To study the variation and significance of plasma coagulation factor VII (FVII) in different kinds of ischemia heart disease (IHD) and examine its relation with plasma lipid and gene polymorphism. FVIIa was determined with one stage clotting assay by using a recombinant soluble tissue factor (rsTF). FVIIc was measured with one stage clotting assay. FVIIag was quantified with an enzyme-linked immunosorbent assay (ELISA). Polymorphism was analyzed with PCR-urea-polyacrylamide gel electrophoresis. Our results showed that FVIIa in stable angina (SA), unstable angina (UA), obsolete and acute myocardial infraction (OMI, AMI) patients was higher than those of normal group with the differences being significant within any two groups. FVIIag in UA, OMI and AMI was higher than those in SA and normal groups. There were positive correlations between FVIIa and serum triglycerides, FVIIa and FVIIc, FVIIc and FVIIag. FVII-323 0/10 bp polymorphism analysis was performed in 60 patients and 0/10 bp polymorphism was found in 5 cases. FVIIc and FVIIag were much lower in cases of 0/10 bp groups than those in cases of 0/0 bp groups. It is concluded that there was activation of extrinsic coagulation pathway in every kind of IHD to different extent. FVIIa was the risk factor in the development of IHD, and more sensitive in reflecting the severity of cardiovacutar disease than FVIIc or FVIIag. FVIIa was higher in OMI, which may be one of the risk factors of re-infraction. Serum triglyceride may indirectly lead to the development of IHD by increasing the level of FVIIa. FVII-323 0/10 bp polymorphism was present in Chinese patients with IHD and it was correlated with the level of FVIIc, FVIIag in plasma. 10 bp allelomorphic gene was a protective factor against thrombogenesis.

OBJECTIVETo investigate the effects of 3,4-dichloroaniline (3,4-DCA) on activities of testicle enzymes as biological markers in rats.

METHODSFifty male rats were randomly divided into 5 groups (n=10). One group was left untreated and used as a solvent control (administered orally by corn oil), while the other 4 groups were treated with 3, 4-DCA. Corn oil was used as a solvent, and 3,4-DCA was diluted into tested concentrations (39, 81, 170, and 357 mg/kg). All the groups orally administered 3,4-DCA or corn oil, once a day for 4 weeks. The testicle tissue was homogenized in a 0.1 mol/L potassium phosphate buffer (0.1 mol/L, pH 7.2). The crude homogenate was centrifuged at 6000 rpm for 5 min at 4 degrees C. The supernatant obtained was used as an enzyme extract for determination of the enzyme activities.

RESULTSCompared with the control, the activities of ALP, ACP, and SDH were increased significantly at a lower level of 3,4-DCA, and decreased at a higher level of 3, 4-DCA, whreas the activities of LDH, LDH-X, and G6PDH were inhibited significantly with the increased 3,4-DCA concentration. Organ coefficient "organ weight/total body weight x 100" of testis, liver, and spleen increased significantly with the increased 3,4-DCA concentration. These results suggest that 3,4-DCA toxicity to the male reproductive system was associated with the activities of testicular enzymes which are the sensitive biochemical endpoints reflecting 3,4-DCA toxicity to the male reproductive system.

CONCLUSION3,4-DCA has toxicity to the reproductive system in male rats.

Aniline Compounds ; toxicity ; Animals ; Biomarkers ; analysis ; Body Weight ; drug effects ; Male ; Organ Size ; drug effects ; Rats ; Rats, Wistar ; Testis ; drug effects ; enzymology ; Toxicity Tests

OBJECTIVETo develop a recombinant single domain antibody against hTERT, human telomerase catalytic subunit.

METHODSA previously prepared His-tagged hTERT fusion protein was used as the antigen, and the variable regions in heavy chain (VH) of immunized mice were RT-PCR amplified and cloned into the pCANTAB 5E, a phagemid vector. By transfection, the display library of mouse VH was developed. The candidate clones were selected by affinity panning, and soluble VH were obtained after expression in E. coli, HB2151. The resultant single VH antibodies were characterized on their binding potentials by western blotting.

