Objective:To explore the impact of various fixatives on the nuclear-cytoplasmic localization of Yes-associated protein (YAP) in human corneal epithelial cells under hyperosmotic stress condition.Methods:Immortalized human corneal epithelial cells were divided into control group and hypertonic group.After 1 day of normal culture, cells of the hypertonic group were exposed to hyperosmotic medium at 450 mOsM by adding sodium chloride for 1 hour.No special treatment was given to the control group.Both groups of cells were fixed with four different fixatives, including 4% paraformaldehyde (PFA), -20 ℃ precooled absolute ethanol, -20 ℃ precooled methanol-acetone 1∶1 mixture, and Zamboni fixative solution for 20 minutes.Subsequent to fixation, immunofluorescent staining procedures were performed to identify the intracellular localization of YAP in the two groups.Results:After fixation with 4% PFA, human corneal epithelial cells showed normal morphology with YAP mainly in the nucleus in both groups, and there was no significant difference in the mean nuclear YAP fluorescence intensity between the two groups ( t=1.803, P=0.121).After fixation with absolute ethanol, cells showed some degree of shrinkage and deformation, diffuse YAP fluorescence staining with YAP-positive signals mainly localized in the cytoplasm in both groups, and the mean nuclear YAP fluorescence intensity was slightly decreased in the hypertonic group compared with the control group, but the difference was not statistically significant ( t=0.803, P=0.453).After fixation with methanol-acetone 1∶1 mixture, cells were crenulated with YAP mainly in the cytoplasm, and the mean nuclear YAP fluorescence intensity in the hypertonic group was slightly decreased compared with the control group, but the difference was not statistically significant ( t=1.067, P=0.327).After fixation with Zamboni solution, the cell structure was complete and clearly outlined, and the YAP nucleoplasmic translocation phenomenon could be clearly observed in cells in different states.The mean nuclear YAP fluorescence intensity in the hypertonic group was 197.5±34.5, which was significantly higher than 62.2±10.0 in the control group ( t=7.530, P<0.001). Conclusions:In the immunofluorescence staining experiment, the nucleoplasmic localization of YAP in corneal epithelial cells is affected by different fixative treatments.Zamboni fixative is better than 4% PFA, absolute ethanol, and methanol-acetone 1∶1 mixture in observing nuclear translocation of YAP after hypertonic stimulation.

Objective:To explore the impact of various fixatives on the nuclear-cytoplasmic localization of Yes-associated protein (YAP) in human corneal epithelial cells under hyperosmotic stress condition.Methods:Immortalized human corneal epithelial cells were divided into control group and hypertonic group.After 1 day of normal culture, cells of the hypertonic group were exposed to hyperosmotic medium at 450 mOsM by adding sodium chloride for 1 hour.No special treatment was given to the control group.Both groups of cells were fixed with four different fixatives, including 4% paraformaldehyde (PFA), -20 ℃ precooled absolute ethanol, -20 ℃ precooled methanol-acetone 1∶1 mixture, and Zamboni fixative solution for 20 minutes.Subsequent to fixation, immunofluorescent staining procedures were performed to identify the intracellular localization of YAP in the two groups.Results:After fixation with 4% PFA, human corneal epithelial cells showed normal morphology with YAP mainly in the nucleus in both groups, and there was no significant difference in the mean nuclear YAP fluorescence intensity between the two groups ( t=1.803, P=0.121).After fixation with absolute ethanol, cells showed some degree of shrinkage and deformation, diffuse YAP fluorescence staining with YAP-positive signals mainly localized in the cytoplasm in both groups, and the mean nuclear YAP fluorescence intensity was slightly decreased in the hypertonic group compared with the control group, but the difference was not statistically significant ( t=0.803, P=0.453).After fixation with methanol-acetone 1∶1 mixture, cells were crenulated with YAP mainly in the cytoplasm, and the mean nuclear YAP fluorescence intensity in the hypertonic group was slightly decreased compared with the control group, but the difference was not statistically significant ( t=1.067, P=0.327).After fixation with Zamboni solution, the cell structure was complete and clearly outlined, and the YAP nucleoplasmic translocation phenomenon could be clearly observed in cells in different states.The mean nuclear YAP fluorescence intensity in the hypertonic group was 197.5±34.5, which was significantly higher than 62.2±10.0 in the control group ( t=7.530, P<0.001). Conclusions:In the immunofluorescence staining experiment, the nucleoplasmic localization of YAP in corneal epithelial cells is affected by different fixative treatments.Zamboni fixative is better than 4% PFA, absolute ethanol, and methanol-acetone 1∶1 mixture in observing nuclear translocation of YAP after hypertonic stimulation.

