Objective To study the role of the chimeric hsp70/CD80 DNA vaccine for treatment for TB. Methods C57BL/6N mice challenged with H37Rv were immunized with the chimeric hsp70/CD80 DNA vaccine, hsp70 DNA vaccine and BCG in order to compare the therapeutic role of these vaccines. Results The levels of interferon ? (IFN-?) in the serum of mice in hsp70/CD80 group (1336.98?129.64) pg/ml was significantly higher than in group BCG (121.54?56.39) pg/ml, pcDNA3 (192.00?64.36) pg/ml and pcDNA3 hsp70 (542.33?99.77) pg/ml. The growth of Mycobacterium tuberculosis in live and spleen were inhibited significantly in hsp70/CD80 vaccinated mice. Conclusions The chimeric hsp70/CD80 DNA vaccine was more efficient than BCG and hsp70 DNA vaccine alone in treating TB.

BACKGROUND: Angiogenesis attracts much attention in tissue engineering field. Previous research has proved that a two-dimensional culture of vascular endothelial growth factor (VEGF) promotes angiogenesis.OBJECTIVE: To evaluate the effect of VEGF on three-dimensional angiogenesis.METHODS: Endothelial progenitor cells were separated from the SD rat bone marrow. At about 70%-80% fusion, rat tail collagen gel was added to establish three-dimensional models. Samples in the experimental group were incubated in complete culture solution containing M199 culture media, fetal bovine serum, VEGF, and double antibody. The samples in the control group were incubated with VEGF-free culture media. In vitro culture and amplification of bone marrow-derived endothelial progenitor cells were determined at 1, 4, 7, and-20 days after incubation. Morphology and quantitative analysis were performed at 3, 6, 9, and 12 days after three-dimensional model establishment.RESULTS AND CONCLUSION: Endothelial progenitor cells grew from three-dimensional matrix into collagen matrix in the experimental group. Budding and infiltration were observed in the collagen within 24 hours, and branching-like structure was then gradually formed. Cells in the control group grew slowly, with slowing budding, small tubiform structure, superficial infiltration into COllagen, sparse network structure, and non-intact. Numbers of newborn vessels in the expedmental group were significantly greater than control group (P<0.01). A detection on gel block showed positive expressions of endothelin-1 and endothelial nitric oxide synthase-3 on the 3~(rd), 6~(th), 9~(th), and 12~(th) days. The results demonstrated that VEGF mobilized and induced endothelial progenitor cells in order to promote angiogenesis. Rat tail collagen gel induced endothelial progenitor cells which behaved migration, proliferation, and pullulation of angiogenesis.

Objective To explore the suitable range of PT-INR for Chinese people with acute deep venous thrombosis (DVT) treated by warfarin anticoagulation therapy. Methods Eighty seven DVT patients with indications to warfarin anticoagulation therapy were enrolled into the study and divide into two groups randomly. Patients from group A (n=47) took warfarin to adjust the PT-INR to range 1.7-2. 5,and patients from group B (n =40) took warfarin to adjust the PT-INR to range 2. 0-3. 0. The therapeutic effectiveness and the incidence of bleeding complications were compared between two groups. Results Forty-six patients (46/47,98%) had limb swelling symptoms relief in group A with one exception,which was diagnosed as pelvic tumor by ultrasonography,CT and tumor markers examination later. No patient underwent bleeding in group A Thirty eight patients (38/40,93%) had limb swelling symptoms relief in group group B with two exceptions,of which one case had Cockett syndrome and the other one had unknown aetiology. The total effective rate of group B was 95% . As to the complications of this group,3 patients had slight gum and nasal mucous membrane bleeding, 1 patient developed gastrointestinal bleeding. No patients had pulmonary embolism in both groups. Conclusion For Chinese people,anticoagulation therapy of acute deep venous thrombosis to adjust the range of PT-INR to 1.7-2. 5, shows good effectiveness and significantly reduced bleeding complications.

