Objective To get a high affinity peptide of vascular endothelial growth factor receptor 3 (VEGFR3) as a potent carrier targeted to lymphangiogenesis of ovarian cancer via the technology of phage display. Methods Solid-phase was panned with direct VEGFR3 extracellular protein coating, then the unbound phage was washed away and the eluted phage was amplified. The positive phage clones were identified by ELISA and sequenced, and the affinity and specialty were identified by competent ELISA. Results After four-round bio-panning, the enriched positive phage clones were identified by ELISA. Eight positive phage clones were sequenced and 5 were consensus (WHGSLKQNLWWY). The short peptide displayed on screened positive phage could bind specifically to VEGFR3, and the binding could be inhibited by natural antibody VEGF-D. Conclusion The phage clone (phage-WHGSLKQNLWWY) obtained via bio-panning of peptide library has a high affinity with VEGF receptor 3. The peptide could be a potent carrier targeted to VEGFR3.

Objective To assess the function of the peptide (WHGSLKQNLWWY) through measuring the a functional affinity constant between humanized VEGF receptor 3 and peptide, then comparing it to that between VEGF D and peptide. Methods After coating the peptide by BSA binding with glutaral couple, the best concentration, best time of peptide to coat the plate and the coating coefficient were determined. With cognizance of the advantages of solid phase method in time consumption and convenience, we assessed the functional affinity constant of engineered peptide and VEGF-D with non-competitive ELISA method. We plotted the D(410) standard curve of the binding reaction of peptide and VEGFR3/Fc using four grades of concentration of VEGFR3/Fc and a series of consistency of peptide. We got the consistency of peptide at the half of maximal OD value through the standard curve and calculated the affinity constant by law of Mass Action. Results The affinity constant of peptide was between 10 7-10 8 mol/L -1, which was only smaller by 10 times than that of VEGF-D. Conclusion The peptide we got from the peptide liberary can strongly bind to the target (VEGFR3), thus providing a theoretical basis for its pharmacal use.

Objective To study the correlation between serum human cytomegalovirus (HCMV) DNA and peripheral T cell subsets as well as islet function in type 1 diabetic patients.Methods HCMV DNA levels in sera from 20 type 1 diabetic patients and 40 controls were measured by quantitative polymerase chain reaction (PCR),then the comparison and correlation analysis were made between HCMV DNA and T cell subsets,blood glucose (BG),insulin (Ins) and C peptide (C P).Results The positive rate and levels of HCMV DNA in type 1 diabetic patients were significantly higher than those of controls respectively.The percentage of CD 8 and the levels of fasting BG and BG 2 hours after meal in type 1 diabetic patients with positive HCMV DNA were by far higher than those in controls,while the percentage of CD 4,the ratio of CD 4/CD 8,the levels of fasting Ins,Ins and C P 2 hours after meal were significantly lower than those of controls.There existed the linear positive correlation between HCMV DNA and CD 8 or fasting BG,negative correlation between HCMV DNA and CD 4/CD 8,fasting Ins or fasting C P.Differences were not so significant between type 1 diabetic patients with negative HCMV DNA and controls compared with those between patients with positive HCMV DNA and controls.Conclusion HCMV infection is associated with dysfunction of islet and T cell mediated immunity in type 1 diabetic patients.

Objective:Human capillary lymphatic endothelial cells (LECs) were isolated and cultured to assist further investigation of the function of lymphatic vessels generation during cancer metastasis. Methods:Human skin was digested by type I collagenase. Cells were isolated using magnetic beads which were marked by monoclonal antibodies against the extracellular domain of VEGFR-3, and purified by cloning col-umn. The morphology and structure of cells were observed by microscopy. Immunophenotype was identified by immunofluorescence. Cell growth curve was recorded to measure the effect of VEGF-C protein. Results:LECs exhibited the typical cobblestone morphology as monolayer growth pattem under microscopy. Enlarged nucleus and rich cytoplasm with bubbles were found under electromicroscopy. LECs specific markers inclu-ding VEGFR-3, LYVE-1, Podoplaninand D2-40 all were positive. VIII factor as specific marker of blood ves-sel endothelium cells (BVECs) was negative. VEGF-C induced a marked increase of cell proliferation. Con-clusions:Human dermal LECs could be harvested successfully using collagenase digestion, immunomagnet-ic beads sorting and clonic column purification.

