Objective To investigate the effect and the possible mechanism of osthole on proliferation and apopotosis of human cervical carcinoma Hela cells and its passible mechanism.Methods After cervical carcinoma Hela cells were incubated with different concentrations osthole,the cell proliferation activity was examined by MTT assay.The apoptosis rate and cellular ROS level were measured by flow cytometry.The Bcl-2 and Bax mRNA expression was determined by semi-quantitative RT-PCR.Results In comparison with the control group,osthole with different concentrations could obviously inhibit the Hela cells proliferation and accelerated the cellular apoptosis,lowered the expression rate of Bcl-2/Bax,raise the cellular ROS level in a osthole dose-dependent manner.Conclusion Osthole may inhibit Hela cell proliferation and accelerates the cells apoptosis,which might be associated with the increasing the cellular ROS level,promoting Bax expression and inhibiting Bcl-2 expression.

Objective To detect the expression of BAT2L protein in developmental brain of rats.Methods Expression,distribution and location of BAT2L protein in developmental brain rats on embryonic day 18,postnatal days 1,7 and 15 and in adult at different time points were detected by Western blotting analysis,immunohistochemistry and immunofluorescence,respectively.Results Western blotting showed that the BAT2L protein expression level was higher in brain of rats on embryonic day 18,postnatal days 1 and 7 than on postnatal day 15 and in brain of adult rats.Immunohistochemistry and immunofluorescence showed that the BAT2L protein was distributed and located in cell membrane and cytoplasm of neurons but not in nuclei and extra-cellular space.Positive neurons were widely distributed in cerebral and cerebellar cortex,hippocampus and cerebral ganglion.The morphology of neurons was significantly different in newborn and adult rats.Positive prominences and branches of certain neurons were found in brain tissues of newborn rats but hardly in those of adult rats.Conclusion BAT2L protein is specifically expressed in cell body,membrane and prominence of neurons in brain of rats.

To identify whether a highly repeated GT sequence from DCA1 promoter from Dunaliella salina,which have been proved to be a salt-inducible promoter in our previous study,would be a salt-inducible regulation element,different primers were designed to amplify 6 different-length fragments of DCA1 promoter from D.salina by PCR.After these fragments were respectively inserted into the HindⅢ-BamH I sites of the vector pU?GUS,serial expression vectors containing the gus gene were generated.D.salina cells transformed with these recombinant plasmids by electroporation were grown in liquid media containing different concentrations of sodium chloride respectively.GUS enzyme activity was measured histochemically and fluorometrically.The results revealed that 3 fragments containing GT repeated sequence drove the external gus gene expression and the expression pattern of the gus gene was regulated by the concentrations of sodium chloride.Additionally,the 2 fragments without tandem GT sequence drove the gus gene expression,but the expression pattern of the gus gene wasn't regulated by the concentration of sodium chloride;Also,the upstream fragment of the tandem GT sequence wasn't able to drive the gus gene expression.In conclusion,the highly repeated GT sequence from the DCA1 promoter plays an important role in the salt-inducible regulation of DCA1 promoter from D.salina and might be a novel salt-inducible element.

Applications of network pharmacology are increasingly widespread and methods abound in the field of drug development and pharmacological research. In this study, we choose rosiglitazone compound as the object to predict the targets and to discuss the mechanism based on three kinds of prediction methods of network pharmacology. Comparison of the prediction result has identified that the three kinds of prediction methods had their own characteristics: targets and pathways predicted were not in accordance with each other. However, the calcium signaling pathway could be predicted in the three kinds of methods, which associated with diabetes and cognitive impairment caused by diabetes by bioinformatics analysis. The above conclusion indicates that the calcium signaling pathway is important in signal pathway regulation of rosiglitazone compound, which provides a clue to further explain the mechanism of the compound and also provides a reference for the selection and application of methods of network pharmacology in the actual research.
Calcium Signaling ; Cognitive Dysfunction ; Computational Biology ; Diabetes Mellitus ; Humans ; Pharmacology ; methods ; Thiazolidinediones ; pharmacology

OBJECTIVETo study the effect of substrate stiffness on proliferation, migration of fibroblast and integrin β(1) expression in fibroblast.

METHODSFibroblasts were inoculated on silicon substrate with stiffness of (16.2 ± 0.5), (19.8 ± 1.1), and (200.1 ± 2.6) kPa. After being cultured for 5 days or 6 days, cells were counted and cell proliferative activities (recorded as absorbance value) were assessed with methyl thiazolyl blue (MTT). After being cultured for 3 days, cell cycle was detected and proliferation index (PI) was calculated. The cell scratch test was used for determination of cell migration rate on post scratch day (PSD) 0 (the day of scratch), 1, 2, and 3. After being cultured for 2 days, the expression of integrin β(1) was determined by flow cytometry with fluorescence. Data were processed with one-way analysis of variance.