Objective:To study the antisense oligonucleotide mediated inhibition on telomerase activity and cell proliferation of GBC-SD cell.Methods:We design the antisense,sense,and random oligonucleotide with phosphoric acid modification for the hTR(Human Telomerase RNA)template sequence.MTT and PCR methods were used to observe the inhibition on telomerase activity and cell proliferation of GBC-SD cell ,and fibroblast cells were used as control group.Results:PS-ODN can lead to the reduction of cell survival rate of GBC-SD cell,wich dosage dependence.Tne experimental group cell detected by scanning electron appeared apoptotic feature.Conclusion:PS-ODN can inhibit telomerase activity of GBC-SD cell effectively and induce the cell apoptosis.

Objective To investigate the role of Wnt/β-catenin signaling pathway in impaired wound healing of diabetes mellitus.Methods The back skin defect was produced in rats with type1diabetes.All of these rats were divided into normal group, diabetes group, lithium chloride group, and epidermal growth factor (EGF) group.The back wound healing and β-catenin expression were observed.Results There were no signs of infection in the wound of rats after injury.Compared with diabetic group, the wound healing time was shorter,wound healing rate was higher, wound cavity volume was smaller, granulation tissue was more mature, and β-catenin positive cell rate was higher in normal group, lithium chloride group, and EGF group(P＜0.05 or P＜0.01).Conclusions Wnt/beta-catenin signaling pathway is involved in the process of wound healing in diabetic rats.

OBJECTIVEUtilizing the hypoxia inducible factor 1/hypoxia reaction element (HIF-1/ HRE) gene regulation system to construct antisense vascular endothelial growth factor (VEGF165) cDNA eukaryotic expression vector promoted by HRE, and investigate its targeted inhibiting VEGF expression of osteosarcoma cells in hypoxia environment.

METHODSEukaryotic expression plasmid with HRE promoter was constructed containing luciferase reporter gene and antisense VEGF165 cDNA by using PCR and recombinant DNA techniques. The recombinant vectors were transfected into osteosarcoma cells with lipofectin method. Hypoxia-inducible reporter gene expression was determined by liquid scintillation analyzer and the expression of VEGF protein was detected by ELISA method.

RESULTSThe eukaryotic expression plasmid containing antisense VEGF165 and luciferase promoted by HRE was constructed successfully. After being transferred into MG63 cells, luciferase expression was increased 3.5 x 10(2) times and VEGF protein expression decreased 45% under hypoxia condition.

CONCLUSIONAntisense VEGF165 cDNA expression, efficiently realized by HRE promoter under hypoxia condition, provides an experimental basis for targeted antiangiogenesis of tumors.

Bone Neoplasms ; metabolism ; pathology ; Cell Hypoxia ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; Luciferases ; genetics ; metabolism ; Oligodeoxyribonucleotides, Antisense ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; Promoter Regions, Genetic ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.

METHODSEukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations.

RESULTSThe eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis.

CONCLUSIONSAntisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.

Apoptosis ; Bone Neoplasms ; metabolism ; pathology ; Bystander Effect ; Cell Hypoxia ; Cell Line, Tumor ; Cell Proliferation ; DNA, Neoplasm ; biosynthesis ; Ganciclovir ; pharmacology ; Genetic Vectors ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; Oligodeoxyribonucleotides, Antisense ; Osteosarcoma ; metabolism ; pathology ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Abstract】 【Objective】To investigate the effect and mechanism of ginsenoside Rb1 attenuating human umbilical vein endothelial cells(HUVEC) senescence induced by high glucose through Sirt3/SOD2 pathway.【Methods】The senescence of HUVEC induced by high glucose(40 mmol/L)was assessed by senescence-associated β-galactosidase(SA-β-Gal)staining,and the expression of plasminogen activator inhibitor 1(PAI-1)and P16. Annexin V-FITC/PI was performed to measure apoptotic effect. The expression of sirtuins 3(Sirt3)and superoxide dismutase 2(SOD2)was detected by western blot. Meanwhile,the level of intracellular malondialdehyde(MDA)and the activity of SOD2 were measured.【Results】Treatment of HUVEC with high glucose for 24 hours induced premature senescence instead of apoptosis,as indicated by a larger proportion of the cells stained with SA-β-Gal and the up-regulated expression of PAI-1 and P16. Pretreatment of HUVEC with ginsenoside Rb1(40 μmol/L)could reverse endothelial cell senescence,as indicated by the reduced SA-β-Gal positive cells and the down-regulated expression of PAI-1 and P16. Furthermore,ginsenoside Rb1 pretreatment upregulated the protein expression of Sirt3 and SOD2,and eventually increased the activity of SOD2 and decreased the level of MDA.【Conclusion】Ginsenoside Rb1 could antagonize high glucose-induced premature senescence of HUVEC via Sirt3/SOD2 signaling pathway.