Objective To discuss the effect of CO2 artificial pneumoperitoneum on the invasion of the endometrial cancer in nude mouse resulted from the transplantation of the cancer cells and its mechanism.Methods Thirty nude mice were divided into 3 groups based on the time in the CO2 pneumoperitoneum circumstance.Control group: the small intestine of the nude mouse was exposed in air for 5 min,and the cancer cells were injected into right lower quadrant after suture.The gas was depleted after 40 min.40 min group: CO2 gas was poured into abdominal cavity to form a 4 mmHg artificial pneumoperitoneum for 5 min before cancer cells were injected into right lower quadrant.The gas were depleted after 40 min.80 min group: CO2 gas was poured into abdominal cavity to form a 4 mmHg artificial pneumoperitoneum for 5 min before cancer cells were injected into right lower quadrant.The gas were depleted after 80 min.The time that each group took to form a solid tumor was recorded.Four weeks later,the transplantation tumors were taken out and sliced into frozen sections and paraffin-embedded sections.The expression of ?1-intergrin and E-cadherins was detected by IMF.Results The time taken to form the solid cancer was shorter in the 40 min group and 80 min group than in the control group,with more blood vessel found(P0.05).Conclusion The CO2 pneumoperitoneum could enhance the abilities of invasion and adhesion of endometrial cancer cells,which is associated with the expression changes of ?1-intergrin and E-cadherins in the cancer cells.

Objective:To observe the clinical effect of treatment of burning mouth syndrome in women by hormone replacement therapy (HRT).Methods:21 cases of climacteric women with burning mouth syndrome (BMS) were treated by HRT with nilestriol and medroxyprogesterone acetate.Another 21 cases were treated with vitamin B,C and oryzanol as the controls.The treatment and follow-up were conducted for 3～6 months.Results:The ratio of effectiveness in HRT group and control group was 85.71% and 14.27% (P

Objective To construct a cDNA library of human epithelial ovarian carcinoma tissue for screening ovarian carcinoma specific-antigen.Methods The total RNA was separated from human epithelial ovarian carcinoma tissue.The mRNA from total RNA was isolated to synthesize the first and second strand cDNA.The ds-cDNA termini were blunted with pfu DNA polymerase.The blunted cDNAs were added EcoR Ⅰ adaptor and then digested by XhoⅠ.Small cDNA molecules(less than 400 bp) were removed through size fraction.After the cDNAs were ligated into ZAP expression vector,the ligated products were packaged in vitro and the bacteriophage particles infected the host strains XL1-Blue MRF′.Results The efficiency of the primary library was 5.5?10~(6)pfu/ml and the amplified library was 3.0?10~(11)pfu/ml with 96% clones positive.The average length of the inserted fragment was over 1 kb.Conclusion The quality of the constructed human epithelial ovarian carcinoma tissue cDNA library is excellent and helpful to screen ovarian carcinoma specific-antigen.

Objective To explore the methods of culture and identification of human placental microvascular endothelial cells(HPMVEC) in vitro,aiming to construct the polar model of human placental barrier.Methods By proteolytic enzymes,the tissular and cellular suspensions were collected from the chorionic villi of pregnant and centrifugated over a preformed discontinuous Percoll gradient.The highly purified human placental microvascular endothelial cells were obtained and cultured in HG-DMEM with fetal calf serum and identified by immunocytochemistry(SP methods) and transmission electron microscope.Results The highly purified human microvascular endothelial cells were cultured in vitro and passaged for three to five generations.Factor Ⅷ-related antigen and CD34 were both positive by immunocytochemistry.Weibel-Palade body(W-P body) was observed by transmission electron microscope.Conclusion Human placental microvascular endothelial cells can be isolated and cultured successfully in vitro by our method.

Objective To screen rhEndoglin-binding peptides from phage displayed 12-peptide library. Methods The rhEndoglin was used as target protein for biopanning of phage-displayed 12-peptide library. After three rounds of screening, 16 phage clones were randomly selected and identified by sandwich ELISA. The positive phage clones were sequenced, and the fuse peptides were deduced by the DNA sequence. Further we identified the affinity and speciality by competitive inhibition test. Results Six of 16 phage clones were identified as positive clones by competent ELISA which could bind to rhEndoglin. Five sequences were obtained, and the amino acid sequence in two of these five was AHKHVHHVPVRL. Conclusion The rhEndoglin-binding peptides can be obtained by screening phage random peptide library. It could play an important role in early diagnosis of ovarian cancer.

Objective To assess functional affinity of rhEndoglin conjugated eptide in order to identify the affinity.Methods We measured the functional affinity of rhEndoglin conjugated eptide with non-competitive ELISA method.After coating the peptide by BSA binding with glutaral couple,affirming the best concentration,best time of peptide to coat the plate and the coating coefficient,and the proper binding time of peptide to Endoglin to reach an equilibrium,we plotted the standard curve of the binding reaction of peptide and Endoglin.Results The affinity constant of peptide and anti-hEndoglin IgG is respectively(2.956?0.749)?106mol/L and(7.403?10.76) ?108mol/L.Conclusion The peptide we got from the peptide liberary can strongly bind to Endoglin,providing a theoretical basis for its clinical use.