 5000 mg/kg.CONCLUSION: Bombax buonopozense root bark aqueous extract possesses potent antiplasmodial activity and may therefore, serve as potential sources of new antimalarial agents.

Prognostic value of human papillomavirus 16/18 genotyping in low-grade cervical lesions preceded by mildly abnormal cytology

Histological low-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grade 1 （LSIL/CIN1） preceded by normal or mildly abnormal cytology is recommended for conservative follow-up, with no separated management. In this study, we assessed the triage value of human papillomavirus （HPV） 16/18 genotyping in 273 patients with LSIL/CINI. HPV16/18 genotyping was performed at baseline and follow-up was at 6-monthly intervals for up to 2 years. At each follow-up, women positive for cytology or high-risk HPV （hrHPV） were referred for colposcopy. Enrollment cytology, HPV16/18 genotyping, and questionnaire-obtained factors were linked to the 2-year cumulative progression rate. Univariate and multivariate analyses were performed taking into account time-to-event with Cox proportional hazard regression. The results showed that 190 cases （69.6%） regressed, 37 （13.6%） persisted, and 46 （16.8%） progressed. HPV16/18 positivity （hazard ratio （HR）, 2.708; 95% confidence interval （CI）, 1.432-5.121; P=-0.002） is significantly associated with higher 2-year cumulative progression rate. Sub-analysis by enrollment cytology and age restricted the positive association among patients preceded by mildly abnormal cytology and aged 30 years or older. Immediate treatment is a rational recommendation for the high-risk subgroup, when good compliance is not assured.