Expression of PD-1 shRNA enhances the killing ability of CD19-targeting CAR-T cells on tumor cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：设计和构建表达PD-1 shRNA的靶向CD19 CAR-T细胞并验证其体外肿瘤细胞杀伤能力。方法：设计并构建表达PD-1 shRNA的CD19 CAR分子基因，将其包装成逆转录病毒载体，通过qPCR法检测病毒载体拷贝数。将慢病毒转导人原代T细胞，获得三种CAR-T细胞，分别为RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞。采用qPCR法检测三种CAR-T细胞中PD-1 mRNA的表达水平，流式细胞术检测三种CAR-T细胞中PD-1表达水平，萤光素酶报告基因实验、流式细胞术检测在不同效靶比时CAR-T细胞对CD19阳性靶细胞（人淋巴瘤daudi细胞）的杀伤功能。结果：RNAU6-CD19 CAR、PD-1 shRNA1-CD19 CAR、PD-1 shRNA2-CD19 CAR三种CAR分子成功包装成逆转录病毒载体，病毒载体拷贝数均高于1×107拷贝/mL，转导人原代T细胞获得CAR-T细胞，RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞转导效率分别为43.1%、55.1%、41.7%。与RNAU6-CD19 CAR-T细胞相比，PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞中PD-1 mRNA表达水平均显著降低（均P<0.01）、细胞表面PD-1表达水平更低（均P<0.01）、体外对daudi细胞的杀伤率更高（P<0.05或P<0.01）。结论：成功构建表达PD-1 shRNA的靶向CD19 CAR-T细胞，其对CD19阳性靶细胞的杀伤率显著提高，PD-1 mRNA及其翻译产物PD-1的表达减少，CAR-T细胞的耗竭减缓。

Construction of anti-CD38 CAR-T cells with targeted inhibition of CD38 by shRNA and preliminary identification of its functions / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨shRNA靶向抑制CD38的方法能否增强抗CD38 CAR-T细胞的抗癌功能。方法：构建shRNA靶向抑制CD38的抗CD38 CAR-T细胞的CAR分子，利用逆转录病毒载体包装成功后转导人原代T细胞，制备CAR-T细胞。实验分为shRNA1 CD38 CAR-T组、shRNA2 CD38 CAR-T组和对照组（shR-NC-CD38 CAR-T细胞）。采用qPCR法检测CAR-T细胞CD38 mRNA相对表达水平，计算CAR-T细胞培养0～14 d的增殖倍数，CFSE法检测CAR-T细胞与人Burkitt淋巴瘤细胞Raji-luc或人多发性骨髓瘤外周血B淋巴细胞RPMI-8226-luc共培养时的增殖情况，荧光素酶化学发光法检测CAR-T细胞在不同效靶比（1∶1、1∶2、1∶4、1∶8）时对Raji-luc和RPMI-8226-luc细胞的杀伤效率，ELISA法检测CAR-T细胞杀伤Raji-luc或RPMI-8226-luc细胞时上清液中IFN-γ水平，FCM检测CAR-T细胞表面耗竭T细胞生物标志物PD-1的表达水平。结果：shR-NC-CD38 CAR、shRNA1-CD38 CAR和shRNA2-CD38 CAR逆转录病毒载体的滴度均为1´107拷贝/mL，转导T细胞后，shR-NC-CD38 CAR-T、shRNA1-CD38 CAR-T和shRNA2-CD38 CAR-T细胞的转导效率（CAR的阳性率）分别为60.3%、67.0%和57.4%。与对照组比较，shRNA2-CD38 CAR-T组细胞中CD38 mRNA的表达水平显著降低（P<0.01），显示shRNA-CD38 CAR-T细胞构建成功。shRNA2-CD38 CAR-T组细胞在体外培养增殖能力更强（P<0.05），对2种CD38阳性的肿瘤细胞的杀伤效率更高（均P<0.05）、IFN-γ释放水平更高（均P<0.05）、细胞表面PD-1的表达水平更低（P<0.05）。结论：成功构建一种shRNA靶向抑制CD38的抗CD38 CAR-T细胞，其抗癌功能表现出明显的优势。

Relationship between body mass index, waist circumference and waist circumference height ratio and bone metabolism markers in Yinchuan middle school students / 中国学校卫生

Objective@#To analyze the relationship between body mass index (BMI), waist circumference (WC), waist to height ratio (WHtR) and bone metabolism markers.@*Methods@#By using the method of cluster sampling, a total of 1 084 adolescents aged 12 to 18 from one middle school in Yinchuan were investigated by questionnaire, physical examination and laboratory examination during sept. to Dec., 2018.@*Results@#BMI, WC and WHtR were significantly negatively correlated with Ca (r=-0.10, -0.15, -0.15, P<0.05), and WC was negatively correlated with β-CTX(r=-0.06 P<0.05). After adjusting for age, sex and vitamin D supplementation, WC had significant effects on Ca and β-CTX(β=-0.33, -0.22, P<0.05). Logistic regression analysis showed that there was no significant relationship between different types of obesity and the level of OC(P>0.05). The level of serum β-CTX in obese students was 1.93 times higher than that in normal students, Ca serum levels of obese students was 0.31 times higher than that in normal students that in abdominal obese students (defined as WC) was 0.54 times higher than that in normal students.@*Conclusion@#BMI, WC and WHtR were correlated with OC, β-CTX and Ca in adolescents in Yinchuan city, and WC was more closely related to Ca and β-CTX, suggesting that obesity has a negative effect on bone absorption.

Association of cardiovascular disease risk factors with fat mass percentage and waist circumference in children and adolescents in Yinchuan City / 中国学校卫生

Objective@#To analyze the association between fat mass percentage (FMP) and waist circumference (WC) with cardiovascular risk factors in children and adolescents in Yinchuan City, and to provide a reference for preventing and contyolling the risk factors of cardiovascular diseases among children and adolescents.@*Methods@#A questionnaire survey, physical examination, body composition determination and biochemical tests were conducted among 1 043 children aged 12-18 years in Yinchuan selected through a convenient cluster sampling method.@*Results@#Among boys and girls, both FMP and WC were associated with high rate of high blood pressure and high TG in children and adolescents(P<0.01). The increased risk of metabolic abnormalities by FMP and WC were 1.14-2.36 times and 1.32-2.09 times, respectively. About 4.9%-22.1% of cardiovascular disease risk was explained by the combination of FMP and WC, which was significantly higher than separate interpretation of FMP or WC (3.5%-22.0%). Standardized regression coefficient for TC and LDL-C (0.25 and 0.22) was greater than WC (0.17 and 0.14) by FMP, and the absolute value of the standardized regression coefficient for SBP, DBP, TG, HDL-C was less than WC by FMP. The predictive risk for high blood pressure, high TG, high TC, low HDL-C and high LDL-C by combination of FMP and WC was 3.67(95%CI=2.42-556), 3.09(95%CI=2.04-4.69), 3.37(95%CI=1.38-8.28), 2.00(95%CI=1.35-2.98) and 4.73(95%CI=2.15-10.44) times higher than different combinations risk groups(P<0.01).@*Conclusion@#The ability of fat mass percentage and waist circumference to predict the risk of cardiovascular risk factors is similar. It might be used as a predictor of cardiovascular risk factors in children and adolescents. It is recommended to combine FMP with WC to predict the risk of cardiovascular disease among children and adolescents.