Objective: To construct subtracted cDNA libraries of stomach tumors and normal stomach tissue using suppression subtractive hybridization(SSH).Methods: cDNA Library subtraction was performed using the protocol described in the Clontech PCR-Select cDNA Subtraction Kit. cDNA was synthesized from 2 μg of poly A+RNA from the tumor and normal tissues using AMV reverse transcriptase. The tester and driver cDNAs were digested with RsaⅠ, a four-base-cutting restriction enzyme that yields blunt ends. The tester cDNA was then subdivided into two portions, and each was ligated with different cDNA adaptor. Two hybridizations were performed. In the first, an excess of driver was added to each sample of tester. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. During the second hybridization, the two primary hybridization samples were mixed together without denaturing and thus the templates were generated from differentially expressed sequences for PCR amplification. Using suppression PCR, only differentially expressed sequences were amplified exponentially and after second PCR amplification the background was reduced and differentially expressed sequences were further enriched. The cDNAs were then directly inserted into a T/A cloning vector to generate a stomach tumor-specific subtracted cDNA library. Results: The amplified library contained 800 positive clones. Plasmid inserts were PCR amplified and showed 250－700 bp inserts. Conclusions: The successfully constructed subtracted cDNA library of gastrocarcinoma and normal tissue enables us to compare two populations of mRNA and obtain clones of genes that expressed in one population but not in the other.The characterization of these genes will allow them to be exploited for their diagnostic and therapeutic potentials.

BackgroundResearches demonstrated that the levels of soluble CD44 (sCD44)molecule in aqueous is significantly higher in primary open-angle glaucomous(POAG) eye than normal eye,but how the sCD44 would affect the expression of apoptosis protein in trabecular meshwork cells is below understanding.Objective The present study was to investigate the effect of sCD44 on the expression of regulatory proteins bcl-2 associated death factor bad in trabecular meshwork cells in the patients with POAG.MethodsHuman scleral tissue with trabecular meshwork were obtained from POAG patients during the surgery.The trabecular meshwork cells were primarily cultured by explant culture method and identified by immunochemistry.The third generation of cells were incubated with free-serum DMEM/F12 medium added differnt dosages of sCD44 (0,1,5,10,25,50 mg/L) for 48 hours.The expression of bad protein in cultured cells was detected using cell counting kit-8 (CCK-8) as the absorbance values at 490 nm(A,90 value),and the bad protein level in cultured cells was assayed by ELISA.ResultsThe cultured cells showed the positive response for laminin ( LM ),neuron specific enolase ( NSE ),fibronectin ( FN ) monoclonal antibodies.The CCK-8 assay showed that the A490 values of the trabecular meshwork cells in 0,1,5,10,25,50 μg/L of sCD44 groups were 0.2460±0.0019,0.1874±0.0015,0.1570±0.0016,0.1302±0.0019,0.1084±0.0018,0.0940±0.0020 respectively with a statistically significant difference among the 6 groups( F =14.922,P =0.000 ),and the A490 values in various dosages of sCD44 groups were significantly lower than the 0 μg/L sCD44 group (P=0.013,0.008,0.011,0.005,0.004).The ELISA assay showed that bad protein levels in 0,1,5,10,25,50 μg/L of sCD44 groups were ( 114.8461 ± 2.9560 ),( 137.8270 ± 2.4259 ),( 161.4194 ± 3.7381 ),( 170.9453 ± 3.2006 ),( 221.2252 ±4.3738 ),( 324.6167±4.4220) ng/L,showing a total difference among them ( F =16.610,P =0.000 ),and the bad protein levels in various dosages of sCD44 groups were significantly lower than the 0 μg/L sCD44 group( P =0.017,0.013,0.008,0.007,0.006).ConclusionssCD44 can contribute to the apoptosis of the trabecular meshwork cells in patients with POAG in certain dose range by regulating the apoptosis regulatory proteins bcl-2 associated death factor bad.

