目的:研究红景天苷对人肺癌A549细胞裸鼠移植瘤生长的作用,并研究其对丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)/细胞外信号调节激酶(extracellular signal regulated kinase,ERK1/2)信号通路的调控作用。方法:培养...目的:研究红景天苷对人肺癌A549细胞裸鼠移植瘤生长的作用,并研究其对丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)/细胞外信号调节激酶(extracellular signal regulated kinase,ERK1/2)信号通路的调控作用。方法:培养人肺癌A549细胞制备浓度为3×10^7 mL^-1的细胞悬液,取200μL皮下注射于裸鼠右前肢腋窝皮下。待注射部位出现肿瘤结节时,为建模成功。挑出肿瘤结节大小均为5 mm左右裸鼠共40只,随机分为5组:阴性对照组、阳性对照组、红景天苷高剂量组、红景天苷中剂量组和红景天苷低剂量组,每组8只。阳性对照组裸鼠腹腔注射3 mg·kg^-1顺铂,每3天注射1次;红景天苷高剂量组、中剂量组、低剂量组裸鼠分别腹腔注射红景天苷50 mg·kg^-1、25 mg·kg^-1、12.5 mg·kg^-1,每天1次;阴性对照组裸鼠注射等体积的生理盐水,每天1次。连续给药2周。取肿瘤组织称取质量,计算瘤体生长抑制率;通过苏木精伊红(HE)染色观察瘤体组织病理变化;通过末端标记法观察肿瘤细胞凋亡情况并计算凋亡指数;通过RT-qPCR检测肿瘤细胞中ERK1/2、细胞周期蛋白D1(cyclinD1)、c-Myc、Bcl-2和Bcl-2相关蛋白X(Bax)mRNA相对表达量;通过蛋白免疫印迹检测肿瘤细胞中ERK1/2、cyclinD1、c-Myc、Bcl-2和Bax蛋白表达水平及p-ERK1/2水平。结果:各组瘤质量比较显示:阳性对照组和红景天苷中剂量组比较,差异无统计学意义(P>0.05),其余各组间比较,差异均有统计学意义(P<0.05);其中组间两两比较显示:阴性对照组>红景天苷低剂量组>阳性对照组和红景天苷中剂量组>红景天苷高剂量组。各组抑瘤率比较显示:阳性对照组和红景天苷中剂量组比较,差异无统计学意义(P>0.05),其余各组间比较,差异均有统计学意义(P<0.05);组间两两比较显示:红景天苷高剂量组>阳性对照组和红景天苷中剂量组>红景天苷低剂量组>阴性对照组。HE染色结果比较显示:阴�展开更多
Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners.Unlike other members in the family,the kinase domain of HER3 lacks key amino acid re...Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners.Unlike other members in the family,the kinase domain of HER3 lacks key amino acid residues for catalytic activity.As a result,HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR,HER2 and HER4.To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways,we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain(HER3-2-3)or both the kinase domain and the C-terminal tail(HER3-2-2)with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary(CHO)cells.While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration,CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1(NRG-1).In contrast,CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1,but maintained partial activation of ERK1/2.The results demonstrate that the intracellular domains play a crucial role in HER3’s function as an allosteric activator and its role in downstream signaling.展开更多
基金partially funded by grants from the Texas Emerging Technology Fund and the Welch Foundation.
文摘Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners.Unlike other members in the family,the kinase domain of HER3 lacks key amino acid residues for catalytic activity.As a result,HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR,HER2 and HER4.To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways,we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain(HER3-2-3)or both the kinase domain and the C-terminal tail(HER3-2-2)with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary(CHO)cells.While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration,CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1(NRG-1).In contrast,CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1,but maintained partial activation of ERK1/2.The results demonstrate that the intracellular domains play a crucial role in HER3’s function as an allosteric activator and its role in downstream signaling.
