OBJECTIVETo establish an implanted model of human colonic carcinoma on chick embryo, and to study the effects of anti-angiopoietin-2 antibody on its vascularization.

METHODSThe human colonic adenocarcinoma cell line HT-29 was transplanted on the chick embryo's chorioallantoic membrane(CAM), and the angiogenesis characteristics were observed by stero-microscope, light microscope and immunohistochemistry. Furthermore, the effects of anti-angiopoietin-2 antibody on angiogenesis and tumor growth were also investigated.

RESULTSThree to seven days after HT-29 cell line was implanted into CAM, tumors grew rapidly and new blood vessels grew toward tumors. Five days after anti-angiopoietin-2 antibody was given, the number of blood vessels in anti-angiopoietin-2 antibody group was significantly down-regulated than that in tumor control group observed by stero-microscope (37.2+/-4.6 vs 56.8+/-7.4, P<0.01), but was up-regulated than that in normal control group (37.2+/-4.6 vs 9.6+/-2.4, P<0.01). Microvessel density(MVD) in anti-angiopoietin-2 antibody group was much lower than that in tumor control group by histological examination (9.6+/-2.4 vs 20.2+/-5.8, P<0.01).

CONCLUSIONAngiopoietin-2 antibody is able to inhibit the angiogenesis induced by colorectal cancer cell line HT-29 obviously. The anti-angiopoietin-2 antibody may be potentially useful for clinical treatment of colonic carcinoma.

Angiopoietin-2 ; immunology ; Animals ; Antibodies, Monoclonal ; pharmacology ; Chick Embryo ; Colonic Neoplasms ; blood supply ; HT29 Cells ; drug effects ; Humans ; Neoplasm Transplantation ; Neovascularization, Pathologic

OBJECTIVETo investigate the effects of high glucose on expression of matrix metalloproteinase-2 (MMP-2) and extracellular matrix metalloproteinase inducer (EMMPRIN) in human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs were cultured in DMEM media containing high glucose with or without polyphenols for 24 hours respectively. The activity of MMP-2 in the supernatant was detected by zymography. The expression of MMP-2 mRNA and protein in HUVECs were detected by RT-PCR and Western blot respectively. The expression of EMMPRIN mRNA and protein in the cells were determined by RT-PCR as well as immunocytochemistry and Western blot respectively.

RESULTSThe expression of MMP-2 and EMMPRIN mRNA were suppressed by a high concentration of glucose. Both the MMP-2 activity and protein level of EMMPRIN expression were also significantly decreased. Polyphenols abolished all the above changes of HUVECs induced by a high glucose level (P < 0.05).

CONCLUSIONSAn acute high glucose stimulation down-regulates the activity of MMP-2, the expressions of MMP-2 and EMMPRIN at RNA and protein levels in the endothelial cells, which may play an important roles in diabetic vascular complications in the early phase. Polyphenols treatment can diminish the detrimental effects of high glucose on HUVECs.

Basigin ; genetics ; metabolism ; Cells, Cultured ; Endothelial Cells ; metabolism ; Flavonoids ; isolation & purification ; pharmacology ; Glucose ; metabolism ; Humans ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Phenols ; isolation & purification ; pharmacology ; Polyphenols ; RNA, Messenger ; metabolism ; Tea ; chemistry ; Umbilical Veins ; cytology

OBJECTIVETo study the protective effects of salidroside on injury induced by hypoxia/hypoglycemia in cultured SH-SY5Y cell.

METHODApoptosis and intracellular free calcium concentration ([Ca2+]i) were measured by flow cytometry, morphological changes and neuronal necrosis were observed with fluorescence microscope, and the lactic dehydrogenate (LDH) release was measured by LDH kits.

RESULTHypoxia/hypoglycemia cultures for 2 hours induced neuronal apoptosis and necrosis. They were 18.59% (P < 0.01) and 4.94% (P < 0.01) respectively. There were morphological changes including chromatin condensation, nuclear fragmentation and formed apoptotic bodies after exposed to hypoxia/hypoglycemia for 2, 4, 6, 12 hours. After 2 hours of hypoxia/hypoglycemia, neuronal [Ca2+]i and the release of LDH were significantly increased. They were 8.46 nmol/L (P < 0.01) and 16.59% (P < 0.01) respectively. The effects were enhanced with the extending time of hypoxia/hypoglycemia. Salidroside might have significantly decreased the percentage of neuronal apoptosis and necrosis, reduced neuronal [Ca2+]i and the release of LDH. The effects of salidroside were strengthened with the increasing of Salidroside dosage.

