With the analyses of correlative techniques, such as ODBC and ASP, and the application characteristics of HIS, this paper develops an object-oriented design project and its application system.

BACKGROUND: It is difficult to in vitro isolate and culture the ovarian surface epithelium with high purity, strong vitality and stable biological characteristics. Tissue adherence and enzymatic digestion are commonly used for primary culture, but there are certain problems in cel col ection, cel viability and cel purity. OBJECTIVE: To establish a method for primary isolating, culturing and identifying human ovarian epithelium. METHODS: The ovarian surface epithelium was obtained with cel brush innovatively, and then the cells were isolated and purified with erythrocyte spal ation and differential adherence. The epidermal growth factor was added into the serum-free Dulbecco’s modified Eagle’s medium-F12 medium for cel culture. The cel morphology was observed under inverted microscope, and hematoxylin-eosin staining and immunocytochemical staining were used to identify the cells, then the growth curve was draw. RESULTS AND CONCLUSION: The ovarian surface epithelium became adherent after cultured for 24 hours, and reached fusion basical y after cultured for 6-12 days. The cells were polygonal or flat with strong transparency and refraction. The morphological characteristics of the cells were in line with those of the normal epithelial cells, and almost al the isolated cells could express the epithelial cells surface marker CK19. The cells could be passaged for 6-8 generations with wel growth and the cel growth curve was in “S” shape. The purity of the cells was more than 95%. The results suggest that cel brush method is simple to operate and can obtain a large amount of ovarian surface epithelium rapidly. The purity of the isolated cells can reach to 95% after treated with erythrocyte spal ation and differential adherence method and the cells are in stable growth.

BACKGROUND: Combined use of multiple interventions for different targets play superimposed or synergistic effects,which has become the current idea for spinal cord injury treatment.OBJECTIVE: To investigate the synergistic effects of low doses of 17-β estradiol combined with bone marrow mesenchymal stem cells (BMSCs) transplantation on the recovery of motor function and inflammatory reactions after spinal cord injury in rats.METHODS: The 10 of 70 male Sprague-Dawley rats served as sham group in which the spinal cord was only exposed but with no treatment, and the rest 60 rats were used to make animal models of spinal cord injury using modified Allen's method and then randomized into four groups (n=15 per group): model, estrogen, stem cell and combined treatment groups. Rats in the stem cell and combined treatment groups were given BMSCs transplantation at injured side; rats in the estrogen and combined treatment groups were given intramuscular injection of 17-β estradiol at 1 and 24 hours after modeling. At 1, 3, 5 and 7 days after modeling, rat functional recovery was evaluated by the Basso, Beatlie, Bresnahan score. The expressions of interleukin-1β and tumor necrosis factor-α in the injured spinal cord were detected by ELISA at 6, 12, 24, and 72 hours after modeling. Apoptosis in nerve cells was observed using TUNEL staining. RESULTS AND CONCLUSION: The Basso, Beatlie, Bresnahan scores were declined significantly after modeling,increased at 5 and 7 days after stem cell transplantation, estrogen treatment or their combined treatment (P < 0.05),especially in the combined treatment group (P < 0.05). The levels of interleukin-1β and tumor necrosis factor-α were elevated gradually after spinal cord injury (P < 0.05), but the levels decreased significantly at 12 and 24 hours in stem cell,estrogen and combined treatment groups (P < 0.05), and this decrease trend was more significant in the combined treatment group compared with the stem cell and estrogen groups (P < 0.05). At 72 hours after modeling, the rate of TUNEL positive cells was highest in the model group (P < 0.05) and lowest in the combined treatment group (P < 0.05).To conclude, the combined use of low doses of 17-β estradiol and BMSCs transplantation can facilitate the recovery of motor function after spinal cord injury by effectively inhibiting apoptosis in nerve cells.