OBJECTIVETo observe the effects of angiotensin converting enzyme inhibitors (ACEI) and angiotensin II receptor blocker (ARB) on tumor growth and angiogenesis in implanted gastric cancer mouse model, and to explore the probable mechanism of ACEI and ARB anticancer effect.

METHODSNude mouse model with human gastric cancer was established by subcutaneously inoculating human gastric cancer cell line SGC-7901. One week later, 60 mice were randomly divided into 5 groups: control group, perindopril group, captopril group, losartan group, and valsartan group. These groups respectively received the normal saline, perindopril (2 mg/kg), captopril (5 mg/kg), losartan (50 mg/kg), valsartan (40 mg/kg) by gavage once a day. Twenty-one days after treatment the tumors were removed and the tissues were stained by immunohistochemistry method to observe the expression of VEGF, MMP-7 and microvessel density (MVD).

RESULTSIn all the ACEI and ARB groups, tumor volumes were significantly inhibited and MVD also decreased significantly as compared with control group (all P<0.01). In captopril and perindopril groups, the expression of VEGF and MMP-7 decreased significantly as compared with control group(all P<0.05). In losartan and valsartan group, the expressions of VEGF were significantly decreased as compared with control group (all P<0.05). The expressions of MMP-7 between ARB groups and control group were not significantly different.

CONCLUSIONACEI and ARB can inhibit the tumor growth in gastric cancer model and suppress the angiogenesis of the tumor.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Cell Line, Tumor ; Female ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neovascularization, Pathologic ; Stomach Neoplasms ; blood supply ; pathology

OBJECTIVETo study the influence of CD44 a cell-matrix adhesion molecule on the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines, in order to investigate the growth and invasion mechanism of osteosarcoma.

METHODSThree osteosarcoma cell lines MG-63, HOS and U2-OS were routinely cultured. Flow cytometry and Western blot analysis were used for detecting the positive rates and relative amount of CD44 protein in the three cell lines. RT-PCR method was also used to compare the differences in the expression of CD44 mRNA among the 3 cell lines. Then, MTT method, adhesion detection, and Microcon-migration assay were used to detect the changes of the cells' proliferation rate, adhesive and invasive abilities after blocking the role of CD44 by using a special neutralizing antibody.

RESULTSThe results of flow cytometry showed that the percentage of CD44 positive cells were both over 99% in HOS and U2-OS, while that in MG-63 was only (2.10 +/- 0.46)%. The average fluorescence density of CD44 in HOS was significantly higher than in U2-OS. Western blot also showed that the relative content of CD44 protein in HOS was notably higher than that in U2-OS, while CD44 was negatively expressed in MG-63. The expression of CD44 mRNA was significantly lower in MG-63 than in both HOS and U2-OS, which were consistent with the expression of CD44 protein. The proliferation rates and adhesive abilities of MG-63 and HOS have no significant difference, but both were significantly higher than that of U2-OS. The invasive abilities of HOS was dramatically higher than MG-63 and U2-OS. After the role of CD44 was blocked by anti-CD44 neutralizing antibody, the proliferation rates of the 3 cell lines did not change significantly. While the HOS and MG-63 adhesion indices decreased dramatically (P < 0.05), the invasive abilities of HOS and U2-OS also decreased notably (P < 0.01).

CONCLUSIONSCD44 could promote the adhesiveness and invasiveness of osteosarcoma cell line HOS. CD44 may take part in promoting the process of U2-OS invasion and the adhesion of MG-63. On the other hand, CD44 could not affect the osteosarcoma cell proliferation rates.

Bone Neoplasms ; metabolism ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Hyaluronan Receptors ; biosynthesis ; genetics ; physiology ; Neoplasm Invasiveness ; Osteosarcoma ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics