Objective To investigate the effect of gastrodin on rat vascular smooth muscle cell (VSMC) migration induced by platelet-derived growth factor-BB (PDGF-BB) and its possible mechanisms.Methods Enzyme digestion method wasused to obtain rataorticVSMCs and be purified bypassage.Immunofluorescence staining was used to identify VSMC marker proteins.A PDGF-BB induced cell migration model was established.Transwell chamber assay was used to evaluate the effect of gastrodin on PDGF-BB induced VSMC migration.Western blots were performed to detect the phosphohorylation levels of c-jun N-terminal kinase (JNK).Results The purity of primary cultured VSMC was more than 99％.The VSMC migrated number in the PDGF-BB group was 85.2 ± 3.486 per field.It was significantly more than 42.5 ± 1.927 per field in the control group (t =9.981,P＜0.001),and gastrodin was enable to make PDGF-BB induced the number of VSMC migration significantly reduce to 71.3 ± 1.783 per filed (t=3.550,P =0.002).Western blots analysis showed that gastrodin inhibited PDGF-BB induced JNK phosphorylation (0.190 ± 0.015 vs.0.190 ± 0.015; t =14.548,P =0.000).Conclusions Gastrodin inhibits PDGF-BB induced VSMC migration,its mechanisms may be associated with the inhibition of the JNK signaling pathway activation.

Objective To evaluate the efficacy and safety of 308-nm monochromatic excimer light for the treatment of amelanotic nevus.Methods Eighteen patients with amelanotic nevus were treated with 308-nm monochromatic excimer light twice a week for five consecutive weeks.Melanin content index (MCI) was measured in the lesions before and after the treatment.After the final exposure,a three-month follow-up was carried out.Results After the start of treatment,the MCI in lesions increased in a session-dependent manner and approximated to 100％ of that in perilesional normal skin after six sessions of treatment.The follow-up revealed a decrease trend in MCI after the end of the treatment,which was nearly equal to that in perilesional normal skin one month later,and dropped to about 90％ of that three months later.No side effects such as blisters or scar were observed during the treatment.Conclusions The 308-nm monochromatic excimer light is safe and effective for the treatment of amelanotic nevus.Re-exposure to 308-nm monochromatic excimer light after three months is recommended as consolidation therapy.

Objective: To investigate influence of isometric exercise (IE) training on number of endothelial progenitor cells (EPCs) and level of vascular endothelial growth factor (VEGF) of circulating blood in patients with coronary chronic total occlusion (CTO). Methods: A total of 20 CTO patients were divided into training group (n=10) and control group (n=10). Both groups received routine medication for three months, training group also received three-month IE training (maximal handgrip was used to induce maximum IE of upper limb muscles, leading to temporary physiological ischemia of skeletal muscle),while control group remained sedentary without exercise training. Flow cytometry was used to measure number of blood EPCs, and enzyme linked immunosorbent assay (ELISA) was used to measure serum concentration of VEGF. Results: Before treatment, the differences of levels of blood EPCs and VEGF between two groups were no significant（P>0.05）. Compared with before treatment, there were significant increase in blood number of EPCs [(0.028±0.009)% vs. (0.044±0.016)%] and VEGF concentration [(65.3±15.1) pg/ml vs. (98.5±17.4)pg/ml] after three-month treatment in training group (P=0.015, P<0.01), and they were significantly higher than those of control group after treatment. Compared with before treatment, there were no significant difference in blood number of EPCs and concentration of VEGF after treatment in control group, P>0.05. The blood numbers of EPCs were positively correlated with VEGF concentration in training group and control group (r=0.727, r=0.785, P<0.05 both). Conclusions: Isometric exercise training can increase blood number of EPCs and VEGF concentration in coronary CTO patients, which may contribute to collateral angiogenesis in remote ischemic myocardium.

Objective: To investigate the Effects of entinostat on the expression of NKG2D ligands in the non-small cell lung cancer (NSCLC) cell lines, A549 and HCC-827, and to detect the effect of entinostat-mediated NK cell killing of A549 and HCC-827 cells. Meth-ods: The effect of entinostat on A549 and HCC-827 cell proliferation was measured by MTT assay. Flow cytometry was used to detect the expression of NKG2D ligands. mRNA levels of the ligands were detected by RT-PCR . The level of soluble MICA in cell culture super-natant was evaluated by ELISA. The cytotoxicity of NK cells against A549 and HCC-827 cell lines (treated with entinostat) was assessed using lactate dehydrogenase release assay. Results: Entinostat showed a time-and dose-dependent inhibition effect on the prolifera-tion of A549 and HCC-827 cell lines. The expression of NKG2D ligands and mRNA transcription levels of MICA and MICB were en-hanced after treatment with 0.5, 1μmol/L entinostat for 48 h. The soluble MICA level in A549 cell culture supernatant was increased by 1μmol/L entinostat. The sensitivity of HCC-827 cells to NK cells was enhanced upon treatment with 0.5, 1μmol/L entinostat. Con-clusions: entinostat enhanced the killing effect of NK cells on non-small cell lung cancer cells by up-regulating the expression of NKG2D ligands. This provides a new method and theory for the treatment of NSCLC.

