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Objective@#To explore the current status related to behavioral problems among children and adolescents aged 12-16 years old in Guizhou Province, China, and to provide a reference for timely prevention and intervention measures.@*Methods@#To capture different economic development levels, a multistage stratified cluster sampling method was used. A total of 3 217 children and adolescents aged 12-16 years old from six middle schools and six primary schools in Guiyang, Duyun, Kaili City from Guizhou Province participated in this study. The Achenbach Child Behavior Checklist (CBCL) and a self designed questionnaire were administered between August and December 2021.@*Results@#The overall detection rate of behavioral problems among the students was 16.60% , and the rate was 14.41% and 18.62% among male and female students, respectively. The results of the Logistic regression analysis showed that good peer relationships ( OR=0.25, 0.16, P <0.05) were associated with a lower risk of behavioral problems in children and adolescents. However, the following factors were associated with an elevated risk of behavioral problems: nonparents as the main caregivers( OR = 1.44 ); children s daily screen time was 1-2 h ( OR =1.47) or more than 2 h( OR =2.75); children who had no sports hobbies( OR = 1.36); father parenting for doting or neglect( OR =1.85, 2.01); maternal parenting for doting( OR = 2.12 ); father had bad life habits( OR =1.69); father and mother were occasionally sick( OR =1.30, 1.38)( P <0.05).@*Conclusion@#Behavioral problems among children and adolescents in Guizhou Province is related to children s unhealthy living habits and various factors in the family environment. It is suggested that families, schools and relevant functional departments give full play to their own advantages and take comprehensive measures to prevent the occurrence of behavioral problems among children and adolescents.
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@#Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.
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Objective@#To analyze the prevalence and associated factors of suicide ideation among adolescent students in Guizhou Province, and to provide scientific evidence for developing strategy. @*Methods@#Multistage stratified cluster sampling method was used to recruit 4 903 adolescents from 14 middle schools in Guizhou Province. China Youth Health Risk Behaviors Questionnaire was used and data were analyzed by using univariate analysis and Logistic regression analysis. @*Results@#Totally 14.3%(703/4 903) middle school students had suicide ideation during the past 12month period, in which 13.2% were boys and 15.4% were girls, more girls were prone to suicide than boys(χ2=5.20, P<0.05); 13.7% junior students, 15.0% senior students, 14.5% Han ethnic students and 14.1% minority students suicide ideation, and the differences between grades and ethnic groups were of no statistical significance (P>0.05). The multivariates Logistic regression analysis showed that risk factors for suicidal ideation included being girl(OR=1.49),higher maternal education(college and above: OR=2.26), suffering psychological bullying over the past 30 days(OR=1.30), taking part in fighting for the past 12 months (OR=1.33), often feeling lonely(OR=1.86), sleeplessness(OR=1.38), deliberate selfinjury(OR=3.58),decreases of daily activities due to depression(OR=1.87), ideation of running away(OR=3.31), internet addiction(OR=1.37), walk violating regulations(OR=2.42), but occasional low mood from academic pressure(OR=0.73) was a protective factor for suicidal ideation(P<0.05).@*Conclusion@#Suicide ideation of middle school students is closely related to campus violence, adverse inner metal experience and unhealthy behavior pattern. All sectors of society should attach great importance and targeted measures to prevent suicide and protect the students.
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@#[Abstract] Objective:To prepare the third generation CAR-T cells targeting EGFRvⅢ (EGFRvⅢCAR-T) and to detect its specific killing effect against EGFRvⅢ+ U87 cells in vitro and in vivo. Methods: Human CD3+ T cells were transfected with lentiviral EGFRv Ⅲ/3CAR, which was generated by calcium phosphate co-precipitation of three plasmids. The expression of EGFRvⅢ/3CAR in T cells was detected by Western blotting and flow cytometry. In vitro killing effect of EGFRvⅢ/3CAR-T cells on EGFRvⅢ+ U87 cells was detected by 51Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA. Nude mouse xenograft model was constructed to detect the in vivo cytotoxicity of EGFRvⅢ/3CAR-T cells on xenograft tumor. Results: The EGFRvⅢ/3CAR lentivirus was successfully packaged with an average titer of 5×106 TU/ml. Western blotting showed that a protein band of approximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells. Flow cytometry indicated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3%. 51Cr release assay showed that the specific killing effect of EGFRvⅢ/ 3CAR-T cells was positively correlated with E/T ratio (E∶T=4∶1, 8∶1, 16∶1, 32∶1). ELISA showed that cytokine IFN-γ secretion was (1 836±148.2) pg/ml, which was significantly different from that of NTT and GFP+ T cells (P<0.01). The specific killing activity of EGFRvⅢ/3CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tumor growth monitoring results showed that the tumor volume of EGFRvⅢ/3CAR-T cell group was significantly different from that of GFP+ T cell group and PBS group around 3 weeks after injection (P<0.01). Conclusion: EGFRvⅢ/3CAR-T cells demonstrated specific antitumor effectagainstEGFRvⅢ+U87cellsbothinvitro and in vivo, providing basis for immunotherapyofgliomainfuture clinical use.
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@#[Abstract] Objective： ： To explore the impact of γ-chain (γc) family cytokines (IL-2, IL-7, IL-15, IL-21) on T cell phenotypes in ex vivo culture to provide experimental evidence for ex vivo cell preparation in adoptive immunotherapy. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers; nylon column sorting, CD3+ magnetic beads sorting, CD3- magnetic beads sorting and natural sedimentation were used to sort T cells from PBMCs. The purity, recovery rate and viability of T cells sorted by the above methods were compared. The CD3/CD28 magnetic beads-activated CD3+T cells were cultured inAIMV medium with IL-2 or mixed cytokines (IL-7, IL-15, IL-21). The expansion fold and phenotypes of T cells in ex vivo culture were detected by flow cytometry. Results： ： The purity of T cells sorted by CD3- magnetic beads sorting was significantly higher than that sorted by nylon column, CD3+ magnetic beads sorting and natural sedimentation ([94.06±1.07]% vs [86.74±1.06]%, [89.61±1.40]%, [88.48 ± 1.86]%, P<0.05); The recovery rate of T cells sorted by natural sedimentation was significantly higher than that by other three methods ([60.29±1.53]% vs [45.03±2.79]%, [20.15±3.41]%, [42.98±2.82]%, P<0.05). Comprehensively, the natural sedimentation method is the best option. The ex vivo expansion fold of T cells in IL-2 group was significantly higher than that in mixed group ([262.6±143.2] times vs [73.0±25.8] times, P<0.05). The proportions of early memory T cells, Tscm+Tscm-like and Tcmin the mixed group were significantly higher than those in the IL-2 group ([55.6±1.82]% vs [39.6±1.52]%, [16.6±1.82]% vs [9.8±1.30]%, [39.0±1.58]% vs [29.2±1.79]%; all P < 0.05). Conclusion： ： Natural sedimentation sorting has advantages of low cost, high recovery and purity. Mixed cytokines of IL-7, IL-15 and IL-21 are beneficial for production of early memory T cells. This study provides an experimental data of ex vivo T cell preparation for cancer adoptive immunotherapy.