Objective: Based on physical activity (PA) and sedentary behavior (SB) variables on weekdays and weekends, the study aims to cluster the physical activities inside and outside kindergartens and to explore the cluster characteristics of different children using physical fitness indicators, so as to provide new strategies and methods for early childhood education and health. Methods: From March to June 2019, 291 children aged 3-6 years from 6 kindergartens in Nanchang were recruited by a stratified cluster random sampling method. The ActiGraph GT3X-BT triaxial accelerometer was used to measure and analyze the PA and SB levels inside and outside the kindergarten. A twostep clustering algorithm model was employed for cluster analysis. Physical fitness were measured and evaluated according to the "National Physical Fitness Measurement Standard Manual (Preschool Section)". Differences in physical fitness among different clusters of children were compared, and the cluster characteristics of different children were analyzed. Results: The clustering algorithm model indicated that based on six indicators, including PA and SB inside the kindergarten on weekdays, and PA and SB outside the kindergarten on both weekdays and weekends, children could be divided into three categories:active inside (high PA, low SB inside), active outside (high PA outside), and inactive (low PA, high SB both inside and outside). The average silhouette coefficient of the model was 0.3, indicating good clustering results. Both the active inside and active outside children showed significantly higher PA inside on weekdays, PA outside on weekdays and weekends, daily low intensity physical activity (LPA) and moderate-to-vigorous physical activity (MVPA) than the inactive children ( F=157.91, 80.79 , 95.86, 95.52, 124.74, P <0.05). After adjusting for gender and age, the physical fitness scores of both active outside ( 19.03 ±0.47) and active inside (19.11±0.40) were significantly higher than those of the inactive children (17.94±0.31). Additionally, active inside children (3.91±0.14) also showed significantly better performance in continuous double-leg jumps, compared to inactive children (3.45±0.11) ( P <0.05). Conclusion Children active inside and those active outside perform well in PA. Future research should focus on the proportion of structured and unstructured PA time to enhance the overall physical fitness of children.

Objective:To study the role of a novel brain-derived peptide hypoxic-ischemic brain damage associated peptide (HIBDAP) in regulating pyroptosis of oxygen-glucose deprived (OGD) microglia.Methods:The sequence of HIBDAP was coupled with the sequence of cell-penetrating peptide transactivator of transcription (TAT) to form TAT-HIBDAP. Fluorescein isothiocyanate (FITC) labeled TAT-HIBDAP was added to microglia cells and observed under fluorescence microscope. Microglia cells were treated with different concentrations of TAT-HIBDAP (1, 5, 10, 20 μmol/L) and then OGD process. Cell pyroptosis was analyzed using lactate dehydrogenase (LDH) assay. The concentration of TAT-HIBDAP with the most prominent inhibiting effects was determined and selected for subsequent experiments. The pyroptosis morphology of the control group, the OGD group and the HIBDAP group (5 μmol/L TAT-HIBDAP+OGD) was observed using transmission electron microscope. The mRNA and protein expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes were examined using real-time quantitative PCR and Western Blot analysis.Results:Fluorescence microscope showed FITC-labeled TAT-HIBDAP could successfully enter microglia cells. Compared with the OGD group, low concentrations of TAT-HIBDAP (1, 5, 10 μmol/L) could significantly reduce microglia pyroptosis and the concentration of 5 μmol/L showed the most prominent effects. Compared with the control group, OGD group showed typical pyroptosis morphology and HIBDAP group showed significantly improved morphology. The mRNA and protein expression of NLRP3 inflammasomes in the OGD group were significantly higher than the control group and also the HIBDAP group.Conclusions:The novel brain-derived peptide HIBDAP may reduce the expression of NLRP3 inflammasomes and inhibit the pyroptosis of OGD microglia.

Objective: To obtain chimeric antigen receptor macrophages ( CAR-M) targeting HER2 stably transfected． Methods : CAR lentivirus vector targeting HER2 was constructed and infected with human monocytic leukemia cell line (THP-1) ．CAR THP-1 cells with green fluorescent labeling were selected by sorting flow cytometry and continued to be cultured in vitro．The CAR THP-1 cells targeting HER2 were co-cultured with the endometrial cancer cell line Ishikawa with negative and positive HER2 expression，and their targeted phagocytosis of CAR-M to HER2 positive tumor cells was detected by imaging flow cytometry ，and the targeted phagocytosis efficiency of CAR-M to HER2 positive tumor cells was detected by flow cytometry. Results : CAR lentivirus infection with THP- 1 cells was less efficient ; After co-culture with cancer cells，flow cytometry and imaging flow cytometry showed that CAR THP-1 cells had enhanced phagocytosis of HER2 positive Ishikawa cells compared with the empty body group (P＜0. 01) ． Conclusion In this experiment，CAR THP-1 cell line targeting HER2 was established by constructing CAR lentivirus vector and transfecting THP-1 cells ，and it was proved that CAR THP-1 could phagocytize HER2 positive Ishikawa cells through specific targeting.

