目的探究皮层肌动蛋白结合蛋白(Cortactin)对异丙肾上腺素(isoprenaline,ISO)诱导的病理性心肌肥大的调控作用及其机制。方法采用ISO刺激新生大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs)24 h,在细胞水平建立心肌肥大模型;C57BL/6...目的探究皮层肌动蛋白结合蛋白(Cortactin)对异丙肾上腺素(isoprenaline,ISO)诱导的病理性心肌肥大的调控作用及其机制。方法采用ISO刺激新生大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs)24 h,在细胞水平建立心肌肥大模型;C57BL/6小鼠皮下注射ISO 1周,在动物水平建立心肌肥大模型。采用RT-qPCR检测mRNA的变化;免疫印迹法检测相应蛋白含量的变化;免疫荧光法检测Cortactin的亚细胞定位及表达量的变化;采用腺病毒感染的方法过表达Cortactin,通过转染小干扰RNA敲低Cortactin。结果在细胞和动物水平上,成功建立ISO诱导的心肌肥大模型,均观察到ISO引起Cortactin和N型钙黏连蛋白(N-cadherin)水平降低;过表达Cortactin可逆转ISO导致的N-cadherin蛋白水平的降低及心肌细胞肥大反应;敲低Cortactin则显示相反的效应。结论Cortactin可能联合N-cadherin通过增强心肌细胞之间的连接,发挥抗心肌肥大的作用。展开更多
Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1,Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis(Bt)in the United States.Laboratory-selected Cry1Ac/Cry2Ab cross resistance a...Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1,Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis(Bt)in the United States.Laboratory-selected Cry1Ac/Cry2Ab cross resistance and fieldevolved practical dual resistance of H.zea to these two toxins have been widely reported.Whether the widespread Cry1Ac/Cy2Ab dual resistance of H.zea has resulted from the selection of one shared or two independent resistance mechanisms by pyramided Bt crops remains unclear.Cadherin is a well-confirmed receptor of Cry1Ac and a suggested receptor of Cry2Ab in at least three Lepidopteran species.To test whether cadherin may serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab,we cloned H.zea cadherin(HzCadherin)cDNA and studied its functional roles in the mode of action of Cry1Ac and Cry2Ab by gain-and lossof-function analyses.Heterologous expression of HzCadherin in H.zea midgut,H.zea fat body and Sf9 cells made all three of these cell lines more susceptible to activated Cry1Ac but not activated Cry2Ab,whereas silencing HzCadherin of H.zea midgut and fat body cells significantly reduced the susceptibility to Cry1Ac but not Cry2Ab.Likewise,suppressing HzCadherin with siRNA made H.zea larvae resistant to Cry1Ac.These results clearly demonstrate that HzCadherin is not a receptor for Cry2Ab,and thus it is unlikely to serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab.展开更多
目的了解肿瘤抗原肽的HLA限制性以及其诱导的T细胞能否杀伤肿瘤细胞,探索无关供者来源细胞用于过继性抗肿瘤T细胞治疗。方法使用16个肿瘤抗原肽诱导18名无关供者外周血单个核细胞(PBMC)分化为反应性T细胞,并分析HLA型别;利用NetMHC数据...目的了解肿瘤抗原肽的HLA限制性以及其诱导的T细胞能否杀伤肿瘤细胞,探索无关供者来源细胞用于过继性抗肿瘤T细胞治疗。方法使用16个肿瘤抗原肽诱导18名无关供者外周血单个核细胞(PBMC)分化为反应性T细胞,并分析HLA型别;利用NetMHC数据库预测肽和HLA分子亲和力;选择HLA-A2限制性的肿瘤抗原肽诱导第二组17名无关供者的PBMC进行杀瘤实验,反应性T细胞作为效应细胞,T2细胞及肿瘤抗原肽同源的肿瘤细胞作为靶细胞,测量LDH(乳酸脱氢酶)释放量或者RTCA(实时无标记细胞分析仪)检测效应细胞杀瘤效率,比较HLA-A2+和A2-T细胞杀瘤效率。结果筛出和HLA-A2具有高亲和力的肿瘤抗原肽LM7,可诱导5/11 HLA-A2+为反应性T细胞,其中HLA-A2+纯合子则为3/3,而HLA-A2-者则为2/7。LM7诱导反应性T细胞杀伤肿瘤百分比A2+组明显强于A2-组(60.72±11.28 vs 47.2±4.46,P=0.03)。结论本研究显示NetMHC预测对于纯合子样品更有帮助,肿瘤抗原肽LM7具有HLA-A2限制性,可诱导部分HLAA2+PBMC分化为反应性T细胞,可杀伤肿瘤,应对供者进行HLA筛选并分析其细胞功能,其诱导的反应性T细胞可作为过继性T细胞抗肿瘤治疗的细胞来源。展开更多
