Objectives To express human TFPI-2 gene in E.coli and get the recombinant protein;to investigate the recombinant activities of antiplasmin and antitumor.Methods (1) Encoding region of mature protein of human TFPI-2 was amplified by PCR.The 645 bp PCR product was cloned into expression vector pET28a and transformed into BL21 strain to get expression.(2)Identifying the DNA segment of TFPI-2 with enzyme digestion and colony PCR and the TFPI-2 fusion protein with western blot respectively.(3)To establish the kinetics assay for detecting the TFPI-2 and test it′s inhibiting plasmin activities.(4)To assess ovarian tumor cell migratory and invasive behaviors with the Boyden chamber in vitro.Results (1)Expression plasmid of recombinant TFPI-2 was constructed and high-level expression of TFPI-2 was produced as fusion protein. (2) The TFPI-2 fusion protein was identified by western blot. (3) Antiplasmin activity of the TFPI-2 was confirmed in the fluid and on the cell surface. (4) In invasion assay, the number of the A2780 and A2780-TFPI-2 groups transferred the Matriged matrix-coated PVPF membrane was obvious decreased compared with that of A2780 group(P0.05).Conclusion (1) Prokaryotic expression of TFPI-2 gene was got, which can provide the rich experimental material for investigating the role of TFPI-2 in relative fields; (2)Recombinant TFPI-2 has antiplasmin activity, which provides the experiment basis for investigating the role of recombinant TFPI-2 in human ovarian tumor migration and invasion in vitro; (3) The recombinant TFPI-2 inhibits the invasive ability of human ovarian tumor cells in vitro, but has no effect on the migration, which may provide a target basis for treating human ovarian tumor with TFPI-2 protein therapy.

Objective To determine the relationship and significance between the acute ischemia, hypoxia and the production of VEGF in rat myocardium and the influence of EDS on expression of VEGF protein in myocardium Methods To establish the model of AMI in rats, Wistar rats were randomly divided into control group, AMI group (1,3,7 day) and EDS group (0, 40 mg/day) Myocardial enzymes, VEGF protein, microvascular density and infarct size were detected Rat cardiac myocytes cultured primarily received EDS 100 ?g/ml in normal and hypoxia condition After 24 hours, VEGF was detected with immunohistochemical technique Results The production of VEGF was higher with ischemia and hypoxia time, the positive relationship was found between the time of AMI and the production of VEGF EDS reduced the concentration of serum myocardial enzymes (CK MB), enhanced the formation of collateral circulation,microvascular density and cardiac function; decreased infarct size In addition, EDS could enhance expression of VEGF protein in cardiac myocytes of rat in normal and hypoxia condition Conclusion Acute ischemia strongly stimulated the production of VEGF in myocardium, which played an important role for autoprotection of ischemic myocardium EDS could promote the establishment of cardiac collateral circulation and enhance microvascular density in infarct zone by upregulation of VEGF protein expression

Objective　To explore the effect of hypoxia on the apoptosis of cultured human umbilical vein endothelial cells（HUVECs） and the role of vascular endothelial growth factor（VEGF） in inhibition of apoptosis． Methods　①Culture and identification of HUVECs．②Establishment of hypoxic model（0，12，24，48 h）in HUVECs．③Incubation of HUVECs with VEGF(0 ng, 100 ng) under hypoxic condition for 24 h. ④Detection of apoptosis of HUVECs with TUNEL method． Results　The percentages of apoptosis were different under different hypoxic conditions． The longer hypoxic time was，the higher apoptosis percentage was．VEGF reduced the apoptosis of HUVECs induced by hepoxia． Conclusion　Over-apoptosis EVCs in one of the important factors for the impairment of endothelial function. HEGF inhibits the apoptosis of HVCs and having a pretive function on them.