Objective: To evaluate the efficacy and safety of mesenchymal stem cells in allogeneic hematopoietic stem cell transplantation for patients with refractory severe aplastic anemia (R-SAA) . Method: The clinical data of 25 R-SAA patients receiving co-transplantation of mesenchymal stem cells combined with peripheral blood stem cells from sibling donors (10 cases) and unrelated donors (15 cases) from March 2010 to July 2018 in Zhengzhou University Affiliated Tumor Hospital were retrospectively analyzed. Antithymocyte globulin (ATG) treatment was ineffective/relapsed in 11 cases, and cyclosporine (CsA) treatment ineffective/relapsed in 14 cases. Results: There were 13 male and 12 female among these patients. One patient had a primary graft failure, one patient had a poorly engraftment of platelets, and the remaining 23 patients achieved hematopoietic engraftment. The median time of granulocyte engraftment was 12.5 (10-23) days and 15 (11-25) days for megakaryocyte. Incidences of grade Ⅰ/Ⅱ acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD) were 37.5% (9/24) and 21.7% (5/23) , respectively. There was no severe GVHD and no severe complications that related to transplantation. 21 of 25 (84%) patients were alive with a median follow-up of 22.9 (1.6-107.8) months. The 5-year overall survival rate after transplantation was (83.6±7.5) %. Conclusion The combination of mesenchymal stem cells is reliable and safe in the treatment of R-SAA in peripheral blood stem cell transplantation of unrelated donors and sibling donors, which could significantly reduce the incidence of GVHD and severe transplantation-related complications.

Abstract Brain Breaks is a physical activity program that combines cultural classroom based physical activity with modern technology while providing children with multi level guidance. As an intervention for intermittent sedentary activities, Brain Breaks can improve students physical activity level, thereby improving their physical fitness and positively affecting their motivation to participate in physical activities and positive learning behaviors. The paper understands this intervention from the connotation, implementation basis, and application effect of Brain Breaks, and then proposes practical application suggestions and future research directions.When Brain Breaks in the cultural classroom is promoted and practiced in China in the future, attention should be paid to the means of implementation by the teachers, the selection of representative target groups, and the precise implementation plan.At the research level, the effects of motor skills, special group interventions, gender differences, environmental changes, and physiological mechanisms of the Brain Breaks are to be explored.

