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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.