AIM To explore the effect of hypoxia on apoptosis in human umbilical vein endothelial cells(HUVECs) and the role of ecdysterone(EDS)in inhibition of apoptosis. METHODS ① Culture and identification of HUVECs;② Hypoxic model(0,12,24,48 h) in HUVECs was established. While HUVECs was incubated 24 h with EDS(100 mg?L -1 ) under hypoxic condition. Apoptosis in HUVECs was detected by TUNEL;③ HUVECs received EDS 100 mg?L -1 in normal and hypoxic condition. After 24 h, vascular endothelial growth factor (VEGF) was detected with immunohistochemical technique. RESULTS The apoptosis percentages are different under different hypoxic condition. The longer hypotic the time was, the higher the apoptosis percentage was. EDS reduced apoptosis of HUVECs. EDS could enhance expression of VEGF protein in cardiac myocytes of rat in normal and hypoxic condition. CONCLUSION The role of hypoxia in HUVECs apoptosis is more significant along with prolonging of hypoxic time. VEGF play an important role in protective effect of EDS on endothelial cell apoptosis.

Objective To investigate factors affecting the metastasis of lymph nodes around the root of inferior mesenteric artery(IMA) in rectal cancer,and the significance of root lymph nodes dissection of IMA in radical surgery for rectal cancer.Methods Clinicopathological data of 105 rectal cancer patients undergoing root lymph node dissection of IMA during radical resection in Peking University First Hospital from January 2005 to December 2008 were analyzed retrospectively.Rectal cancer patients without root lymph node dissection of IMA during the same period served as control.Results were compared between these two groups for survival and local recurrence rates.Results The rate of lymph node metastasis around the origin of IMA was 9.5％ (10/105).The five-year survival rate in patients with IMA root nodal dissection was 71.3％,and that without was 70.6％ (P =0.995),while the local recurrence was respectively 1.9％ and 7.4％ (P ＜ 0.05).In multivariate analyses,IMA root nodal metastasis occurred more frequently in patients with pT3 and pT4 tumor(Wald =5.764,P ＜ 0.05) and poorly differentiated tumor(Wald =7.818,P ＜ 0.05).Conclusions Root lymph nodes dissection of IMA could not increase five-year survival rate,but it could reduce local recurrence rate in patients with rectal cancer.In radical surgery of rectal cancer,lymphadenectomy of IMA root should be performed in patients with T3 and T4 tumor with poorly differentiated tumor,so as to reduce local recurrence rate.

Objective To evaluate total pelvic exenteration (TPE) in the treatment of locally recurrent rectal cancer (LRRC). Methods Clinical data of 35 patients with LRRC who underwent TPE between 1989 and 2003 were analyzed retrospectively. Results Thirty patients underwent TPE, the remaining 2 did sphincter-preserving TPE, 2 with lower sacrectomy and 1 with hemipelvectomy, among them 80% cases received radical resection. Operative mortality rate was 3%, and morbidity rate was 51%. The overall post TPE tumor local recurrence rate was 48%. The 5-year survival rate was 16% in all cases and 19% in radical resection group. The 5-year survival rate in patients without lymph node metastasis was 24%, and 0 in patients with metastasis. Conclusion Effective TPE treatment lies in strict patient selection and radical resection.

OBJECTIVETo develop a method of quantitative analysis of multi-components by single marker (QAMS) for simultaneously determining paeoniflorin and albiflorin in Paeoniae Radix Alba.

METHODUsing paeoniflorin as the internal reference substance, the relative correction factor (RCF) of albiflorin was determined by HPLC and UPLC with good reproducibility. The contents of paeoniflorin in 16 samples of Paeoniae Radix Alba were authentically determined by the external standard method, and the content of albiflorin was calculated according to the RCF. The contents of these two components in the samples were determined with the external standard method.

RESULTNo siginificant differences between the quantitative results of QAMS method and external standard method were observe.

