OBJECTIVETo evaluate the therapeutic effect of vascular endothelial growth factor (VEGF) and tumor necrosis factor receptor (TNFR) on avascular necrosis of the femoral head in rabbits.

METHODSAvascular necrosis of the femoral head was induced in 26 New Zealand white rabbits by injections of horse serum and prednisolone. The rabbits were then divided into VEGF/TNFR treatment group, VEGF treatment group, and untreated model group, with another 4 normal rabbits as the normal control group. In the two treatment groups, the therapeutic agents were injected percutaneously into the femoral head. Enzyme-linked immunosorbent assay was performed to determine the concentration of TNF-alpha in rabbit serum followed by pathological examination of the changes in the bone tissues, bone marrow hematopoietic tissue and the blood vessels in the femoral head.

RESULTSCompared with the model group, the rabbits with both VEGF and TNFR treatment showed decreased serum concentration of TNF-alpha with obvious new vessel formation, decreased empty bone lacunae in the femoral head and hematopoietic tissue proliferation in the bone marrow cavity.

CONCLUSIONPercutaneous injection of VEGF and TNFR into the femoral head can significantly enhance bone tissue angiogenesis and ameliorate osteonecrosis in rabbits with experimental femoral head necrosis.

Animals ; Drug Therapy, Combination ; Female ; Femur Head Necrosis ; chemically induced ; drug therapy ; Male ; Rabbits ; Random Allocation ; Receptors, Tumor Necrosis Factor ; therapeutic use ; Tumor Necrosis Factor-alpha ; blood ; Vascular Endothelial Growth Factor A ; therapeutic use

The GFP(green fluorescence protein)-streptavidin(SA) bi-functional fusion protein was generated and characterized in order to demonstrate novel platform for efficiently and durably modifying the cell surface with SA-tagged bi-functional proteins.The GFP-SA/pET24d construct was generated and expressed in BL21(DE3) host bacteria at the high level.The recombinant protein GFP-SA was purified through the Ni-NTA affinity chromatography,and then refolded.After biotinylation B16 tumor cells were modified with GFP-SA bi-functional fusion protein and then subjected to fluorescent microscopy and FACS analysis.The effect of surface modification on the viability and growth of B16.F10 tumor cells was evaluated by MTT staining.The GFP-SA recombinant fusion protein was expressed in BL21(DE3) at about 20 % of total bacterial proteins.The GFP-SA bi-functional fusion protein exhibited the bi-functionality,i.e.,SA-mediated high-affinity binding to biotinylated cell surfaces and GFP-emitted green fluorescence.The cell surface modification with GFP-SA bi-functional fusion protein did not affect the viability and growth of the modified B16.F10 tumor cells significantly.The GFP-SA bi-functional fusion protein was obtained and could be displayed efficiently on the surface of the biotinylated B16.F10 tumor cells through the specific and tight interaction between streptavidin and biotin,thus can be used as good trace protein and experimental control in the development of surface-modified tumor vaccine.

Vascular endothelial growth factor 121 (VEGF121) was expressed as inclusion bodies by recombinant Escherichia coli. High concentrations of both biomass (46 g dry cell/L) and VEGF121 inclusion bodies (4.5 g/L) were obtained by applying a high-cell-density culture. After the inclusion bodies were washed and dissolved, VEGF121 was refolded at 0.2 mg/ml by ultrafiltration in refolding buffer with a yield of 81%. Renatured VEGF121 was purified by anion chromatography and Sephacry S-100 chromatography with purity higher than 95% and fnal purification yield of 31%. The purified VEGF121 could stimulate the proliferation of human umbilical vein endothelial cells as demonstrated by a biological activity assay.

This study was aimed to clone human intercellular adhesion molecule-1 (ICAM-1) gene, to transfect the constructed eukaryotic expression vector ICAM-1-GFP into CHO cells, as well as to detect ICAM-1-GFP expression in CHO cells binding with Molt-4 cells. ICAM-1 cDNA gene was amplified by RT-PCR and inserted in PMD(18)-T vector. Then ICAM-1 cDNA from pMD18-ICAM-1 vector was subcloned into eukaryotic expression vector pEGFP-C1 to construct recombinant ICAM-1-pEGFP-C1 vector. Restriction analysis and DNA sequencing were used to confirm the recombinant vector. After stable transfection of CHO-K1 cells with the recombinant vector, the expression and subcellular localization of ICAM-1-GFP were detected by RT-PCR, flow cytometry and fluorescence microscopy. The function of ICAM-1-GFP fusion protein was assessed by the binding of ICAM-1-GFP/CHO cells to Molt-4 cells. The results showed that 1622 bp full-length ICAM-1 cDNA obtained and was successfully ligated with pMD(18)-T-vector, subcloned to construct recombinant ICAM-1-pEGFP-C1 vector. Restriction analysis and DNA sequencing indicated that recombinant ICAM-1-GFP was successfully constructed and ICAM-1-GFP was expressed stably in CHO cells. ICAM-1-GFP expression was only observed in the cytoplasm of ICAM-1-GFP/CHO cells by fluorescence microscopy. The ICAM-1-GFP/CHO cells were bound to PMA-treated Molt-4 cells. The expression of MEM-148 was very weak in PMA-treated Molt-4 cells. It is concluded that the ICAM-1-GFP eukaryotic expression vector has been constructed successfully and expresses stably in CHO cells. PMA can increase the binding of Molt-4 cells to ICAM-1-GFP/CHO cells by inducing specialized form of ICAM-1 clustering.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; DNA, Complementary ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; Recombinant Proteins ; genetics ; Transfection

