Objective To investigate the effect of miR-20b on cell proliferation and cell cycle in gastric cancer because of up-regulation of miR-20b in gastric cancer.Methods miR-20b mimics and its inhibitor were respectively transfected into MGC 803 gas-tric cancer cell and methyl thiazolyl tetrazolium ( MTT ) and fluorescence-activated cell sorting ( FACS ) were used to analyze cell growth and cell cycle.Western blot was used to explore the molecular basis of miR-20b.Results Compared with its control, cell growth was obvious elevated and the cell cycle transition was also increased from G 1 to S phase after miR-20b mimics transfection .After transfecting miR-20b inhibitor, cell growth was markedly decreased and cell cycle transition was also delayed from G 1 to S phase.Fur-thermore, miR-20b induced the expression of cyclin D1 (CCND1) and C-Myc, decreased the expressions of p21 and p15.Conclu-sions miR-20b was considered as a potential oncogene to modulate cell growth and cell cycle transition through regulating the expres -sion of cell cycle-related genes .

Treated 28 cases of obesity by electroacu-puncture plus auricular-press therapy, and 15 cases got significant effect, 10 cases got effectiveness, 2 cases got improvement and 1 case had no effect.

Objective To evaluate the relationship between cavitas pelvis fluidify and infertility.Methods The clinical data of 129 infertility patients which ultrasound hint cavitas pelvis fluidify but hysterosalpingography hint both fallopian tubes to be unobstructed were analyzed retrospectively. The patients were divided into two groups, after 3 months cure with traditional Chinese medicine, the cavitas pelvis fluidify of 86 cases were obsolescent as group Ⅰ , the cavitas pelvis fluidify of 43 cases were no obsolescent as group Ⅱ. Compared their pregnancy rates. Results The pregnancy rate of group Ⅰ was 30.23%(26/86),group Ⅱ was 6.98%(3/43 ), there was significant deviation between the two groups (P < 0.05 ). Eighty-seven patients who were no pregnant were diagnosed laparoscopy, there were 55 cases with endometriosis (EMS), 25 cases with cavitas pelvis accretion, 1 case with tuberculosis of peritoneum, 6 cases with carmoisine cavitas pelvis fluidify without other abnormal. Thirty-one of these patients were pregnant through in vitro fertilization and embryo transfer. Conclusion Cavitas pelvis fluidify is very important in clinic, search for the cause of a disease and cure actively is needed, so that they can cure utility.

Objective To detect the expressions of Numb and α-catenin in the gastric cancer and matched normal gastric mucosa,and to analyze their relationship with clinicopathological factors of gastric cancer and explore their roles in gastric carcinogenesis and progression.Methods Immunohistochemical method was used to detect the expressions of Numb and α-catenin in 109 cases of tumor specimens and 97 cases of matched normal gastric mucosa.The correlations between Numb,α-catenin and clinicopathological factors were analyzed.Results The positive rates of Numb and α-catenin in gastric cancer were significantly lower than those in normal gastric mucosa (60.6％ ∶ 100.0％,x2 =48.361,P =0.000;76.1％ ∶ 100.0％,x2 =26.480,P=0.000).The expression of Numb protein was correlated with Lauren type (x2 =17.018,P =0.000) and tubular adenocarcinoma differentiation (x2 =17.586,P =0.000).The expression of α-catenin protein was correlated with Borrmann type (x2 =6.700,P =0.035),Lauren type (x2 =11.098,P =0.001),tubular adenocarcinoma differentiation (x2 =8.203,P =0.017) and lymph node metastasis (x2 =6.402,P =0.011).The expressions of Numb and α-catenin were statistically correlated in 109 cases of gastric cancer (rk =0.184,P =0.028).Conclusion Compared with normal gastric mucosa,both Numb and α-catenin expressions are down-regulated and the two expressions are correlated in gastric cancer.Numb and α-catenin may be involved in the regulation of histological differentiation of gastric cancer by certain passages,Numb protein may be adjusted by α-catenin pathway which is involved in Wnt-β-catenin pathway,and thus plays an important role in promoting cell proliferation,invasion and metastasis in human gastric cancer.