CONCLUSIONSalidroside has effect of anti-neuronal apoptosis. This effect might be related to its function of decreasing intracellular free calcium concentration.

Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Hypoxia ; drug effects ; Glucosides ; isolation & purification ; pharmacology ; Humans ; Hypoglycemia ; metabolism ; pathology ; L-Lactate Dehydrogenase ; metabolism ; Neurons ; pathology ; Neuroprotective Agents ; pharmacology ; Phenols ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Rhodiola ; chemistry

AIMTo separate and purify the anti-myocardial ischemic polysaccharide fraction with a homogenous molecular weight from Ophiopogon japonicus, then study the chemical structure of the parts.

METHODSCrude polysaccharides were prepared by extracting the tube root fraction of Ophiopogon japonicus with water, then precipitation with ethanol. From the crude polysaccharides, the polysaccharide of MDG-1 was separated and purified using ultrafiltration, DEAE Sepharose FF and Sephadex G-25 column chromatography. Its structure was studied by complete hydrolysis, periodate oxidation, Smith degradation, methylation analysis, 1H NMR and 13C NMR analysis etc.

RESULTS AND CONCLUSIONMDG-1 was a water-soluble beta-D-fructosan, containing a backbone composed of Fruf (2 --> 1), and a branch of Fruf (2 --> 6) Fruf (2 --> per average 2. 8 of main chain residues. Mn, Mw and Mp of MDG-1 were 3 400, 4 800 and 5 000, respectively. MDG-1 contains trace of Glc, which maybe connect to its reducing terminal. Molar ratio of Fru and Glc is approximately 35: 1.

Methylation ; Molecular Structure ; Molecular Weight ; Ophiopogon ; chemistry ; Plant Tubers ; chemistry ; Plants, Medicinal ; chemistry ; Polysaccharides ; chemistry ; isolation & purification

Objective: To evaluate the inhibitory effect of tumor vaccines in colon carcinoma model mice. Methods: Mouse bone marrow⁃derived dendritic cells（BMDCs）were stimulated by using CpG β⁃glucan nanoparticles（CNP）in vitro. The BMDCs were divided into PBS group，NP group（without CpG nanoparticles），Lysate group（MC38 cell lysate）and CpG group（CpG1826），which were determined for the expression of marker molecules on the surface by flow cytometry and for the contents of interleukin⁃6（IL⁃6）and IL⁃12p40 in the culture supernatant by ELISA. The tumor lysate nano⁃vaccine was pre⁃ pared by mixing 50 mg／mL tumor lysate（MC38 cell lysate）with 200 mg／mL CNP in a volume ratio of 1∶1，with which mice were subcutaneously immunized as Vaccine group. Vaccine group，PBS group，CNP group and Lysate group were im⁃ munized once a week，for three times in total. Mice were subcutaneously inoculated with MC38 cells，2 × 105 cells for each， in the right lower limb 1 h after the last immunization，and measured for tumor volume once every three days to plot the tumor growth curve. The ratios of CD3+ CD4+ T and CD3+ CD8+ T cells in the blood were analyzed by flow cytometry and the levels of tumor necrosis factor⁃α（TNF⁃α）and interferon γ（IFNγ）in the blood and spleen of mice were determined by ELISA. Results: CNP effectively increased the expression of CD11c+ CD80+，CD11c+ CD86+，CD11c+ MHC⁃Ⅱ+ and the secretion of IL⁃6 and IL⁃12p40 in BMDCs in vitro，which were significantly higher than those in other 4 groups（t = 4. 3 ~ 46. 2，each P < 0. 05）. Compared with that of the other three groups，the tumor volume of mice in Vaccine group decreased significantly（t =2.6~3.4，eachP <0. 05）；TherewasnosignificantdifferenceinCD3+ CD8+ TandCD3+ CD8+ Tcellratios（t = 0.5~ 1. 9，each P > 0. 05）；The content of IFNγ in blood increased significantly（t = 3. 8 ~ 4. 6，P < 0. 05），while thatof TNF⁃α showed no significant difference（t = 0. 4 ~ 2. 0，each P > 0. 05）；However，the contents of IFN γ and TNF⁃α in spleen increased significantly（t = 6. 3 ~ 13. 0，each P < 0. 001）. Conclusion The prepared nano⁃vaccine of tumor lysate improvedtheimmune level in mice and effectively inhibited the growth of colon carcinoma.