OBJECTIVETo investigate the changes of the cardiac hemodynamics after acute high altitude exposure in healthy young males and the relationship with acute mountain sickness(AMS).

METHODSLeft ventricular function and oxyhemoglobin saturation (SaO2), heart rate (HR), blood pressure (BP) were measured in 218 healthy young males before and after high altitude exposure within 24 h respectively. According to the lake louise score criteria, the subjects were divided into two groups: acute mountain sickness group (AMS group) and non acute mountain sickness group (non-AMS group).

RESULTSHR, diastolic blood pressure (DBP), mean arterial pressure (MAP), left ventricular ejection fraction (LVEF), stroke volume (SV), stroke index (SI) cardiac output (CO), cardiac index (CI) were significantly increased upon acute high altitude exposure (P < 0.05). Whereas SaO2 and end-systolic volume (ESV) were significantly decreased (P < 0.05). In addition, HR, systolic blood pressure (SBP) and MAP in AMS group were significantly higher than those in non-AMS group (P < 0.05). But stroke index (SI) and end-diastolic volume (EDV) in AMS group were significantly lower than those in non-AMS group (P < 0.05).

CONCLUSIONCardiac function in healthy young males upon acute high altitude exposure was enhanced. EDV, HR and SI might become the indexes of predicting the acute mountain sickness in the future.

Acute Disease ; Adult ; Altitude ; Altitude Sickness ; physiopathology ; Humans ; Male ; Ventricular Function, Left ; physiology

objective To investigate the tissue localization of CD4+T cells producing IL-17,namely Th17 cells.in patients with systemic lupus erythematosus (SLE),as well as its relationship with the activity of lupus.Methods By using H&E staining.double-label immunofluorescence.immunohistochemistry and confocal microscopy.the localization of Th17 cells was carried out in peripheral blood mononuclear cells (PBMCs).affected tissue of skin and lung obtained from 4 patients with active SLE and 2 normal human controls.Flow cytometry.reverse transcription PCR.ELISA were used to detect the proportion of Th17 cells in PBMCs,the mRNA expression of interleukin-17(IL-17)A and IL-17 F,and serum level of interleukin 17,respectively,in 50 consecutive adult patients with SLE and 15 normal human controls.Results Th17 cells were detected in PBMCs of patients with active SLE.and the fuorescence intensity of IL-17 was significantly higher in patients with active SLE than in normal human controls(127.6±20.5 vs 40.6±11.1,P＜0.001).Infiltrates of Th17 cells were noted in both skin and lung tissues of patients with active SLE.but not in those of normal human controls.The proportion of Th17 cells in PBMCs was increased in patients with active SLE.and the proportion positively correlated with SLE disease activity index(SLEDAI) (r=0.725,P＜0.01).Further more.a significant increase was observed in the mRNA expression of IL-17 A and IL-17 F and serum level of IL-17 in patients with active SLE compared with normal human controls.The amount of Th17 cells was positively correlated with the development of vasculitis.and it experienced a decrease with the remission of SLE.Conclusions A proliferation of Th17 cells is noted in patients with active SLE.which seems to closely correlated with the activity of SLE and may take part in the development of vasculitis in SLE.

Objective To investigate the relationship between the level of serum uric acid(SUA) and carotid atherosclerosis in male patients with type 2 diabetes mellitus(T2DM). Methods A collection of 579 male T2DM patients with or without carotid atherosclerosis were grouped based on quartiles of SUA. Age, SUA, smokers, duration, body mass index(BMI), blood pressure, total cholesterol(TC), triglyceride(TG), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C), blood urea nitrogen(BUN), creatinine(Cr), and HbA1C were determined in all subjects. The plaques in carotid arteries and intima-media thickness(IMT) were measured with high-resolution ultrasound. Results BMI, systolic and diastolic blood pressures, TG, and Cr showed a gradual increase, while HDL-C and HbA1C showed a gradual decrease according to the higher SUA quartiles in male T2DM(P＜0.05). Nevertheless, the detectable rates of smokers, duration, age, TC, LDL-C, and BUN had no relationships with the SUA quartiles(P＞0.05). The detectable rate of carotid atherosclerosis and the thickness of carotid plaque were positively associated with the levels of SUA in male patients with T2DM(P＜0.05). However, intima-media thickness of carotid arteries did not illustrate the correlation with the levels of SUA in male T2DM patients(P＞0.05). Age, HbA1C, and SUA were independent factors of carotid atherosclerosis in these patients by logistic regression(P＜0.05). Conclusion The levels of SUA seems to be associated with the occurrence and development of carotid atherosclerosis in male patients with T2DM.