Objective:To investigate the proliferation and apoptosis characteristics of polyploid non-small cell lung cancer (NSCLC) cell model induced by docetaxel (Doc), and to analyze the potential role of polyploid tumor cells in chemotherapy resistance and tumor recurrence.Methods:NSCLC A549 cells were treated with dimethyl sulfoxide (DMSO) or 1 μmol/L Doc for 24 h. After drug removal, the cells were cultured in complete medium until the third day or the 5th day, and then they were recorded as the control group, Doc 24 h group, Doc 24 h+ 3 d group, Doc 24 h + 5 d group, respectively. The cell morphology was detected by using immunofluorescence staining. Flow cytometry was used to determine cell ploidy and cell cycle. Dil labeling and CFSE labeling were applied to detect cell proliferation. Flow cytometry by Annexin-V/PI double labeling was used to detect apoptosis. The changes of cyclin and apoptotic protein were analyzed by using Western blot.Results:Immunofluorescence staining results showed that compared with the control group, the volume of a small number of surviving cells in Doc 24 h group was increased slightly and the cells showed multinuclear status; while the cell volume in Doc 24 h+ 3 d group and Doc 24 h+ 5 d group continued to increase, and the nucleus remained multinuclear. The results of cell ploidy analysis also showed that the percentage of polyploid cell subsets was (3.40±0.95)%, (20.80±2.87)% in Doc 24 h group, (55.67±3.85)% in Doc 24 h+3 d group and (76.20±2.51)% in Doc 24 h+5 d group. With the prolongation of withdrawal time, the percentage of polyploid cell subsets was increased, and the difference was statistically significant ( F= 478.054, P < 0.05). The percentage of G 1 and S phase cell subsets in Doc 24 h group was lower than that in the control group, and the percentage of G 2/M phase cell subsets was higher than that in the control group, and the difference was statistically significant (both P < 0.05). The protein expression level of cdc2, P-cdc2 (Thr14), P-cdc2 (Tyr15), P-cyclin B1 (Ser128), P-cyclin B1 (Ser147) in the cells of the control group, Doc 24 h group, Doc 24 h+ 3 d group and Doc 24 h+ 5 d group was down-regulated in sequence, while the expression level of cyclin B1 was up-regulated, and cdc25c was down-regulated in Doc 24 h + 3 d group and Doc 24 h+ 5 d group. Dil staining results showed that the fluorescence of cell-labeled Dil in Doc 24 h group, Doc 24 h+ 3 d group and Doc 24 h + 5 d group did not decrease significantly. CFSE staining showed that the fluorescence intensity of CFSE labeled by polyploid A549 cells did not change significantly with the prolonged withdrawal time. Annexin-V/PI double staining showed that the percentage of apoptotic cell subsets in Doc 24 h group was higher than that in the control group ( P < 0.05), but the percentage of apoptotic cell subsets in Doc 24 h + 3 d group and Doc 24 h + 5 d group was lower than that in Doc 24 h group, while there was no statistically significant difference when compared with the control group ( P > 0.05). Western blot results showed that the expression of bcl-xl and mcl-1 in the control group, Doc 24 h group, Doc 24 h + 3 d group and Doc 24 h + 5 d group was up-regulated in sequence, while the expression of bax and bak in Doc 24 h + 3 d group and Doc 24 h + 5 d group was up-regulated, but down-regulated in Doc 24 h+5 d group. Conclusions:Doc can induce polyploidy of A549 cells in vitro. The cell cycle is blocked in G 2/M phase. After doc treatment, the proliferation of A549 cells is significantly decreased, and the apoptosis of A549 cells is promoted. However, with the prolongation of withdrawal time, apoptosis resistance occurs, and the expression levels of corresponding pro-apoptosis and anti-apoptosis proteins show significant changes. This may be helpful for polyploid tumor cells to produce drug resistance and tumor recurrence after chemotherapy intervention.

The efficacy and safety of co-transplantation of unrelated donor peripheral blood stem cells (UD-PBSCs) combined with umbilical cord mesenchymal stem cells (UC-MSCs) in refractory severe aplastic anemia-Ⅱ(RSAA-Ⅱ) were analyzed retrospectively. Fifteen patients with RSAA-Ⅱ underwent UD-PBSCs and UC-MSCs co-transplantation, among whom 14 cases had hematopoietic reconstitution without severe graft versus-host disease (GVHD). The 5-year overall survival rate was 78.57%. Combination of UD-PBSCs and UC-MSCs transplantation could be a safe and effective option for RSAA-Ⅱ.

Objective:To investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) and their conditioned medium on proliferation, migration and apoptosis of human non-small cell lung cancer (NSCLC) polyploid A549 cells.Methods:A549 cells in logarithmic phase were selected. After induction treatment with 1 μmol/L docetaxel for 24 h, DMEM/F12 medium with 10% fetal bovine serum was used to culture the cells for 3 d, finally the polyploid A549 cells model was successfully established. After finishing the separation and culture of hUC-MSC, hUC-MSC conditioned medium was prepared. Normally cultured polyploid A549 cells were treated as the control group, conditioned medium cultured polyploid A549 cells were treated as the conditioned medium group. hUC-MSC was co-cultured with polyploid A549 cells, and the ratio of the total number of cells was 2:1 and 5:1, respectively, which were recorded as MSC 1 group and MSC 2 group. Cells in each group were continually cultured for 48 h or 72 h. Proliferation and apoptosis of polyploid A549 cells in each group were detected by using flow cytometry, cell migration ability was detected by using Transwell assay, and the expressions of migration and apoptosis-related proteins were detected by using Western blotting.Results:Polyploid A549 cells model was successfully established and hUC-MSC was cultured separately. The result of cell proliferation detected by flow cytometry showed that at 48 h, the mean fluorescence intensity of the control group, conditioned medium group, MSC 1 group and MSC 2 group was 1 695±305, 2 020±85, 1 259±35 and 1 356±33, respectively, and the difference was statistically significant ( F = 14.00, P < 0.05); at 72 h, the mean fluorescence intensity of the control group, conditioned medium group, MSC 1 group and MSC 2 group was 1 052±77, 1 309±24, 864±201 and 1 103±237, respectively, and the difference was statistically significant ( F = 3.90, P > 0.05). The result of Transwell assay showed that at 48 h, the number of cell migration in the control group, conditioned medium group, MSC 1 group and MSC 2 group was 52±9, 57±12, 68±8 and 75±11, respectively, and the difference was statistically significant( F = 32.16, P < 0.05); the number of cell migration in each experimental group was all higher than that in the control group (all P < 0.05). The percentage of apoptotic cells in the control group, conditioned medium group, MSC 1 group and MSC 2 group was (15.53±4.27)%, (13.77±1.75)%, (3.60±0.50)% and (2.33±0.06)%, respectively, and the difference was statistically significant ( F = 182.36, P < 0.05); there was no statistically significant difference between the control group and conditioned medium group ( P > 0.05); there were statistically significant differences between MSC 1 group and the control group, MSC 2 group and the control group (both P < 0.05). Western blotting results showed that compared with the control group, the expression of migration-related protein matrix metallopeptidase 9 (MMP-9) was increased, the expression of pro-apoptotic protein bax was reduced, the expression of anti-apoptotic protein bcl-xL was increased in conditioned medium group, MSC 1 group and MSC 2 group. Conclusions:hUC-MSC can improve the migration and anti-apoptotic ability of polyploid A549 cells, suggesting that hUC-MSC may affect the survival of tumor cells during the process of chemotherapy damage and repair.