CONCLUSIONIt is a convenient and accurate method to determine multi-components when some authentic standard substances were unavailable. It can be used to control the quality of Paeoniae Radix Alba

Benzoates ; analysis ; Bridged-Ring Compounds ; analysis ; Chromatography, High Pressure Liquid ; methods ; Glucosides ; analysis ; Monoterpenes ; Paeonia ; chemistry

Objective To evaluate whether the protein SP70 could be used as a serum biomarker for the diagnosis of non-small cell lung cancer (NSCLC).Methods Polyclonal antibody was prepared by immunizing New Zealand rabbit with SPC-A1 cells.Sandwich ELISA was carried out by using newly-prepared polyclonal antibody(PcMb) coating assay plates,monoclonal antibody (McAb) NJ001 and HRP goat antimouse antibody as primary antibody and labeling antibody respectively.After optimizing the experiment conditions,serum from 175 lung cancer patients [ 80 NSCLC adenocarcinoma,70 NSCLC squamous carcinoma and 25 small cell lung cancer ( SCLC) ],25 benign lung disease ( BLD) patients and 300 healthy controls (HC) were examined.CEA,NSE,CYFRA21-1 were measured by ECLIA for comparison.Results Positive rates of NSCLC adenocarcinoma,NSCLC squamous carcinoma,SCLC and BLD were 68.8％,51.4％,16.0％ and 12.0％ respectively,obviously higher than that of HC (7.3％).NSCLC (adenocarcinoma,Squamous carcinoma) had significantly higher positive rate than SCLC (60.7％ υs 16.0％,x2 =17.23,P＜0.05)and BLD(60.7％ υs 12.0％,x2 =20.41,P ＜0.05).Among 68 NSCLC patients who had definite staging,positive rates at early stage ( Ⅰ/Ⅱ,n=30) reached up to 76.7％.Meanwhile,positive rates of CEA,NSE and CYFRA21-1 (32.7％,18.0％ and 37.3％) were significantly lower than the targeting antigen to McAb NJ001 in NSCLC(60.7％ υs 32.7％,x2 =23.63,P ＜0.05;60.7％υs18.0％,x2 =57.22,P＜0.05;60.7％ υs37.3％,x2=16.34,P＜0.05).Conclusions It showed high positive rates of SP70 in the serum of NSCLC patients,which suggested thai SP70 might be a potential valuable biomarker for the diagnosis of NSCLC.

OBJECTIVETo establish a HPLC method of characteristic chromatographic profile for the quality control of Paeoniae Radix.

METHODThe 67 batches of samples were analyzed on a Polaris C18-A column with a gradient elution of acetonitrile and phosphate solution (pH 3.0) at a flow rate of 1.0 mL x min(-1) and detected at 230 nm.

RESULTNine main marker peaks were identified and semi-quantificated. By the similarity evaluation software for chromatographic fingerprint of traditional Chinese medicine (Version 2004A) and hierarchical clustering analysis, 67 batches of Paeoniae Radix were classified.

CONCLUSIONThe method can be applied for quality assessment of Paeoniae Radix.

Chromatography, High Pressure Liquid ; methods ; Paeonia ; chemistry ; Quality Control

Objective To investigate the diagnostic value of detection of protein SP70 in differentiating benign and malignant pleural effusion.Methods A case-control study was conducted from July 2011 to February 2012.108 cases of pleural effusion from patients with clinically proven lung cancers and 122 cases of benign pleural effusion were collected.SP70 was detected by Sandwich ELISA,while CEA,CYFRA21-1,NSE were measured by electrochemiluminescence immunoassay for comparison.Meanwhile,protein SP70 on exfoliated cells in pleural effusion was detected by direct immunofluorescence,and was compared with the results of HE staining.The differences between the groups were evaluated by the chisquare test Fisher' s exact test.Results Positive rates of SP70,CEA,CYFRA21-1,NSE were 72.2％,58.3％,52.8％ and 30.6％ in malignant pleural effusion,obviously higher than benign pleural effusio (9.8％,13.1％,23.0％ and 19.7％).The specificity of SP70,CEA,CYFRA21-1,NSE were 90.2％,86.9％,77.0％ and 80.3％,NSCLC had significantly higher positive rate than SCLC(74.3％ ＞0.0％,P =0.02 ＜ 0.05),detection of protein SP70 in malignant pleural effusion had significantly higher coincidence rate than HE staining(72.2％ vs 47.2％,x2 =14.03,P ＜ 0.05).Conclusion Determination of the protein SP70 in pleural effusion and in exfoliated cells,can improve the sensitivity and specificity of the diagnosis of malignant pleural effusion.