Objective:Hyperoxia is a necessary therapy in some neonatal diseases, and long-term therapeutic hyperoxia may induce severe damaging effects on intestinal epithelial cells.The aim of this study was to investigate whether hyperoxia could promote the expression of ASK1 and P38 in intestinal epithelial cells through ROS.Methods:The human colon adenocarcinoma cell line Caco-2 cells were treated with different concentrations of H 2O 2(100 μmol/L, 200 μmol/L and 400 μmol/L)and 85% oxygen in vitro.The expression of ASK1 was detected by immunofluorescence, and the expression of P38 and p-P38 were detected by Western Blot and Real-time PCR. Results:With the increase of H 2O 2 concentration, the fluorescence intensity of ASK1 increased.The fluorescence intensity of ASK1 in the hyperoxia group was significantly stronger than that of the control group and the H 2O 2 groups.With the increase of H 2O 2 concentration(100 μmol/L、200 μmol/L、400 μmol/L), the expression of P38 protein(0.21±0.02, 0.28±0.13, 0.44±0.07)and p-P38 protein(0.09±0.02, 0.19±0.03, 0.37±0.07)gradually increased.The expression of P38 mRNA in 200 μmol/L and 400 μmol/L H 2O 2 groups(4.03±0.68、3.94±0.71)was significantly higher than that in 100 μmol/L H 2O 2 group(3.05±0.47)( P<0.01). The expressions of P38 protein, p-P38 protein and P38 mRNA in the hyperoxia group were significantly higher than those in the H 2O 2 group( P<0.01). Compared with the control group, the expressions of P38 protein, p-P38 protein and p38 mRNA in the hyperoxia group and H 2O 2 groups increased significantly( P<0.01). Conclusion:The expression of ASK1 and P38 in intestinal epithelial cells increased significantly under hyperoxia, which indicated that hyperoxia might activate ASK1 and thereby regulate the expression of downstream P38 through ROS, resulting in intestinal epithelial cells damage.

BACKGROUND:Human mesenchymal stem cells (hMSCs) become aging and even die after several passages. Some investigations have shown that telomere has a close correlation with life span of the cells. Whether the ectopic expression of human telomerase reverse transcriptase (hTERT) could induce the activity of the telomerase, maintain the length of telomere, and finally prolong the life cycle of MSCs without losing their multipotent differentiation capacity is still uncertain.OBJECTIVE: To observe the influence of the ectopic expression of hTERT on the telomerase activity and cell life cycles of hMSCs.DESIGN: Repetitive measurement trails.SETTING: Research Center of Stem Cell, Zhengzhou University Medical College.MATERIALS: The experiment was conducted in the Research Center of Stem Cell, Zhengzhou University Medical College from October 2003 to December 2005. hMSCs were obtained from 20 healthy donators from the Department of Pediatric Surgery and Outpatient, the Third and First Affiliated Hospitals of Zhengzhou University. Enhanced green fluorescent protein plasmid (pEGFP-C1) and pEGFP-hTERT were provided by Dr. Chantal Autexier of Canada. DH5α strain provided by Dr. Hou Wei-hong, the Key Molecular Medical Laboratory of Zhengzhou University Medical College.METHODS.: Under sterile condition, 2 mL bone marrow of sternum of healthy donors were harvested, and prepared after centrifugalization,dilution and passage.① Transfection of pEGFP-hTERT into hMSCs and the screening and amplification of resistance cloning:The 5th passage cells were seeded in a 24-well plate,and transfected by pEGFP-hTERT with lipofectamine method.The cells were divided into four groups including untransfected group,lipofectamine group,pEGFP-C1 group and pEGFP-hTERT group. Resistance cloning screen and amplification was performed by G418. ②hTERT mRNA expression and detection of telomerase activity:RT-PCR and PCR-ELISA were used to detect the hTERT mRNA expressions of the fifth passage hMSCs transfected with pEGFP-hTERT, and pEGFP-C1, the untransfected tenth passage hMSCs and K562 cells (positive control), and the telomerase activity of the fifth and thirtieth passage hMSCs transfected with pEGFP-hTERT,the fifth pEGFP-C1-transfected cells and the tenth passage untransfected cells. ③Karyotype analysis of hTERT-transfected MSCs: Chromosome analysis was performed by conventional Giemsa staining.④Inducement of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification:The transfected MSCs were cultured in a medium containing epidermal growth factor and basic fibroblast growth factor, which could induce the cells differentiate into neuron-like cells. The culture solution was changed every 3 days, and the changes in cell growth condition and morphologic characteristics were observed under an inverted microscope. The microtubule associate protein (MAP2) and neurofliament subunit M (NF-M) were identified by RT-PCR.MAIN OUTCOME MEASURES:①hMSCs transfection with different kathion liposomes and the screening and amplification of resistance cloning; ②hTERT mRNA expressions of each group and detection of telomerase activity; ③Karyotype analysis of pEGFP-hTERT-transfected MSCs; ④ Induction of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification.RESULTS: ①With the decrease of G418 concentration, the cells in the untransfected and lipofectamine groups died, and stably EGFP expressed MSCs were obtained; after G418 screening, there was a cell clone undergone 35 passages and continued to proliferate, whose appearance and growth characteristics were similar to the untransfected MSCs observed under inverted microscope. ②The fifth passage pEGFP-C1-transfected hMSCs and tenth passage untransfected hMSCs remained telomerase-negative, but the K562 and fifth passage hTERT-transfected cells showed positive telomerase activity. ③The telomerase activity of the fifth and thirtieth passage hTERT-transfected cells was positive. ④The hTERT-MSCs at passage 10, 20 and 30 had 23 pairs of chromosomes, and two X chromosomes. So they were still normal diploid with normal chromosome appearance and number. ⑤Many hTERT-transfected MSCs had the typical appearance of neuron-like cells. RT-PCR analysis showed that th expressions of MAP2 and NF-M were increased.CONCLUSION:Ectopic expression of the hTERT gene is found in hMSCs,and can induce the telomerase activity of hMSCs.The ectopic expression of the hTERT gene in hMSCs could extend the life spans of cells and maintain their multipotent differentiation capacity.