RESULTS(1) The proliferative speed and proliferative activity of fibroblasts were all increased along with the increase in substrate stiffness. PI of fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5), (19.8 ± 1.1), and (200.1 ± 2.6) kPa was respectively 24.8%, 27.4%, 32.4%. On PSD 2, migration rate of fibroblasts inoculated on silicon substrate with stiffness of (19.8 ± 1.1) and (200.1 ± 2.6) kPa was respectively (91.4 ± 5.1)%, (100.0 ± 1.3)%, which were higher than that of fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5) kPa [(55.8 ± 6.8)%, with F value respectively 3.5, 4.0, P values all below 0.01]. (3) The expression rate of integrin β(1) in fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5) kPa was the lowest (43.22%), and that in fibroblast inoculated on silicon substrate with stiffness of (200.1 ± 2.6) kPa was the highest (81.26%).

CONCLUSIONSSubstrate stiffness may have a great effect on proliferation and migration of fibroblast during the process of wound healing and scar formation, which can be related to regulation of integrin β(1) expression.

Cell Movement ; Cell Proliferation ; Cells, Cultured ; Fibroblasts ; cytology ; metabolism ; pathology ; Humans ; Integrin beta1 ; metabolism ; Mechanical Phenomena ; Silicon

Objective To identify the expression of DLK1 protein in different types of renal cell carcinomas and its correlations with pathological characteristics and metastasis. Methods Immunohistochemistry analysis was performed to evaluate the expression of DLK1 protein in 94 cases of primary clear cell renal cell carcinoma, 76 cases of papillary renal cell carcinoma, 45 cases of chromophobe renal cell carcinoma, 71 cases of distal metastatic and 24 cases of lymph node metastatic clear cell renal cell carcinoma, as well as 18 cases of normal renal tissue. The correlations of DLK1 protein expression with pathological characteristics were analyzed. Results DLK1 protein was expressed in proximal and distal renal tubular epithelial cells in all the normal renal cases. In contrast, DLK1 protein expression was lower in different types of renal cell carcinoma. The low or negative expression of DLK1 protein in clear cell renal cell carcinoma, papillary renal cell carcinoma and chromophobe renal cell carcinoma was 33.0% (31/94), 27.6% (21/76) and 33.3% (15/45), respectively. Compared to normal renal tissue, DLK1 protein expression was significantly down-regulated in renal cell carcinomas (P＞0.05), whereas there was no significant difference on DLK1 protein expressions among the different types (P＞0.05) of renal cell carcinomas. DLK1 protein expression was not correlated with sex (60 male and 34 female cases), age (≥55, 50 cases and 55, 44 cases), grade (41 cases in grade I, 9 cases in grade II, 21 cases in grade III and 23 cases in grade Ⅳ respectively) and lymph node metastasis (76 cases with and 18 cases without lymph node metastasis) in clear cell renal cell carcinoma (P＞0.05). There was also no significant difference among primary, lymph node and distal metastatic lesions of clear cell carcinoma (P＞0.05). Conclusions DLK1 protein expression is commonly down-regulated in different types of renal cell carcinomas. Down-regulation of DLK1 protein expression is not associated with pathological characteristics and metastasis in clear cell renal cell carcinoma.

Objective To evaluate the effects of different concentrations of sevoflurane anesthesia on longterm learning and memory abilities in neonatal rats.Methods Twenty-seven neonatal Sprague-Dawley rats of both sexes,aged 7 days,weighing 12-20 g,were randomly assigned into 3 groups (n =9 each):control group (C group),2％ sevoflurane group (S1 group) and 3％ sevoflurane group (S2 group).Groups C,S1 and S2 inhaled air,2 ％ sevoflurane and 3 ％ sevofluran for 4 h,respectively.The neonatal rats were reared to 35 days old and underwent open field test,to 36 days old and underwent Morris water maze test,and to 42 days old and underwent continuous multiple-trail inhibition avoidance training.Results Open field test:There was no significant difference in the movement time,movement speed and the time the animals spent in the central square among the 3 groups (P ＞ 0.05).Morris water maze test:Compared with C group,the looking for platform latency on 2nd-5th days in S2 group and on 2nd-3rd days in S1 group was significantly prolonged,and the percentage of time of staying at the platform quadrant was decreased in S1 and S2 groups (P ＜ 0.05 or 0.01).The looking for platform latency on 3rd4th days in S2 group was significantly longer than that in group S1 (P ＜ 0.05).Continuous multiple-trail inhibition avoidance training:The latency detected at 24 h after training was significantly shorter in S1 and S2 groups than in group C (P ＜ 0.05),and in group S2 than in S1 group (P ＜ 0.05).Conclusion Sevoflurane anesthesia decreases the long-term learning and memory function in neonatal rats in a concentration-dependent manner.