Objective:To study the migration of polyploid tumor cells induced by docetaxel, the characteristics of epithelial-mesenchymal transition, and the killing effect of immune cells on them, in order to explore the potential role of polyploid tumor cells in tumor recurrence.Methods:The human non-small cell lung cancer A549 cells were treated with 1 μmol/L docetaxel for 24 h, and the cells were collected as Doc 1 d group. After drug removal, the cells were cultured in fresh and complete medium for 3 or 5 days, then the cells were collected as Doc 3 d group or Doc 5 d group respectively. The A549 cells were treated with DMSO for 24 h as control group. Immunofluorescence staining was used to detect cell morphology, flow cytometry was used to analyze cell ploidy, scratch test was used to detect cell migration, Western blotting was used to detect the expression of epithelial-mesenchymal transition related proteins, and lactate dehydrogenase release method was used to evaluate the killing activity of cytokine-induced killer (CIK) cells.Results:Compared with the control group, most of the cells in the Doc 1 d group, Doc 3 d group and Doc 5 d group were apoptotic, a few of the surviving cells were significantly larger, and the nucleus was polynuclear. The proportions of polyploid cell subset (DNA content > 4N) in the control group, Doc 1 d group, Doc 3 d group and Doc 5 d group were (1.93±0.55)%, (22.97±2.37)%, (51.30±12.51)% and (67.87±8.31)% respectively, and the difference among the four groups was statistically significant ( F=26.521, P<0.001). The proportion of polyploid cell subset in Doc 1 d group, Doc 3 d group and Doc 5 d group was significantly higher than that in the control group (all P<0.001). With the prolongation of withdrawal time, the proportion of polyploid cell subset in Doc 3 d group and Doc 5 d group was significantly higher than that in Doc 1 d group ( P=0.009; P=0.004). After 24 h and 48 h culture, the wound healing rates of the control group were both 100%, and the wound healing rates of the Doc 3 d group were (39.10±2.12)% and (46.13±5.32)% respectively, with no significant difference ( t=2.126, P=0.051). Compared with the control group at 24 h and 48 h, the cell migration abilities of Doc 3 d group were significantly lower ( t=49.756, P<0.001; t=30.825, P<0.001). Compared with the control group, the expression of E-cadherin protein decreased gradually in the Doc 1 d group, Doc 3 d group and Doc 5 d group, the expression of Vimentin protein increased gradually, and the expressions of Snail protein and N-cadherin protein did not change significantly. The killing efficiencies of CIK cells against the cells of the control group, Doc 3 d group and Doc 5 d group were (27.27±1.91)%, (17.87±2.35)%, (9.47±0.51)% respectively, and the difference was statistically significant ( F=11.294, P<0.001). The killing efficiency of Doc 3 d group and Doc 5 d group was significantly lower than that of the control group ( P=0.004; P<0.001). The killing efficiency of Doc 5 d group was significantly lower than that of Doc 3 d group ( P=0.003). Conclusion:The migration ability of polyploid tumor cells induced by docetaxel is weakened, but epithelial-mesenchymal transition is likely to occur, and the killing effect of immune cells on them is reduced.