RESULTSAn about 350 bp VH fragment was amplified from spleen cells of mice immunized by His-tagged hTERT and expressed by phage displayed as VH library. The size of the library was 8 x 10(4). After three rounds of affinity panning, 4 independent clones were chosen and consequently expressed as soluble single domain antibodies (Mr = 16 000). In Western blot analysis, the single domain antibody from 2 of 4 clones proved to react with the His-tagged hTERT fusion protein (Mr = 167 000) without dependence of His-tags and also detect the native hTERT (Mr = 127 000) extracted from the human HeLa cancer cell line. DNA sequencing showed both of the single domain antibodies were encoded by the heavy chain variable region of the mouse.

CONCLUSIONSThe single domain antibodies developed were hTERT recognizable and hTERT specific, thus providing a basis for application of recombinant single domain antibody in inhibition of telomerase activity and anticancer therapy.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Complementarity Determining Regions ; genetics ; DNA-Binding Proteins ; HeLa Cells ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Mice ; Molecular Sequence Data ; Sequence Analysis, DNA ; Telomerase ; immunology

OBJECTIVETo develop monoclonal antibodies against the catalytic subunit of human telomerase hTERT for its expression detection of human tumors.

METHODSA dominant epitope in hTERT (peptide hTERT(9))was automatically synthesized based on Fmoc method, and was used to immunize BALB/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization of which were performed by Western blotting and immunohistochemical staining.

RESULTSAntigenic peptide hTERT(9) was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. Three hybridoma cell lines secreting anti-hTERT(9) antibodies designated as H4, G8 and A11 were established after primary screening and consequent three rounds of limited dilution. Both of H4 and G8 were IgM, while A11 was IgG1 in isotyping. The competitive assay showed that the antibodies were hTERT(9) specific, and the affinity of G8 was stronger than that of H4 and A11 assayed by affinity ranking. However, in Western blotting, both of H4 and G8 stained an about 123 000 protein band with HeLa and 293 cell extracts but not with normal 2BS cells. Besides, positive staining presented in the nucleus of HeLa, while 2BS was non-reactive immunohistochemically. The sections from paraffin-embedded blocks of 127 cases of human cancer, 40 of precancerous and 19 of benign tumors were in situ stained by G8 antibody, the results showed that the human cancer tissues were 80.31% (102/127) positive in specific nuclear reaction, on the contrary, only a minority of precancerous lesions present weak positive (17.5%, 7/40), and negative in benign tumors (0/19).

CONCLUSIONSThe monoclonal antibodies developed against synthetic peptide were hTERT-specific and could recognize both the native and the denatured form. Thus their use in immunoblotting or immunohistochemistry for detecting the telomerase hTERT expression of cancer cell and tissues was promising.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Binding, Competitive ; Blotting, Western ; methods ; Catalytic Domain ; DNA-Binding Proteins ; Female ; HeLa Cells ; Humans ; Immunohistochemistry ; methods ; Mice ; Mice, Inbred BALB C ; Neoplasms ; enzymology ; pathology ; Telomerase ; chemical synthesis ; immunology

To investigate the in vitro and in vivo proangiogenic effects of brain-derived ncurotrophic factor (BDNF),human umbilical vein endothelial cells (HUVECs) were isolated and cultured in primary culture.The effect of BDNF on the proliferation of HUVECs was examined by MTT assay.The effects of BDNF on HUVEC migration and tube formation were studied by modified Boyden chamber assay and tube formation assay,respectively.Matrigel plug assay and chorioaUantoic membrane assay were used to evaluate the effects of BDNF on angiogencsis in vivo.Our results showed that BDNF substantially stimulated the migration and tube formation of HUVECs in vitro,although it did not induce HUVEC proliferation.BDNF also induced angiogenesis both in matrigcl plug of mouse model and in chick chorioallantoic membrane.In conclusion,BDNF can promote angiogenesis both in vitro and in vivo,and may be a proangiogenic factor.