Background Primary open angle glaucoma (POAG) is a major blindness-causing disease,characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork lining the aqueous outflow pathway modulates the aqueous outflow facility. To study the biological characteristics of the trabecular meshwork cells has important significance. Objective This study was to culture the trabecular cells from primary open-angle glaucomatous eye (POAG) and study the biologic characteristics of passaged cells. Methods The deep scleral tissue with trabecular meshwork was obtained during the trabeculectomy from 8 eyes with POAG. The trabecular cells were primarily cultured and passaged in vitro. The generation 3 cells were identified by immunochemistry with the laminin (LM), fibronectin (FN) and neuron specific endolase (NSE)monoclonal antibodies. The ultrastructure was examined to observe the biological characteristics of the cells under the transmission electronic microscope. The experimental results were compared among POAG group, normal control group and blank control group. Results The primarily cultured POAG trabecular cells migrated from the edge of tissue mass about 10 days. The cells of generation 3 presented the logarithmic phase in the first 4 days and fused in the 7th day. FN,LM and NSE were positively expressed in the generated cells in POAG group and normal control group rather than blank control group. The MOD values of the generation 3 cells for FN in POAG group and normal control group were 0. 35 ± 0.06 and 0. 26 ± 0. 01, and those for LM were 0. 34 ± 0. 03 and 0. 25 ± 0. 02 respectively, showing statistically significant difference between these two groups ( FN: t = 14. 446, P＜0.001; LM: t = 9. 346, P＜0. 001 ). The microvilli, cytolysosome and phagocytic vesicle were obviously decreased in the trabcular cells of POAG group compared with normal control group under the transmission electron microscope. Conclusion The trabecular meshwork cells from POAG can be successfully cultured and passaged in vitro. It provides a cytology basis for further glaucoma research.

Adult ; Humans ; Laparoscopy ; methods ; Male ; Retrocaval Ureter ; surgery ; Ureterostomy ; methods ; Young Adult

Autoimmune Diseases ; diagnosis ; etiology ; therapy ; Child, Preschool ; Encephalitis ; diagnosis ; etiology ; therapy ; Female ; Humans ; Receptors, N-Methyl-D-Aspartate ; immunology

Objective To identify the potential risk factors affecting mortality rate of ALl in severe trauma population. Method It was a retrospective cohort study treating trauma as a single cause for emergency depart-ment (ED)) and emergency intensive care unit (EICU) admissions. Eighteen potential risk factors affecting the mortality of ALI were examined by univariate and multivariate logistic analyses in these severe trauma patients. Re-sults There were 343 severe trauma patients with post-traumatic ALI admitted to ED and EICU the Second Affili-ated Hospital Medical College,Zhejiang University,during the study period. The five risk factors that affected the mortality with unadjusted odd ratios (ORs) and 95% confidence intervals (CIs) were (1) APACHE Ⅱ score, (2)duration of trauma, (3) age, (4) aspiration of gastric contents, and (5) DIC. Specific risk factors also affected different patients subpepulations at different degrees. Conclusions Factors of APACHE Ⅱ score and aspiration of gastric contents that can predict the mortality of ALl may exist in the early stage of trauma. Duration of trauma and DIC that greatly affect the short- and long-term development of ALI deserve special attention. Elderly patients (aged beyond 65 years) are the independent risk factor for the secondary sepsis and deterioration of pulmonary function. Patients with these risk factors need aggressive supportive care as early as possible in order to prevent fur-ther aggravation.

Lactic acid production and antagonistic property of five strains of LAB isolated from piglet intestine were investigated. The results showed that among all strains L5 exhibited the most rapid production and highest amount of lactic acid in the culture. Consequently, the pH in L5 culture showed the fast decline, with the final value significantly lower than those of other cultures. Strain L1 showed the least production of lactic acid and highest pH among all strains. Culture supernatants of the five strains showed different degrees of antagonistic effect against pathogenic E. coli K88, K99, 987P, O141, E1, and S. aureus. When taking out the effect of the acid, the culture supernatants still showed 22%～53% inhibitory effect, suggesting that the bacteria produced other inhibitory substances apart from lactic acid. The inhibitory effect of the culture supernatant was above 92% after heat treatment and above 85% when treated with proteases.