Objective: To establish a hypertension risk assessment model among the middle-aged and elderly populations based on residents' electronic healthcare records of the basic public health service program, so as to provide insights into prevention of hypertension. Methods: Demographic features and physical examinations were collected among residents at ages of 40 years and older from residents' electronic healthcare records of the basic public health service program in a county of Zhejiang Province from 2019 to 2020. The risk factors of hypertension were identified using a multivariable logistic regression model, and the odds ratio (OR) for each risk factor was transformed into approximate relative risk (RR), which was included in the formula for calculation of the disease risk proposed by Harvard School of Public Health to create a hypertension risk assessment model. The predictive value of the model was evaluated using a receiver operator characteristic (ROC) curve. Results: Totally 7 275 subjects were enrolled, with a mean age of (66.15±7.91) years, and the participants included 3 189 males and 4 086 females, with a male-to-female ratio of 0.78∶1. There were 190 cases with new-onset hypertension (2.61%). Multivariable logistic regression analysis revealed that overweight, obesity, central obesity, borderline high triacylglycerol (TG), elevated TG, abnormal fasting plasma glucose (FPG), prehypertension and family history of hypertension were included in the hypertension risk assessment model, with approximate RR values of 1.66, 1.96, 1.54, 1.17, 1.64, 1.45, 1.69 and 1.11. The area under the ROC curve (AUC) of the model was 0.678 (95%CI: 0.641-0.715, P<0.001), and the optimal positive cut-off was 0.899. The model predicted 139 subjects with RR>0.899 for hypertension, with a sensitivity of 73.16% and specificity of 55.79%. Conclusions The hypertension risk assessment model created in this study is feasible to predict the RR for developing hypertension among the middle-aged and elderly populations, which has a predictive value in healthcare management.

Objective : The live cell application was used to observe the process of phagocytosis of endometrial cancer cells by macrophages, and flow cytometry was used to detect the effects of macrophages engulfing tumor cells on activation of cytotoxic T cell. Methods : Ishikawa cells and THP1-induced macrophages were labeled with CFSE fluorescent probe and CD11 b respectively, and then mixed and seeded on a glass imaging dish.The live cell application was performed to record the phagocytosis of Ishikawa cells by macrophages within 120 minutes.Flow cytometry was used to detect the effect of macrophages engulfing tumor cells on activation of cytotoxic T cell. Results : The green fluorescence of Ishikawa cells was taken up by macrophages after the co-cultured two types of cells were in contact with each other, and macrophages were able to engulf multiple Ishikawa cells continuously.Macrophages that engulfed Ishikawa cells could induce activation of cytotoxic T cell. Conclusion The live cell application was successfully conducted to construct an in vitro model of phagocytosis of tumor cells by macrophages, which provided a feasible experimental method for detecting the dual killing process of macrophages and T cells on tumor cells.

Objective: To establish a method for isolation and culture of primary endothelial cells from non-human primate coronary arteries, and to provide a cell model for the study of human coronary endothelial cells. Methods: The coronary arteries of macaca mulattas were separated aseptically. The primary endothelial cells were separatedviatissue adhesion after collagenase digestion. CD31 positive cells were detected and sorted by flow cytometry to determine the purity of endothelial cells. After stimulation with prostaglandin E2(PGE2), the cellular viability and proliferation ability of primary coronary endothelial cells from macaca mulattas were evaluated by high-content cell imaging and CCK-8 assay, and the migration ability and tube function of primary coronary endothelial cells from macaca mulattas were measured by Transwell method and Matrigel glue method, respectively. Results: The confluence percentage of primary coronary artery cells of macaca mulattas was about 80% after 10-14 daysin vitroculture, and the cellular morphology was irregular polygons and paver shape. The purity of endothelial cells was about 31.7% by flow cytometry. After sorting, the purity of endothelial cells was confirmed by flow cytometry, which was more than 95%. PGE2could significantly up-regulate the proliferation, migration and tube formation abilities of primary coronary endothelial cells of macaca mulattas. Conclusion This study successfully established the isolation and culture method of primary coronary endothelial cells from macaca mulattas, and proved that it could be used as anin vitrocell model to simulate human coronary endothelial cells through functional studies.

