Netrin⁃1对T⁃ALL细胞中VEGFA表达的影响及相关机制研究  

Effect of Netrin⁃1 on VEGFA Expression in T⁃ALL Cells and Its Related Mechanism

作　　者：朱瑶 刘海燕[1] 相燕 杨晖[1] 姚新原 安曦洲 张凯楠 黄兰[1,2] 梁绍燕 于洁[1] ZHU Yao;LIU Hai-Yan;XIANG Yan;YANG Hui;YAO Xin-Yuan;AN Xi-Zhou;ZHANG Kai-Nan;HUANG Lan;LIANG Shao-Yan;YU Jie(Department of Hematology&Oncology,Children′s Hospital Affiliated to Chongqing Medical University,Key Laboratory of Child Development and Disorders of Ministry of Education,National Center for Clinical Medical Research on Child Health and Diseases(Chongqing),Chongqing Key Laboratory of Pediatrics;Institute of Pediatrics,Children′s Hospital affiliated to Chongqing Medical University,Chongqing 400010,China)

机构地区：[1]重庆医科大学附属儿童医院血液肿瘤科,儿童发育疾病研究教育部重点实验室,国家儿童健康与疾病临床医学研究中心,儿科学重庆市重点实验室 [2]重庆医科大学附属儿童医院儿科研究所,重庆400010

出　　处：《中国实验血液学杂志》2022年第4期1049-1055,共7页Journal of Experimental Hematology

摘　　要：目的:探讨Netrin⁃1对急性T淋巴细胞白血病(T Cell Acute Lymphoblastic Leukemia,T⁃ALL)细胞中VEGFA水平的影响及可能的作用机制。方法:采用ELISA法检测10例T⁃ALL患儿和23例非恶性血液系统疾病患儿骨髓中Netrin⁃1、VEGFA的水平并进行比较,分析疾病组和对照组患儿骨髓中Netrin⁃1与VEGFA水平的相关性。体外培养T⁃ALL细胞株Jurkat、Molt⁃4,用不同浓度的Netrin⁃1(0,25,50,100 ng/ml)处理细胞24 h后,qRT⁃PCR及Western blot检测不同浓度下各细胞中VEGFA的表达量;qRT⁃PCR检测两种细胞中Netrin⁃1受体的表达,并采用co⁃IP检测Netrin⁃1与T⁃ALL细胞中受体的结合;Western blot检测各组细胞中Akt-mTOR通路中关键蛋白的磷酸化水平。使用Akt/mTOR抑制剂与Netrin⁃1(100 ng/ml)联合处理T⁃ALL细胞后,采用Western blot检测各组细胞中VEGFA的表达量以及Akt/mTOR通路中关键分子的磷酸化水平。结果:T⁃ALL患儿骨髓中Netrin⁃1及VEGFA表达水平显著高于对照组(P<0.05),且在T⁃ALL患者中两者表达水平呈线性相关(r2=0.974)。随着Netrin⁃1浓度的升高,VEGFA的表达水平也升高(P<0.05)。当Netrin⁃1浓度为100 ng/ml时,Netrin⁃1与Integrin⁃β4受体结合。Netrin⁃1浓度为100 ng/ml处理细胞24 h后,Akt、mTOR通路关键分子的磷酸化水平均提高;当Netrin⁃1(100 ng/ml)与Akt抑制剂联合处理T⁃ALL细胞后,VEGFA的表达量下降,Akt、mTOR的磷酸化水平也下降;而与mTOR抑制剂联合处理细胞后,Akt的磷酸化水平升高,mTOR的磷酸化水平降低,VEGFA的表达量也下降。结论:T⁃ALL患儿骨髓中Netrin⁃1及VEGFA表达量存在异常且两者存在线性关系;Netrin⁃1通过Integrin⁃β4受体,激活Akt/mTOR通路诱导T⁃ALL细胞表达VEGFA。Objective:To investigate the effect of the axon guidance factor Netrin⁃1 on the expression of VEGFA in T cell acute lymphoblastic leukemia(T⁃ALL)and its related mechanism.Methods:ELISA assays were applied to detect the levels of Netrin⁃1 and VEGFA in the bone marrow(BM)samples from children in the T⁃ALL and control group.The level of Netrin⁃1 and VEGFA were compared between control children and patients,and the liner correlation between Netrin⁃1 and VEGFA was analyzed.The T⁃ALL cells Jurkat and Molt⁃4 were culture in vitro,and the cells were treated with different concentration of Netrin⁃1(0,25,50,100 ng/ml)for 24 h,quantitative RT⁃PCR(qRT⁃PCR)and Western blot were used to detect the VEGFA expression in Jurkat,Molt⁃4 cells.The expression of Netrin⁃1 receptors in T⁃ALL cells was detected by qRT⁃PCR and the interaction between Netrin⁃1 and receptor in each cells was detected by co⁃IP.Furthermore,Western blot was used to detect the phosphorylation level of key prateins of AKT signal transduction pathway including Akt and mTOR in T⁃ALL cells treated with Netrin⁃1(100 ng/ml).The expression of VEGFA and phosphorylation of AKT pathway transducers were detected by Western blot,after T⁃ALL cells treated with Netrin⁃1(100 ng/ml)combined with inhibitors specific to Akt or mTOR.Results:The expression level of Netrin⁃1 and VEGFA in T⁃ALL patients BM samples were both signi⁃ficantly higher than that of control group.And the expression level of Netrin⁃1 was positively correlated with that of VEGFA(r2=0.974).With the increase of Netrin⁃1 concentration,the expression level of VEGFA also increased(P<0.05).Netrin⁃1 interacted with its receptor,integrin⁃β4 at the Netrin⁃1 concentration of 100 ng/ml.Further,the treatment of Netrin⁃1 could increase the phosphorylation of Akt and mTOR,which were the key transducers of AKT pathway.After treatment of T⁃ALL cells with Netrin⁃1(100 ng/mL)and Akt inhibitor,the expression of VEGFA and phosphorylation of Akt or mTOR decreased.Wh

关 键 词：Netrin⁃1 血管内皮生长因子A AKT/MTOR 急性T淋巴细胞白血病 

分 类 号：R733.[医药卫生—肿瘤]