BACKGROUND: Human transforming growth factor-β1 gene can be used for gene therapy of the degeneration of intervertebral discs, but the key to the experiment is to construct its effective vector.OBJECTIVE: To determine whether or not adult degenerated intervertebrai disc cells cultured in vitro after transfected by eukaryotic expression vector can express the product of human transforming factor-βl, and to provide the experimental basis of gene therapy for intervertebral disc degeneration.DESIGN: Single sample experiment. SETTING: Traumatic Orthopedic Institute of Shandong Province and the Orthopedic Department of Weihai Municipal Hospital.MATERIALS: The experiment was conducted at the laboratory of Traumatic Orthopedic Institute of Shandong Province between October 1999and January 2001. Intervertebral disc samples were from the operated patients with protrusion of irtervertebral disc after the patients were informed.Sample 1 was intervertebral disc at L4/5 from a 30-year-old woman; sample 2 was intervertebral disc at L5/S1from a 30-year-old woman.METHODS: ① Culture of adult degenerated intervertebral disc cells:Samples ex vivo were taken back to the laboratory within 30 minutes; fibrous ring cells and myelin nucleus cells cultured primarily were collected.② Transfection: Cells were put in the 24-well culture plate with 5.5×105cells in each well. Constructed PCI-hTGF-β1 eukaryotic expression vector was used to perform transfection, then transfected PCI group and nontransfected group were set. ③ The expression product of cells transfected for 48 hours was determined with immunohistochemical staining method.MAIN OUTCOME MEASURES: Comparison of absorbance of the positive cell product of eukaryotic expression vector PCI-hTGF-β1 in the primary fibrous ring cells and myelin nucleus cells in each group.RESULTS: ① Sample 1: The absorbance of positive cell product of eukary otic expression vector PCI-hTGF-β1 in the primary fibrous ring cells and myelin nucleus cells was 3.49 and 3.69 times that in PCI group, and 3.55times that in non-transfected group. ② Sample 2: The absorbance of positive cell product of eukaryotic expression vector PCI-hTGF-31 in the primary fibrous ring cells and myelin nucleus cells was 3.56 and 3.46 times that in PCI group, and 3.43 times and 3.33 times that in non-transfected group.CONCLUSION: PCI-hTGF-31, as the effective eukaryotic expression vector in the transfection of transforming growth factor-31 gene to culture degenerated intervertebral disc cells in vitro, can transfect adult degenerated intervertebral disc cells cultured in vitro and obtain the high expression of human transforming growth factorβ1 gene.

Objective To investigate treatment effects of lentivirus mediated RhoA short hairpin RNA(shRNA) on xenograft tumor of ovarian cancer in nude mice in vivo and the underlying mechanism.Methods Human ovarian cancer cell line HO8910 were inoculated to establish subcutaneous xenograft model of human ovarian cancer.Tumor-bearing nude mice were assigned randomizely to three groups:LentiRhoA-sh group,Lenti-negative control (NC) group and phosphate buffered saline (PBS) group.lentivirus mediated RhoA shRNA,negative control lentivirus and PBS were respectively injected in the three groups.Effects of treatment were observed by tumor growth curve,tumor volume,tumor weight,and tumor inhibition rate.Xenograft tissues and liver,spleen,lung,and renal tissues were examined by hematoxylin and eosin (HE) staining or were detected by streptavidin-perosidase (SP)immunochemical method.The changes of RhoA gene expression in xenograft tissues after lentivirus mediated RhoA shRNA treated were also detected by real-time qPCR,immunochemistry and Western blot assay.Cell apoptosis in xenograft tissues were examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL) method and apoptotic index (AI) were counted.Results Compared with Lenti-NC group and PBS group,the growth speed of xenograft in Lenti-RhoA-sh group delayed significantly after injection 9 days(P ＜ 0.01).Tumor volume (338 ± 114) mm3 decreased significantly in the Lenti-RhoA-sh group when compared with those in Lenti-NC group(1190 ± 332)mm3 and PBS group (1101 ± 396) mm3 (P ＜ 0.01).Tumor weight (0.23±0.11)g decreased significantly in the Lenti-RhoA-sh group when compared with Lenti-NC group (0.79 ± 0.19)g and PBS group (0.74 ± 0.17)g (P ＜ 0.01).Real-time qPCR result shown that the expression of RhoA mRNA (0.30 ± 0.05) decreased significantly in the Lenti-RhoA-sh group compared with Lenti-NC group (0.95 ±0.06) and PBS group(1.00 ±0.11 ; P ＜0.01).Western blot result showed that the expression level of RhoA protein decreased significantly in the Lenti-RhoA-sh group (0.14 ± 0.06) compared with those in Lenti-NC group(0.78 ± 0.14) and PBS group (0.75 ± 0.13;P ＜ 0.01).TUNEL staining displayed that AI significantly increased in the Lenti-RhoA-sh group (20.9 ± 3.4) ％ compared with those in Lenti-NC group(5.2 ±±2.0)％ and PBS group(6.0 ±2.1)％ (P ＜0.01).Conclusion Lentivirus mediated RhoA shRNA may be effectively down-regulate of the expression of RhoA,inhibit the growth of subcutaneous xenograft tumor of ovarian cancer in nude mice by increasing the cell apoptosis.