Objective To investigate the frequency of CD4~+CD25~+FoxP3~+regulatory T cells (Treg)and the expression of the functional protein,FoxP3,in patients with active tuberculosis and the relationship between Treg and the pathogenesis of tuberculosis.Methods Forty-five patients with active tuberculosis(including 25 cases of pulmonary tuberculosis and 20 tuberculous lymphadenitis), 20 healthy controls,20 recovered tuberculosis patients and 6 patients with reactive hyperplasia in cer- vical lymph node were enrolled.The frequency of CD4~+ CD25~+ FoxP3~+ Treg in the peripheral blood was measured by flow cytometry.FoxP3 mRNA expression was determined by real-time reverse transcriptase-polymerase chain reaction(RT-PCR)and the expression of FoxP3 protein in lymphoid tissues was measured by immunohistochemistry.Results The frequency of natural Treg in the peripheral blood from the patients with active tuberculosis was 2.91%?0.23%,which was signifi- cantly higher than that of healthy control group(1.22%?0.18%)and recovered tuberculosis patients(1.50%?0.17%,P

OBJECTIVETo assess the larvicidal effects of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or Bacillus thuringiensis subsp israelensis (B.t.i) toxin Cyt2Ba against the second instar larvae of Culex pipiensquinquefasciatus and Aedes albopictus and compare different formulations for their larvicidal effects.

METHODSThe AaIT- or Cyt2Ba-coding sequences were cloned into pET28a(+) and the recombinant plasmids were transformed into E. coli BL21(DE3). After induction with IPTG, the recombinant proteins expressed by the recombinant E. coli were detected and identified by SDS-PAGE and Western blotting, respectively. The larvicidal activity of the bacterial suspension was tested at different concentrations against mosquitoes. The effective engineered bacteria were prepared into dry powder with different formulations, and their larvicidal activity was tested.

RESULTSAaIT and Cyt2Ba proteins were successfully expressed in E. coli. The recombinant AaIT protein showed no virulence to the mosquito larvae. The suspension of the recombinant E. coli expressing Cyt2Ba protein exhibited a stronger killing effect on Aedes albopictus larvae than on Culex pipiens quinquefasciatus larvae at 48 h (P<0.001) with LCof 3.00×10cells/mL and 1.25×10cells/mL, respectively. The dry powder of the engineered bacteria formulated with yeast extract, wheat flour or white pepper powder at the mass ratio of 1:1 showed the strongest killing effect on mosquito larvae (P=0.044), and the formulation with white pepper powder produced a stronger killing effect than formulations with yeast extract or wheat flour (P=0.002).

CONCLUSIONThe B.t.i Cyt2Ba protein expressed in E. coli BL21(DE3) shows a good larvicidal activity against mosquitoes, and appropriate formulations of the engineered bacteria can enhance its efficiency in mosquito control.

OBJECTIVECastrated rats exhibit significant shrinkage of the ventral prostate and apoptosis of prostatic cells, which can be attributed to the reduced blood supply to the prostate. But what causes the blood decrease in the prostate remains unknown. This study aims to explore the molecular mechanism of the changes in the microcirculation of the ventral prostate of rats following castration.

METHODSWe randomized 24 male adult rats into 6 groups of equal number, and collected their ventral prostates at 0, 1/2, 1, 2, 3 and 7 d, respectively, after castration. Then we observed the changes of the microvessels under the transmission electron microscope, detected the apoptosis of endothelial cells by TUNEL, and determined the expressions of VEGF, endostatin, angiostatin and angiopoietin-2 by Western blot.

RESULTSThe castrated rats showed dramatic changes in the microvessels of the ventral prostate, obvious apoptosis of the endothelial cells, down-regulated expression of VEGF, and up-regulated expressions of endostatin and angiostatin, while angiopoietin-2 remained unchanged.

CONCLUSIONThe decreased level of VEGF and increased levels of endostatin and angiostatin might underlie the mechanism of the changes in the microcirculation of the ventral prostate of rats following castration.

