Rap1GAP基因表达上调对HL-60细胞体内及体外侵袭能力的影响

The effect of up-regulated expression of Rap1GAP on the invasion ability of HL-60 cells in vitro and in vivo

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.

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 to the control cells.The invasion rate of R1 and R2 are （55±5）% and （59±4）%,which are significantly higher than （14±4）% of the control cells.The mRNA level of MMP-9 was up-regulated about 12.0 folds in R1 and R2 cells compared to the corresponding control cells.The median survival times of R1 and R2 mice are （32.00± 1.85） d and （33.37±2.50） d,respectively,which are shorter than （43.62±2.32） d of the control group.Three mice of R1 and R2 groups showed leukemic cells infiltration in meninges tissue,and the genes of Rap1GAP and MMP-9 were amplified by PCR method.Conclusion Up-regulated expression of Rap1GAP increased the invasion ability of HL-60 cells accompanied with enhancement of MMP-9 expression in vitro,and the experiment in mouse model also confirmed that Rap1GAP enhanced the invasion of HL-60 cells in vivo.