Objective To investigate the effects of ischemic postconditioning (Post) on myocardial cell apoptosis and expres- sion of Bcl-2 and Bax protein during ischemia/reperfusion period in rabbits.Methods Eighteen rabbits were randomly allocated to three groups (6 in each group),sham operation (group S),ischemia/reperfusion group(group IR) and ischemic postconditioning group(group Post).Group IR and group Post were subjected to 15 minutes of left anterior descending coronary artery occlusion followed for 30 minutes of reperfusion.Ischemic postconditioning was achieved by three 30 seconds cycles of reperfusion,each followed by 30 seconds ischemia.Cardiomyocyte apoptosis were determined by in situ TDT-mediated dUTP nick end labeling (TUNEL) and DNA electrophoresis.The expression of Bcl-2 and Bax proteins in apoptotic myocardial cells were detected by immunohistochemistry sepa- rately.Results Compared with group IR,apoptotic index was significantly reduced in group Post [(28.06?2.92) % vs.(55.70? 13.96)%,P

Objective To investigate the effects of CO_2 pneumoperitoneum on the expression of focal adhesion kinase (FAK) of gastric cancer MKN-45 cells. Methods CO_2 pneumoperitoneum with different pressures was simulated in vitro, and the gastric cancer MKN-45 cells were divided into test and control groups. In the test group, gastric cancer MKN-45 cells were cultured in CO_2 pneumoperitoneum with different pressures [5, 10 or 15 mm Hg (1 mm Hg =0.133 kPa)] for 4 hours. The condition of the cells exposed to CO_2 pneumoperitoneum with a pressure of 15 mm Hg was observed at 0.5, 2 and 4 hours. Gastric cancer MKN-45 cells in control group were cultured at normal atmospheric pressure. The expression of FAK and phosphorylated FAK (FAK Tyr397) of each group was detected by Western blot. Multiple-group analysis was done by one-way ANOVA, and intergroup comparison was done by LSD test. Results In CO_2 pneumoperitoneum with pressures of 5, 10, 15 mm Hg, the expression of FAK was 2.14±0.17, 2.07±0.21 and 2.52±0.26, respectively, and the expression of FAK Tyr397 was 1.82±0.28, 1.93±0.52 and 3.71±0.37, respectively. The expression of FAK and FAK Tyr397 in the control group was 2.43±0.46 and 1.71±0.23, respectively. We found significant differences between the 2 groups (F = 2.171, 26.951, P < 0.01). After gastric cancer MKN-45 cells being treated for 0.5, 2 and 4 hours in CO_2 pneumoperitoneum with a pressure of 15 mm Hg, the expression of FAK Tyr397 was 3.41±0.44, 4.12±0.56 and 5.24±0.41 respectively, which is also significantly different (F =116.119, P < 0.01). The expression of FAK Tyr397 was back to 0.72±0.16 1 hour after the release of CO_2. Conclusions CO_2 pneumoperitoneum with different pressures can not promote the expression of FAK in gastric cancer MKN-45 cells which had been cultured for 4 hours, but can activate FAK through promoting its phosphorylation. The degree of FAK phosphorylation increases with pressure and time, and the activity of FAK decreases to pretreatment level rapidly once pressure is released.

Objective To investigate the radiosensitizing effect of 17AAG-cypate micelles on human non-small cell lung cancer A549 cells and its possible mechanism.Methods (1) A single-hit multi-target model formula was used to analyze the radiosensitizing effects of 17AAG-M and 17AAG-cypate-M.(2) The effects of 17AAG-cypate-M on the viability of A549 cells under laser and X-ray irradiation were analyzed by MTT assay.(3) The effect of the drugs on the cell senescence was observed by β-galactosidase staining assay.(4) The effects of different treatment conditions on DNA damage repair were analyzed by γ-H2AX immunofluorescence staining assay.(5) The expression of p-Erk1/2 and p-Akt was measured by Western blot.The paired t test was used for analyzing the differences between groups.Results Compared with the X-ray irradiation group,the X-ray+17AAG-cypate-M group had a lower mean lethal dose and a sensitization enhancement ratio greater than 1,indicating that 17AAG-cypate-M had a radiosensitizing effect.Compared with the 17AAG-M group,the 17AAG-cypate-M group showed significantly lower cell viability (P<0.01),a significantly higher percentage of aging cells (P<0.01),and significantly further delayed DNA damage repair (P<0.01).And the 17AAG-cypate-M group had lower expression of p-Erk1/2 and p-Akt than the 17AAG-M group.Conclusions Compared with 17AAG-M,17AAG-cypate-M has a higher radiosensitizing effect on A549 cells.The mechanism might be inducing the cell senescence,delaying DNA damage repair,and inhibiting the expression of p-Erk1/2 and p-Akt.