Angiopoietin-2 ; metabolism ; Angiostatins ; metabolism ; Animals ; Endostatins ; metabolism ; In Situ Nick-End Labeling ; Male ; Microcirculation ; Orchiectomy ; Prostate ; blood supply ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism

Objective To reveal the whole-mount distribution pattern of intramuscular nerves in the medial and lateral plantar muscles and to explore its clinical significance. Methods Twenty-four adult cadavers were dissected to remove the medial and lateral groups of the plantar muscles. The distribution pattern of the intramuscular nerves was demonstrated by modified Sihler' s staining. Results The nerve branch for adductor hallucis muscle entered the muscle from the deep surface of the insertion of the muscle, while those nerve branches for abductor hallucis, flexor hallucis brevis, abductor digiti minimi and flexor digiti minimi brevis muscles entered the muscle from the deep side of the origin of the muscle. There were one lunate and one rectangular intramuscular nerve dense regions (INDRs) in the abductor hallucis muscle; two reniform INDRs in the transverse head of the adductor hallucis muscle, one reniform and one rectangular INDRs in the oblique head of the adductor hallucis muscle; there were two rectangle INDRs in the flexor hallucis brevis, abductor digiti minimi and flexor digiti minimi brevis muscles. These five muscles were divided into two neuromuscular compartment, but the percentage position of INDR and the center of INDR on muscle length in each muscle were different. Conclusion These result may provide morphological guidance for surgical operation to avoid nerve injury, the selection and matching of muscle transplantation and the injection of botulinum toxin A to block the spasticity of these muscles.

OBJECTIVE: To observe the role of aminophylline and simvastatin in preventing and curing chronic obstructive pulmonary disease (COPD), and to explore the underlying mechanisms based on airway inflammation and mucus hypersecretion.
 METHODS: The rat model of COPD was established by combination of cigarette smoking with intratracheal lipopolysaccharide (LPS) injection. Male SD rats were randomly divided into 4 groups (n=10 per group): a control group, a COPD group, an aminophylline group and a simvastatin group. The rats in the control group and the COPD group were treated with normal saline once a day via intragastric administration, while the rats in the aminophylline group and the simvastatin group were treated with aminophylline (5 g/L) and simvastatin (0.5 g/L) 1 mL/100 g once a day via intragastric administration, respectively. Pulmonary function and pathological changes in bronchus and lung were observed. The levels of IL-8, IL-17, and TNF-α in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expressions of TLR4 and mucin 5AC (MUC5AC) in bronchi and lung tissues were detected by real-time PCR and Western blot, respectively.
 RESULTS: Pulmonary function and the pathophysiologic changes in bronchi and lung tissues in the COPD rats were consistent with typical phenotype of COPD. Compared with the control group, lung function indexes were significantly attenuated in the COPD group, while the levels of IL-8, IL-17, and TNF-α in BALF as well as the mRNA and protein levels of MUC5AC and TLR4 were significantly increased. Compared with the COPD group, lung function indexes were significantly increased in the aminophylline group and simvastatin group (P<0.01), while pulmonary pathological damages, the levels of IL-8, IL-17, and TNF-α in BALF as well as the mRNA and protein levels of MUC5AC and TLR4 were significantly decreased (P<0.01). Compared with the aminophylline group, the peak expiratory flow as well as the levels of IL-8, IL-17, and TNF-α in the simvastatin group were elevated (P<0.05). There are no significant difference in the mRNA and protein levels of MUC5AC and TLR4 between the 2 groups (P﹥0.05).
 CONCLUSION Aminophylline and simvastatin can decrease IL-8, IL-17, and TNF-α levels in BALF and inhibit the expression of MUC5AC and TLR4 in airway and lung tissues in COPD rats, suggesting that they may have a preventive and therapeutic effect on COPD through reducing the airway inflammation and mucus hypersecretion.
Aminophylline ; pharmacology ; Animals ; Bronchi ; metabolism ; Bronchoalveolar Lavage Fluid ; chemistry ; Cytokines ; chemistry ; Inflammation ; drug therapy ; Lipopolysaccharides ; Lung ; metabolism ; physiopathology ; Male ; Mucin 5AC ; metabolism ; Mucus ; metabolism ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Simvastatin ; pharmacology ; Smoke ; adverse effects ; Smoking ; adverse effects ; Toll-Like Receptor 4 ; metabolism