OBJECTIVE: To evaluate the detection of humen-lung-specific X protein (LUNX) gene in micrometastases of patients with non-small cell lung cancer. METHODS: The expression of LUNX gene in tumor tissue, lung and lymph nodes was detected by reverse transcriptase-polymerase chain reaction(RT-PCR) both in 43 non-small-cell lung cancer patients (the experimental group) and 15 lung benign patients (the control group). LUNX mRNA expression in clinic pathology,stage of cancer cell differentiation, clinic stage, age, sex, smoking history, and 4 lung cancer blood markers (CEA,CA125,NSE, and CYFRA211) were evaluated. RESULTS: The expression of LUNX gene was positive in the 2 groups. LUNX gene expression was positive in 33 of the 87 lymph nodes of the 43 patients in the experimental group (37.93%), and in 2 of the 26 lymph nodes in the control group (7.69%). The LUNX mRNA positive in the lymph nodes was closely related to the pathological type, cancer cell differentiation and clinic stage(r=0.660,0.500,0.460; P=0.011,0.017,0.022, all P<0.05), while not closely related to age, sex, smoking history and 4 lung cancer blood markers (CEA,CA125, NSE, and CYFRA211) (r=0.111, 0.135,0.083,0.354; P=0.739,0.714,0.773,0.125,all P>0.05). CONCLUSION The LUNX mRNA expression detected by RT-PCR is more sensitive than by traditional ways. The expression of LUNX gene mRNA in the lymph nodes is a valuable index for the detection of micrometastases in patients with non-small cell lung cancer.
Adult ; Aged ; Biomarkers, Tumor ; biosynthesis ; genetics ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Female ; Glycoproteins ; genetics ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Lymph Nodes ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; Phosphoproteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Objective:To investigate the incidence, risk factors and prognosis of acute renal injury(AKI)in elderly patients with respiratory distress syndrome(ARDS).Methods:The elderly patients with ARDS treated in the Department of Respiratory Medicine, Emergency Department and Geriatrics of the Second People's Hospital of Lianyungang from July 2016 to July 2019 were divided into AKI group and non-AKl group according to KDIGO diagnostic criteria.The clinical data and the differences were compared and analyzed between the two groups.Binary Logistic regression was used to analyze Risk factors for AKI.Kaplan-Meier cure was used to analyze the influence of different stages of AKI on the prognosis of ARDS patients.The Cox proportional hazards model was used to analysis risk factors for AKI and ARDS on elderly patients＇prognosis.Results:A total of 432 elderly patients with ARDS were enrolled in the study, in which the mean age was 74.7 ± 8.8 years, and AKI occurred in 129 cases(29.9%). Compared with non-AKI group, AKI group showed older age, and higher proportion of the incidences of hypertension, diabetes, atrial fibrillation, consciousness disturbance, mechanical ventilation and a low mean arterial pressure(all P<0.05). The incidence of AKI was increased significantly in patients with moderate to severe ARDS( P< 0.001). The levels of basal creatinine, AST and NT-proBNP were significantly higher in AKI Group than in non-AKI Group( P= 0.001, P< 0.001, P< 0.001). AKI Group patients had the more elevated inflammatory marker level of neutrophil / lymphocyte ratio(NLR)( P= 0.003)and D-dimer( P< 0.001), and the level of high-sensitivity c-reactive protein(hsCRP)( P=0.040). AKI group showed the increased incidence of urine protein( P< 0.001), low ejection fraction( P= 0.040), and positive rate of pleural effusion( P= 0.003). Logistic Regression analysis showed the following independent risk factors for the development of ARDS-associated AKI, included hypertension( OR: 1.789, 95%, CI: 1.105-2.894, P=0.018), diabetes( OR: 1.976, 95% CI: 1.076-3.628, P=0.028), consciousness disturbance( OR: 2.531, 95% CI: 1.203-5.251, P=0.014), mechanical ventilation( OR: 3.421, 95% CI: 1.521-7.694, P=0.003), AST>40 U/L( OR: 2.495, 95% CI: 1.431-4.348, P=0.001), increased basal creatinine levels( OR: 1.014, 95% CI: 1.002-1.027, P=0.024), and NLR( OR: 1.015, 95% CI: 1.001-1.029, P=0.042). Kaplan-Meier survival curve showed that there was a significant difference in the prognosis between patients with different AKI stages( χ2=19.790, P<0.001), and there was no significant difference in the prognosis between stage 1-AKI and non-AKI( χ2=2.188, P=0.139). The risk of poor prognosis was higher in AKI(stage 2-3)group( χ2=18.268, P<0.001; χ2=6.347, P=0.012)than in patients without AKI or stage 1 AKI.Multivariate Cox Proportional Hazard Model Analysis elucidated that AKI( HR: 1.858, 95% CI: 1.207-2.861, P= 0.005)and moderate-severe ARDS( HR: 1.815, 95% CI: 1.167-2.822, P=0.008)were independent risk factors for poor prognosis of ARDS in the elderly. Conclusions:Hypertension, diabetes, disturbance of consciousness, mechanical ventilation, AST>40 U/L, elevated levels of basal creatinine and NLR are independent risk factors for ARDS-associated AKI in elderly patients with ARDS.Patients with moderate-severe ARDS and AKI(2-3 phases)have the increased risk of poor prognosis.