BACKGROUND: Stem cells are relatively primitive cells possessing the capabilities of self-renewal, high proliferation and multi-potential differentiation in vivo under certain conditions. Pancreatic stem cells and umbilical cord blood mesenchymal stem cells (MSCs) may serve therapeutic purpose clinically, but they are still difficult to culture in vitro at present.OBJECTIVE: To explore the method for isolation, purification and culture of pancreatic stem cells and umbilical cord blood MSCs in vitro and observe their morphological changes during culture in vitro.DESIGN: Completely randomized experiment with repeated measurement.SETTING: Stem Cell Research Center, Teaching and Research Division of Physiology, Medical School of Zhengzhou University.MATERIALS: This experiment was conducted in the Stem Cell Research Center, Teaching and Research Division of Physiology, Medical College of Zhengzhou University, between April 2004 and January 2005. Ten to fifteen newborn SD rats (1-3 days) were selected for culture in vitro of pancreatic stem cells, and fresh umbilical cord blood was collected from healthy woman (24-35 years old, with informed consent) at full-term delivery for culture in vitro of umbilical blood SMCs.METHODS: The abdomen of the newborn SD rat was opened under aseptic condition to obtain the pancreas, which was cut into small tissue blocks and digested with type-V collagenase for islet isolation. The isolated islets were purified in continuous roller-bottle culture. Umbilical cord blood was freshly collected for isolating the monocytes by means of density gradient centrifugation in lymphocyte separation medium (with density of 1.077 g/cm3). The islet cells and umbilical cord blood monocytes were cultured in the incubator at 37 ℃ with 5% CO2. The morphological changes of the cells were observed at designed time points and flow cytometry was used to determine the expression of cell surface molecules.MAIN OUTCOME MEASURES: The isolation and culture of pancreatic stem cells and umbilical cord blood MSCs, and their morphological changes during culture in vitro.RESULTS: During culture in vitro, the fusiform islet progenitor cells showed adherent polar growth and continuous proliferation, which covered the whole bottom of the flask after 12-14 days and could be subcultured for passages. However round cells appeared after removal of the growth factor and serum in the culture medium. The monocytes isolated from the umbilical cord blood grew initially into numerous hematopoietic cell clones, most of which proved to be granulocyte clones by Switzerland staining. Seven days later, flat flask wall-adhering epithelial cells and long fusiform fibroblasts were observed mixed with a number of osteoclasts. As the cell culture was prolonged, the cell number increased steadily.CONCLUSION: Pancreatic stem cells and umbilical cord blood SMCs can be cultured in vitro for further experiments.

