Rap1GAP基因表达上调对HL-60细胞体内及体外侵袭能力的影响

目的 探讨上调Rap1 GAP基因表达对白血病细胞株HL-60细胞体外侵袭能力的影响,并构建白血病动物模型验证体外实验的结果.方法 采用实时定量PCR及Western blot法检测已构建的Venus/HL-60细胞（空载体对照组）及Rap1 GAP过表达单克隆细胞株Rap1 GAP/HL-60 （R1、R2）细胞的Rap1GAP表达水平,Transwell方法检测空载体对照组、R1、R2细胞的体外侵袭能力,实时定量PCR检测各组细胞MMP-9 mRNA表达,并通过明胶酶谱法检测MMP-2及MMP-9活性;将4周龄BALB/c裸鼠进行预处理后接种白血病细胞,观察各组裸鼠生存时间及白血病细胞在其...

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Published in中华血液学杂志 Vol. 36; no. 7; pp. 570 - 574
Main Author 邱婷婷 李德鹏 李振宇 徐开林 祁小飞 岑建农 陈子兴
Format Journal Article
LanguageChinese
Published 徐州医学院附属医院血液科 221002%徐州医学院附属医院血液科%215006,苏州大学附属第一医院、江苏省血液研究所 2015
Subjects
HL-60细胞
基因Rap1GAP
基质金属蛋白酶类
白血病浸润
Matrix metalloproteinases
Gene,Rap1GAP
HL-60 cells
白血病浸润
基质金属蛋白酶类
Leukemic infiltration
HL-60细胞
基因,Rap1GAP
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Summary:目的 探讨上调Rap1 GAP基因表达对白血病细胞株HL-60细胞体外侵袭能力的影响,并构建白血病动物模型验证体外实验的结果.方法 采用实时定量PCR及Western blot法检测已构建的Venus/HL-60细胞（空载体对照组）及Rap1 GAP过表达单克隆细胞株Rap1 GAP/HL-60 （R1、R2）细胞的Rap1GAP表达水平,Transwell方法检测空载体对照组、R1、R2细胞的体外侵袭能力,实时定量PCR检测各组细胞MMP-9 mRNA表达,并通过明胶酶谱法检测MMP-2及MMP-9活性;将4周龄BALB/c裸鼠进行预处理后接种白血病细胞,观察各组裸鼠生存时间及白血病细胞在其脏器中的浸润情况.结果 R1、R2细胞的Rap1GAP表达水平分别为空载体对照组的16.2、17.3倍,侵袭率分别为（55±5）％、（59±4）％,显著高于空载体对照组的（14±4）％（P值均＜0.001）.R1和R2细胞的MMP-9 mRNA表达水平增加,约为空载体对照组的12.0倍.动物模型实验结果显示接种R1细胞裸鼠（R1组）生存时间为（32.00±1.85）d,R2组为（33.37±2.50）d,空载体对照组为（43.62±2.32）d,R1组和R2组裸鼠生存时间较空载体对照组缩短（P＜0.05）.R1组和R2组共有3只裸鼠出现脑膜组织浸润,脑膜组织扩增出Rap1GAP和MMP-9基因,空载体对照组裸鼠各脏器白血病细胞浸润不明显.结论 Rap1GAP增强HL-60细胞侵袭能力,同时伴随MMP-9 mRNA表达水平升高,裸鼠体内实验亦证实Rap1GAP提高了HL-60细胞体内侵袭力.
Bibliography:HL-60 cells; Gene, RaplGAP; Leukemic infiltration; Matrix metalloproteinases
Qiu Tingting, Li Depeng, Li Zhenyu, Xu Kailin, Qi Xiaofei, Cen Jiannong, Chen Zixing（ Jiangsu Institute of Hematology, Ist Affiliated Hospital, Soochow University, Suzhou 215006, China）
Objective To investigate the effect of up-regulation of Rap lGAP on the invasion ability of leukemic HL-60 cells in vitro,and to establish leukemia mouse model to verify the effects in vivo.Methods Quantitative RT-PCR and Western blot methods were used to detect the expression of Rap1GAP in Venus/HL-60 （vehicle control） and Rap 1GAP/HL-60 cells （R1 andR2）.Transwell method was used to examine the invasion ability in vitro.Quantitative RT-PCR and gelatin zymograph were used to study the expression of MMP-2 and MMP-9.Four-week-old BALB/c nu/nu mice were pre-treated and inoculated with leukemic cells from different groups,several index including survival time were then monitored.Results Rap1GAP mRNA level of R1 and R2 increased about 16-17 folds as compared
ISSN:0253-2727
DOI:10.3760/cma.j.issn.0253-2727.2015.07.009