Objective: To compare the efficiency of different methods for extracting rhesus monkey lung fibroblasts and their effects on functions, so as to provide a method for obtaining primary lung fibroblasts that are closer to human fibroblasts. Methods: Two extraction methods for rhesus monkey lung fibroblasts were used, direct tissue block adhesion method and collagenase combined digestion with tissue block adhesion method. The cell morphology was observed with the inverted microscope, the purity of isolated rhesus monkey lung fibroblasts was identified by immunofluorescence, cell viability was detected by CCK-8, the expression of α-SMA was detected by flow cytometry and the effect of long-term in vitro culture on cell apoptosis was detected by apoptosis kit. Western blot was used to detect the expression of α-SMA protein. Results: The combined digestion with collagenase and tissue block adhesion method could see small and bright cells crawling out in 24 hours, and cells could be seen crawling out in a large area after 48 hours. The cells were in a long spindle shape, after 4 days to 5 days, a single layer of cells could be formed. Identified by immunofluorescence, all cells expressed α-SMA. Tissue adhesion method showed small and bright cells crawling out after 72 hours. After 4 days to 5 days, the cells crawled out in a small area and showed a long spindle shape. After a week, the cells crawled out in a large area and formed a single layer of cells and the cells are all expressed α-SMA by immunofluorescence. The experimental results showed that the cell viability of the cells crawled out by the collagenase digestion method was significantly higher than that of the tissue adhesion method. After TGF-β1 stimulates the cells, the cells extracted by collagenase digestion method proliferated faster and expressed α-SMA more obviously. Conclusion Both methods can isolate rhesus monkey lung fibroblasts in vitro, but the collagenase digestion method extracts cells in a shorter time and in better condition. The expression of related proteins is more stable after stimulation by stimulants, which is an effective method to obtain rhesus monkey lung fibroblasts, and it is also an effective method to obtain primary lung fibroblasts that are closer to human.

Communication with the family members of donors is an integral part of the organ donation and transplantation, and the core of it lies in building trust through interpersonal communication. Every word and deed from the communicator will directly affect the overall impression of family members of potential donors towards organ donation. Regardless of whether or not granted the donation ultimately, family members may share their personal experiences and feelings with friends and relatives around them, which develops a secondary dissemination. Therefore, "the choice of best candidate for communication with family members of organ donation" has been an issue that organ donation practitioners have been working on in clinical practice. Taking into consideration of the experiences from different countries or regions, various advices and practices on this issue have been proposed due to differences in social systems, cultural background, organizational structure, clinical practice, etc. In this paper, we had a discussion on this topic, summarize the difficulties currently encountered in communication with family members and propose corresponding strategies.

Objective:To investigate the reliability and validity of Stanford attendance scale (sps-6) in the study of attendance among professional groups.Methods:In August, 2018, the 1455 employees from 81 workplaces in Beijing, Shanghai, Jiangsu and Guangdong were randomly investigated as the subjects. The reliability and validity of sps-6 were analyzed by using the internal consistency reliability (Cronbach's coefficient) , half split half coefficient, content validity, integration validity, discrimination validity, cluster analysis and structural validity analysis.Results:Cronbach's coefficients of sps-6 scale, working process and work results were 0.692, 0.918 and 0.907, respectively; Guttman of scales and dimensions The split half coefficients were 0.792, 0.803 and 0.794, respectively; Pearson correlation coefficients of the total score of each item and scale were 0.526-0.673 ( P<0.01) ; the qualification rate of set validity and differentiation validity were 100%; the results of cluster analysis supported the theoretical basis for the formation of the scale. The general non-standard fitting index (TLI) =0.982, approximate error mean square root mean square (RMSEA) =0.071, comparative fit index (CFI) =0.990, fit goodness index (GFI) =0.987, modified fit goodness index (AGFI) =0.965, Norm fit index (NFI) =0.990. The results showed that the scale had higher structural validity, and the results of sps-6 in the occupational population were (21.36±4.04) , and the distribution was normal (deviation was 0.053, peak was 0.023) . The scores of sps-6 scale were statistically different in various charactoristics of gender, age, education level, marital status, annual income, position, position level and industry ( P< 0.01) . Conclusion:Stanford attendance scale has high reliability and validity, and can be applied to the study of attendance in professional groups.

Objective:To investigate the reliability and validity of Stanford attendance scale (sps-6) in the study of attendance among professional groups.Methods:In August, 2018, the 1455 employees from 81 workplaces in Beijing, Shanghai, Jiangsu and Guangdong were randomly investigated as the subjects. The reliability and validity of sps-6 were analyzed by using the internal consistency reliability (Cronbach's coefficient) , half split half coefficient, content validity, integration validity, discrimination validity, cluster analysis and structural validity analysis.Results:Cronbach's coefficients of sps-6 scale, working process and work results were 0.692, 0.918 and 0.907, respectively; Guttman of scales and dimensions The split half coefficients were 0.792, 0.803 and 0.794, respectively; Pearson correlation coefficients of the total score of each item and scale were 0.526-0.673 ( P<0.01) ; the qualification rate of set validity and differentiation validity were 100%; the results of cluster analysis supported the theoretical basis for the formation of the scale. The general non-standard fitting index (TLI) =0.982, approximate error mean square root mean square (RMSEA) =0.071, comparative fit index (CFI) =0.990, fit goodness index (GFI) =0.987, modified fit goodness index (AGFI) =0.965, Norm fit index (NFI) =0.990. The results showed that the scale had higher structural validity, and the results of sps-6 in the occupational population were (21.36±4.04) , and the distribution was normal (deviation was 0.053, peak was 0.023) . The scores of sps-6 scale were statistically different in various charactoristics of gender, age, education level, marital status, annual income, position, position level and industry ( P< 0.01) . Conclusion:Stanford attendance scale has high reliability and validity, and can be applied to the study of attendance in professional groups.