Objective To investigate the effect and mechanism of cysteine-rich protein 61 (Cyr61) on oxidative stress in human kidney tubular epithelial cell line after anoxia.Methods Human kidney tubular epithelial cell line (HK-2 cells) were divided into 5 groups:control group,Cyr61 group,MAPK inhibitor group (Cyr61 +PD98059),p38 inhibitor group (Cyr61 +SB203580) and PI3K inhibitor group (Cyr61+Wortmannin).Each group was pretreated for 12 h and then injured by anoxia.The cell viability was determined by MTT assay and the apoptosis rate of HK-2 cells was determined by flow-cytometry.The cellular ROS level was measured by spectro-fluorometry.The cellular superoxide dismutase (SOD) and catalase (CAT) were measured by nephelometry test.The expression of Nrf2 in HK-2 cells was detected by Western blotting.Results Anoxia enhanced the expression of ROS and Nrf2,decreased the expression of SOD and CAT significantly,meanwhile decreased HK-2 viability and increased HK-2 apoptosis (all P ＜ 0.05).Cyr61 increased the expression of p-Akt,Nrf2,SOD and CAT in HK-2,and decreased the expression of ROS,at the same time increased HK-2 viability and decreased HK-2 apoptosis (all P ＜ 0.05).Wortmannin inhibited the expression of p-Akt,Nrf2,SOD and CAT,meanwhile decreased HK-2 viability and increased HK-2 apoptosis (P ＜ 0.05).PD98059 and SB203580 had no affect on HK-2 compared to Cyr61 group (P＞0.05).Conclusions Cyr61 promotes the expression of Nrf2 through PI3K pathway in HK-2,which enhances the expression of SOD and CAT,and decreases the expression of ROS.Cyr61 exhibits protective effects on HK-2 cells injured by oxidative stress after anoxia.

Objective To investigate the influence of different HBV DNA extraction methods with Micro-Nucleic-Releaser (MNR), polysaccharide deposition method and boiling method in real-time quantitative and provide reference data for clinical lab regarding the PCR kit selection. Methods Three different HBV DNA isolation methods including MNR, polysaccharide deposition method and boiling method were used to study the extraction efficiency and anti-interference ability when HBV DNA virus in lipemia, haemolysis or jaundice serum samples was detected with real-time PCR. The traditional HBV DNA isolation method of Hydroxybenzene-Chloroform was used as control method. Results The quantitative results and amplification efficiency differed among different extraction methods. Complete hemolytic sample brought biggest influence, modest hemolytic sample give minimal influence, whereas the lipemia and jaundice did not bring significant influence to detection. The MNR give the best amplification efficiency and quantitativereproducibility. The extraction efficiency of boiling method was lowest. The fluorescent efficiency of real-timePCR from Hydroxybenzene-Chloroform methods was better than other methods, but it suffered from losingHBV DNA. Conclusions Real-time PCR system of HBV DNA isolation by MNR can provide an accurate,highly sensitive, and rapid method to quantify Hepatitis B virus. It is suitable to use in clinical laboratory.

Objective In this study we discussed the training method for skills of evidence-based nursing(EBN)for nurses in surgical department.Methods We selected 43 nurses in two surgical department as training objects.The trainging procedure was established according to the four steps of evidencebased nursing,putting forward questions,searching for evidences,making observations and applying evidence-based nursing.The nurses were trained according to the procedure.The skills of EBN,ability of obtaining information and satisfaction degree of nurses was evaluated after training.Results The skills of EBN and the ability of obtaining information after training Was statistically different from those before training(P ＜0.01).95% nurses considered that their EBN ability was improved after training. Conclusions The skills of EBN and the ability of obtaining information increased after EBN training in nurses of surgical department.This had instructive significance for the application of evidence-based nureing.