文摘目的探究皮层肌动蛋白结合蛋白(Cortactin)对异丙肾上腺素(isoprenaline,ISO)诱导的病理性心肌肥大的调控作用及其机制。方法采用ISO刺激新生大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs)24 h,在细胞水平建立心肌肥大模型;C57BL/6小鼠皮下注射ISO 1周,在动物水平建立心肌肥大模型。采用RT-qPCR检测mRNA的变化;免疫印迹法检测相应蛋白含量的变化;免疫荧光法检测Cortactin的亚细胞定位及表达量的变化;采用腺病毒感染的方法过表达Cortactin,通过转染小干扰RNA敲低Cortactin。结果在细胞和动物水平上,成功建立ISO诱导的心肌肥大模型,均观察到ISO引起Cortactin和N型钙黏连蛋白(N-cadherin)水平降低;过表达Cortactin可逆转ISO导致的N-cadherin蛋白水平的降低及心肌细胞肥大反应;敲低Cortactin则显示相反的效应。结论Cortactin可能联合N-cadherin通过增强心肌细胞之间的连接,发挥抗心肌肥大的作用。
基金funded by the USDA National Institute of Food and Agriculture (Hatch Grant ARZT-1360890-H31-164 and multi-state grant ARZ-T1370680-R31-172 (NC246))the National Natural Science Foundation of China (NSFC)–Henan Joint Major Grant (U2004206)+2 种基金the State Key Laboratory of Cotton Biology Open Fund, Zhengzhou University, China (CB2020A06)the Henan Agriculture Research System, China (HARS22-09-G3)the earmarked fund for China Agriculture Research System (CARS-27)
文摘Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1,Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis(Bt)in the United States.Laboratory-selected Cry1Ac/Cry2Ab cross resistance and fieldevolved practical dual resistance of H.zea to these two toxins have been widely reported.Whether the widespread Cry1Ac/Cy2Ab dual resistance of H.zea has resulted from the selection of one shared or two independent resistance mechanisms by pyramided Bt crops remains unclear.Cadherin is a well-confirmed receptor of Cry1Ac and a suggested receptor of Cry2Ab in at least three Lepidopteran species.To test whether cadherin may serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab,we cloned H.zea cadherin(HzCadherin)cDNA and studied its functional roles in the mode of action of Cry1Ac and Cry2Ab by gain-and lossof-function analyses.Heterologous expression of HzCadherin in H.zea midgut,H.zea fat body and Sf9 cells made all three of these cell lines more susceptible to activated Cry1Ac but not activated Cry2Ab,whereas silencing HzCadherin of H.zea midgut and fat body cells significantly reduced the susceptibility to Cry1Ac but not Cry2Ab.Likewise,suppressing HzCadherin with siRNA made H.zea larvae resistant to Cry1Ac.These results clearly demonstrate that HzCadherin is not a receptor for Cry2Ab,and thus it is unlikely to serve as one shared mechanism for the cross and dual resistance of H.zea to Cry1Ac and Cry2Ab.
文摘目的了解肿瘤抗原肽的HLA限制性以及其诱导的T细胞能否杀伤肿瘤细胞,探索无关供者来源细胞用于过继性抗肿瘤T细胞治疗。方法使用16个肿瘤抗原肽诱导18名无关供者外周血单个核细胞(PBMC)分化为反应性T细胞,并分析HLA型别;利用NetMHC数据库预测肽和HLA分子亲和力;选择HLA-A2限制性的肿瘤抗原肽诱导第二组17名无关供者的PBMC进行杀瘤实验,反应性T细胞作为效应细胞,T2细胞及肿瘤抗原肽同源的肿瘤细胞作为靶细胞,测量LDH(乳酸脱氢酶)释放量或者RTCA(实时无标记细胞分析仪)检测效应细胞杀瘤效率,比较HLA-A2+和A2-T细胞杀瘤效率。结果筛出和HLA-A2具有高亲和力的肿瘤抗原肽LM7,可诱导5/11 HLA-A2+为反应性T细胞,其中HLA-A2+纯合子则为3/3,而HLA-A2-者则为2/7。LM7诱导反应性T细胞杀伤肿瘤百分比A2+组明显强于A2-组(60.72±11.28 vs 47.2±4.46,P=0.03)。结论本研究显示NetMHC预测对于纯合子样品更有帮助,肿瘤抗原肽LM7具有HLA-A2限制性,可诱导部分HLAA2+PBMC分化为反应性T细胞,可杀伤肿瘤,应对供者进行HLA筛选并分析其细胞功能,其诱导的反应性T细胞可作为过继性T细胞抗肿瘤治疗的细胞来源。