Objective: To evaluate chemiluminescence immunoassay (CLIA) for detecting blood levels of aldosterone and rennin with its diagnostic value of primary aldosteronism (PA) with comparison to radio immunoassay (RIA). Methods: According to American protocols of CLSI, we conducted a veriifcation study between RIA and CLIA for their precision, accuracy, linearity and reference ranges; meanwhile, taking clinical diagnosis as golden standard, examined renin activity or concentration and aldosterone concentration in 20 healthy volunteers and 40 hypertension patients by both RIA and CLIA, compared the ratios of ARR (aldosterone concentration/renin activity) or ADRR (aldosterone concentration/renin concentration) for the speciifcity and sensitivity of PA diagnosis. Results: Within-lot and between-lot accuracies of CLIA for detecting aldosterone levels were below 5% and 10%, the recoveries were 102% and 95% respectively. There was a good linear correlation in the range of aldosterone at (3-74) ng/dl and renin at (0.99-330) μIU/ml. In healthy volunteers, renin level was higher in 2 subjects, while aldosterone level and ADRR ratio were within normal references in all subjects by the manufacturer. In hypertension patients, the sensitivity and speciifcity for aldosterone and rennin detections by CLIA were at 85.7% and 97.0%, by RIA were at 85.7% and 94.0%. Conclusion: CLIA has the superiority of simple performance, repeatable and without radioactive contamination; it is recommended for replacing RIA as necessity.

A strain of P. Aeruginosa,which was seperated from clinical environment,shows a special characteristic. It keeps normal short rod shape when cultured at 37℃, however,it forms filament without pyocyanin producing when cultured at 25℃ overnight. The filaments will divide and form short rods, simultaneously, produce pyocyanin when culture time is prolonged to over 72h or culture temperature is raised to 37℃. The preliminary study indicates that this phenomenin has nothing to do with nutritive conditions and could the inbluenced by inoculating density and irradiating with ultraviolet rays The absence of pyocyanin was not the cause of filamentous formation by the test results.

A white-rot fungi Rigidoporus sp.W-1 which could produce laccase was isolated. The fermentation conditions in shaking flasks were investigated. The optimal carbon source was wheat bran and (NH 4) 2SO 4 was the optimal nitrogen source. The components of the medium were optimized by orthogonal experiment. When W-1 was cultured under the optimum conditions, the activity of laccase could get to 7.1U/mL in 7 days.A great amount of crude laccase could be obtained by adding fresh medium to the 7 days old mycelium.

Objective To investigate the value of ultrasound elastosonography in the diagnosis of liver fibrosis. Methods One hundred and twenty patients with chronic hepatitis B were examined by ultrasound elastosonography and given elasticity scores,the correlation coefficient between the elasticity scores and the histologic fibrosis stage was evaluated and its difference in the diagnosis of liver fibrosis was compared. Results The Spearman's correlation coefficient between the elasticity scores and the histologic fibrosis stage was 0. 875,which was highly significant ( P＜0. 01). There was no significantly difference between elasticity scores and fibrosis staging in the diagnosis of liver fibrosis by Marginal Homogeneity (Z=- 1. 144, P = 0. 149). The sensitivity,specificity and accuracy of ultrasonic elastography for diagnosing liver fibrosis were 92. 5%, 85.0%, 90.0%, respectively. Conclusions Elastosonography is helpful for the diagnosis of liver fibrosis.

Objective To evaluate the value of grey scale ultrasonography (US) combined with elastography in the difference diagnosis of benign and malignant thyroid lesions. Methods Sixty-two patients with 81 lesions were examined by grey scale US and elastography preoperatively. All the lesions were confirmed by pathology. According to their features of grey scale ultrasonograms and elastograms, two receiver operating characteristic (ROC) curves were drawn, one for gray ultrasound and one for the combination methods. The area under curve (AUC) of them were made a comparison. Results The cut-off value of the grey scale group was equal or more than 5, the sensitivity,specificity was 60. 0% ,98. 5%. And the cut-off value of grey scale US combined with elastography group was equal or more than 6, the sensitivity, specificity was 80.0%, 89.4%. The AUC of grey scale US was 0.860 and that of the combination group was 0. 916. The difference between them was statistically significant Z = - 7. 863, P = 0. 000). Conclusions The grey scale US combined with elastography could improve sensitivity and decrease omission diagnose rate. It may be more helpful in the difference diagnosis of malignant from benign thyroid lesions.