To study the variation and significance of plasma coagulation factor Ⅶ (FⅦ) in differ ent kinds of ischemia heart disease (IHD) and examine its relation with plasma lipid and gene polymorphism. FⅦa was determined with one stage clotting assay by using a recombinant soluble tissue factor (rsTF). FⅦc was measured with one stage clotting assay. FⅦag was quantified with an enzyme-linked immunosorbent assay (ELISA). Polymorphism was analyzed with PCR-urea-polyacrylamide gel electrophoresis. Our results showed that FⅦa in stable angina (SA),unstable angina (UA), obsolete and acute myocardial infraction (OMI, AMI) patients was higher than those of normal group with the differences being significant within any two groups. FⅦag in UA, OMI and AMI was higher than those in SA and normal groups. There were positive correlations between FⅦa and serum triglycerides, FⅦa and FⅦc, FⅦc and FⅦag. FⅦ-323 0/10 bp polymorphism analysis was performed in 60 patients and 0/10 bp polymorphism was found in 5 cases. FⅦc and FⅦag were much lower in cases of 0/10 bp groups than those in cases of 0/0 bp groups. It is concluded that there was activation of extrinsic coagulation pathway in every kind of IHD to different extent. FⅦa was the risk factor in the development of IHD, and more sensitive in reflecting the severity of cardiovacutar disease than FⅦc or FⅦag. FⅦa was higher in OMI, which may be one of the risk factors of re-infraction. Serum triglyceride may indirectly lead to the development of IHD by increasing the level of FⅦa. FⅦ-323 0/10 bp polymorphism was present in Chinese patients with IHD and it was correlated with the level of FⅦc, FⅦag in plasma. 10 bp allelomorphic gene was a protective factor against thrombogenesis.

Objective To investigate and analyze the impact of gender difference on outcome and prognosis of ST-segment elevation myocardial infarction (STEMI) in patients treated with primary percutaneous coronary intervention (PCI).Methods This was a prospective and multicentered observation study.All the patients with acute STEMI admitted to the hospitals from June 1st 2009 to June 1st 2010 were continuously recruited.In this study,a unified questionnaire was applied and the 382 patients satisfied the criteria.A unified follow-up questionnaire was used on patients who were discharged from the hospital.Results On average,the female patients were 8 years older than the males.The median “symptom-to-balloon time” was 312.5 minutes in females and 270.0 minutes in males,and it was significantly different (P=0.007).During hospitalization,a higher proportion of female patients developed heart failure,angina and bleeding.No gender differences were found on the in-hospital mortality rates and medical therapy recommended by the guideline.The female patients were more prone to multi-vessel disease than males (P=0.002).Success rates of primary PCI did not show any gender differences.One-month mortality and other cardiovascular events also did not show gender difference when the patients were followed for one month after being discharged.The rates of heart failure and re-hospitalization due to cardiac incidents among female patients were obviously higher than the males,three months after being discharged (P=0.007,respectively).However,the three-month and long-term cardiac mortality did not show differences related to gender.Female patients were associated with higher all-cause mortality than that in males,but there was no statistically significant difference (female 4.2％ vs.male 1.6％;P=0.056).Data from multi-factor regression analysis showed that being female was not an independent predictor related to in-hospital mortality or during the follow-up period.Conclusion Being female was not an independent predictor of in-hospital mortality or during follow-up period among patients who were treated with primary PCI.Worse long-term outcome seen in female patients was likely to be explained by older age or longer pre-hospital delayed time.

Objective To investigate the diagnostic value of fiberoptic ductoscopy (FDS) in pathological milky white nipple discharge.Methods The data of 1688 patients with pathological milky white nipple discharge who underwent FDS examination in Chengdu Third People's Hospital from Oct.2011 to Oct.2016 were analyzed retrospectively.Results Among the 1688 cases,the proportion of patients with milky white nipple discharge was 30％,higher than that of the bloody discharge (15％) and yellow liquid (24.5％).The detection rate of lesions in patients with milk nipple discharge was 9.3％,among whom 6.1％ was breast cancer.Conclusions FDS should be routinely performed in patients with pathological milky white nipple discharge,as an examination tool to exclude the intra ductal lesion.The disease should be paid more attention by physicians.