Objective To investigate the correlation between the biomarkers related to radio-sensitivity and preoperative radiotherapy in rectal cancer patients, and to establish a logistic regression model to predict the effect of the preoperative radiotherapy through detecting the expression levels of the molecular markers. Methods 33 patients with rectal cancer who received preoperative radiotherapy from January 2010 to January 2015 were retrospectively analyzed. Patients' information was also collected including the serum level of carcino-embryonic antigen (CEA), the immune-histochemical expression levels of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), thymidylate synthase (TS) and Ki-67, and image data (CT or magnetic resonance imaging) before radiotherapy, preoperative clinical staging and the postoperative pathologic staging. According to the postoperative pathological remission, the treatment effects of preoperative radiotherapy included effective (CR+PR) and ineffective (PD+SD) were evaluated. The relationship between these molecular markers and the curative effect of preoperative radiotherapy was analyzed by logistic regression analysis using SPSS v17.0 software, and a logistic curative effect prediction model was established. Results As a result of single factor and multiple factors logistic binary regression analysis, CEA, VEGF and Ki-67 were recognized as the interested factors for the radio-sensitivity predicting in patients with rectal cancer who received preoperative radiotherapy. A molecular markers predictive model for radio-sensitivity in preoperative radiotherapy in rectal cancer is as follow: log P=1.700-0.276×CEA-0.238×VEGF-0.135 ×EGFR+1.377 ×TS+0.080 ×Ki-67. Serum CEA level and the expression of VEGF might associate with radio-resistant, and the expression of Ki-67 might associate with better reaction to preoperative radiotherapy. Conclusion The levels of serum CEA, VEGF and Ki-67 may be the predictors of radio-sensitivity in rectal cancer patients who received preoperative radiotherapy.

Objective To investigate the effects of gemcetabine and LY294002 monotherapy or combination on the proliferation and poptosis of pancreatic cancer cell lines BxPc-3 and MiaPaCa-2.Methods Cell proliferation and poptosis were detected by MTT and Annexin V-FTTC,respectively.Results Both gemcetabine and LY294002 could inhabit the proliferation of the two cell lines.Their inhibitory effects were increased accompanied with increased drug concentrations and the cell survival rates was negatively correlated with logarithmic of the drug concentrations (r＜-0.95,P＜0.01).The inhibitory effects of gemcetabine and LY294002 to the BxPc-3 proliferation were significantly stronger than to the MiaPaCa-2(P＜0.05).For BxPc-3 and MiaPaCa-2,the IC50 of gemcetabine were(10.07±1.83),(36.45±2.71)μmol/L(P＜0.05),and the IC50 of LY294002 were(7.84±1.48),(17.89±1.98)μmol/L(P＜0.05),respectively.Gemcetabine and LY294002 could induce cell apoptosis(P＜0.01).Though both the concurrent or consecutive use of these two drugs could promote cell apoptosis,the effect of the concurrent group was significantly stronger(P＜0.05).The order of these two drugs in the concurrent group had no significant influence on their effects(P＞0.05).Conclusion Both gemcetabine and LY294002 could inhibit the proliferation of pancreatic cancer cell lines.Their concurrent application shows a significant inhibitory effect on the cell apoptosis.

As the main medicinal powder for drawing out pus and removing necrotic tissue in external therapies of traditional Chinese surgery, Sheng Powder has made great contributions to the treatment of inflammatory wounds and has the unique bactericidal and decay-discharging function that can not be replaced by antibiotics. However, Sheng Powder has toxicity because it contains mercury. So far, there is no clinical research on the standards of dose and usage of Sheng Powder and there is a lack of objective and quantitative criteria for operating standards and monitoring of toxicity and side effects. Therefore, the authors choose Jiuyi Powder, one of the most commonly used Sheng Powder, to evaluate the safety of its external use, and form a standardization program for clinical implementation.