Aim To look for novel small-molecule inhibitors of CDK9 through structure-based virtual screening and biological activity determination.Methods Homology modeling of CDK9 was based on the 3-D structure of other cyclin-dependent kinase family members,and then virtual screening by DOCK(molecular docking)of database of small molecule was carried on.MTT method was used in inhibition of tumor cell growth in vitro,while Western blot was used for further study of molecular mechanisms.Results From the top 1000 compounds with the best DOCK energy score,27 compounds were selected for biological assay based on the diversity of chemical structure and functional group.12 of 27 selected compounds showed significantly inhibition activity on tumor cell proliferation,and only one compound in 12 with half-maximum inhibition concentration(IC50)values less than 20 ?mol?L-1 named C-21 was selected for further molecular mechanism study.The western blotting data showed C-21 compound could effectively inhibit CDK9 from phosphorylating large subunit C-terminal of RNA polymerase Ⅱ in a dose-dependent manner.Conclusions Through homology modeling,virtual screening by computer,determination of biological activity and experimental studies of molecular mechanism,a new promising lead compound targeted for CDK9 was found and confirmed.

Objective To study the roles of micheliolide on ovarian cancer cells. Methods Firstly, human ovarian cancer cell lines HeyA8, SKOV3 and A2780/DDP were treated with different concentration of micheliolide (0.25, 0.5, 1, 2.5, 5, 10, 20, 50 μmol/L) for 72 hours, then methyl thiazolyl tetrazolium (MTT) assay was used wo detect the growth of the human ovarian cancer cell lines and the stongest inhibited cell line were selected for the following test. Secondly, after HeyA8 cell line was treated with different concentration (5, 10, 20μmol/L) of micheliolide for 24 hours, the HeyA8 cell apoptosis was measured byflow cytometry. Thirdly, the expression of RelA mRNA in HeyA8 cell was detected through real-time PCR, the expressions of nuclear factor κB(NF-κB)signal pathway related protein RelA and the activited cysteinyl aspartate specific proteinase (caspase-9) were detected by western blot analysis. Results (1) The growth of HeyA8, SKOV3 and A2780/DDP cells were all significantly inhibited after being treated with different concentration of micheliolide for 72 hours and the roles of inhibition were all concentration dependant (P<0.05). The half maximal inhibitory concentration (IC50) of HeyA8, SKOV3 and A2780/DDP were (9.8±2.2), (12.0±2.1) and (12.8±1.8)μmol/L, respectively. We chose HeyA8 cell to do the following expreriments because of its best inhibited effect. (2) After HeyA8 cell was treated with micheliolide of different concentrations, as the concentration increased (20 and 0μmol/L, for example), the apoptosis rate of HeyA8 cell raised from (7.2±1.0)%to (17.4±1.1)%, the percentage of survived cells reduce from (92.8 ± 1.3)% to (82.6 ± 1.4)%,and the relative mRNA level of RelA decreased from 1.00 ± 0.13 to 0.18 ± 0.00 (P<0.01); furthermore, the expression of RelA protein was weaken and the activited caspase-9 protein expression was increased gradually. Conclusions Micheliolide plays a significantly inhibited role in HeyA8, SKOV3 and A2780/DDP cells. The inhibited role of micheliolide inovarian cancer cells might through inhibiting nuclear factor-kappa B (NF-кB) signaling pathway, and inducing the expression of activited caspase-9 protein to promoting apoptosis of HeyA8 cell.

Objective To compare diabetes(pre-diabetes) prevalence and awareness between Province Shanxi and Fujian .Meth-ods Study data was from China National Diabetes and Metabolic Disorders Study 2007-2008 .5 926 individuals(Shanxi 3 254 ,Fu-jian 2 672) were included as study participants .Standardized questionnaire was used to obtain demographic and lifestyle data .All participants were administered a glucose tolerance test .Results Overall diabetes prevalence was 9 .6% .The diabetes prevalence in Shanxi was lower compared with Fujian(8 .2% vs .11 .3% ,P<0 .001) .Overall pre-diabetes was 15 .4% and no significant differ-ence was found between the two provinces .The awareness of diabetes and pre-diabetes in Shanxi was lower compared with Fujian , although both provinces were not satisfactory .Conclusion Regional differences were showed in diabetes (pre-diabetes) prevalence and awareness between Province Shanxi and Fujian .