0.05), but the level of albumin dropped significantly(P

Objective : To evaluate the effectiveness of interventions for human papillomavirus ( HPV ) vaccine hesitancy among female university students based on the precaution adoption process model (PAPM), so as to provide the evidence for improving the coverage of HPV vaccine in this population. Methods : HPV vaccine hesitant female students were selected using a cluster sampling method from Zhejiang Chinese Medical University, and randomly assigned to the PAPM group and control group. Students in the PAPM group received PAPM-based interventions for HPV vaccine hesitancy, while students in the control group were given routine interventions. The HPV-related knowledge, HPV vaccine-related knowledge and HPV vaccine hesitancy scores were collected from both groups prior to interventions ( T0 ), 0 ( T1 ), 1 ( T2 ) and 3 months post-interventions ( T3 ), and the effectiveness of interventions was evaluated using analysis of variance for repeated measures. Results : There were 147 students in the PAPM group and 141 students in the control group. In the PAPM group, 36.73% of the students majored in medical sciences, and 48.23% were freshmen; in the control group, 39.72% majored in medical sciences, and 50.35% were freshmen. The mean scores of HPV- and HPV vaccine-related knowledge were significantly greater in the PAPM group than in the control group, respectively ( T1, 5.29 vs. 4.91; T2, 4.27 vs. 4.22; T3, 4.22 vs. 4.04; P<0.05 ); however, no significant differences were detected in the HPV vaccine hesitancy scores between the two groups, respectively ( T1, 2.98 vs. 2.95; T2, 3.07 vs. 3.07; T3, 3.08 vs. 2.97; P>0.05 ). The mean scores of the confidence dimension ( T1, 3.37 vs. 3.23; T2, 3.48 vs. 3.40; T3, 3.38 vs. 3.25 ) and the dimension of influence by others ( T1, 3.44 vs. 3.33; T2, 3.42 vs. 3.37; T3, 3.46 vs. 3.27 ) were significantly greater in the PAPM group than in the control group (P<0.05), while the mean scores of the complacency dimension were significantly lower in the PAPM group than in the control group ( T1, 1.98 vs. 2.03; T2, 2.06 vs. 2.20; T3, 2.18 vs. 2.15; P<0.05 ). Conclusions PAPM-based interventions for HPV vaccine hesitancy may effectively improve the awareness of HPV and HPV vaccines, reduce complacency, and enhance the influence by others among female university students.

Objective To evaluate the dynamic contrast-enhanced MRI (DCE-MRI)in detecting experimentally induced testicular ischemia. Methods Thirty healthy male New Zealand rabbits were randomly assigned into 6 groups. There were 5 rabbits in each of the following experimental groups: ( 1 ) Normal control, (2) Sham-operated, (3) ischemia of 3 h group, (4) ischemia of 6 h group, (5) ischemia of 12 h group, (6) ischemia of 24 h group. In all experiment groups, the right testis served as the internal control while the left testis served as the experimental side. DCE-MRI for each animal lasts about 10 minutes. Signal enhanced ratios (SERs) of ROI for both sides of each group were calculated by a computer, and parameters of SERs of 30 s, 75 s, 120 s and maximal SER were used for statistical analysis.Time intensity curves (TICs) were made for two sides of each group via Excel 2003 software and classified into 4 types. Statistical analysis was performed to compare the differences of SERs between left and right testis by two independent Kolmogorov-Smirnov test. Results In group I and 2, significant enhancement was observed on both testes of 10 rabbits. The enhancement decreased gradually with the elongation of ischemia in torsion groups. Three cases of type Ⅰ and 2 cases of type Ⅱ were observed in group 1,5 cases of type Ⅰ in group two, 2 cases of type Ⅰ and 3 cases of type Ⅱ b in group three, 2 cases of type Ⅰ and 2 cases of type Ⅱ b in group four, 5 cases of type Ⅱ b in group five and 5 cases of type Ⅲ in group six were noticed in the left testes. And in TICs of right testes, all cases showed TICs of type Ⅰ except 2 cases of type Ⅱ a in group six. In four torsion groups, the values for SER75 of the left side were 0. 084%, 0. 076%, 0.164% and 0.065%, while the right side were 0.255%, 0.410%, 0.586% and 0.302% (P ＜0.05). The values for SER120 in group three, five and six were 0.221% , 0.158% and 0.059% for the left side, and 0.405%,0.522% and 0.207% for the right side(P ＜0.05). The values for MSER in group three, five and six were 0.217% ,0.164% and 0.072% for the left side, and 0.405%, 0.586% and 0.302% for the right side(P ＜0.05). Conclusion DCE-MRI technique may be useful in the diagnosis of testicular torsion, which shows potential in the clinical application.