中文摘要：

目的 研究含SH2结构域的肌醇磷酸酶（SHIP）基因碳末端PxxP结构域点突变对细胞体外迁移及侵袭能力的影响及其机制.方法 采用基因转染技术将携带野生型（wt）和突变型（mu）SHIP基因的慢病毒载体感染人白血病细胞系K562细胞；采用实时荧光定量PCR（FQ-PCR）和Western blot法分别在mRNA和蛋白水平检测转染后各组SHIP基因表达水平；采用Transwell小室比较K562/wtSHIP、K562/muSHIP转染后K562细胞跨膜及侵袭能力有无差别；Westem blot法比较各组细胞基质金属蛋白酶（ MMP）-2、MMP-9和磷酸化灶性黏着斑激酶（p-FAK）的表达情况,并检测各组细胞NF-KB活性改变.结果 感染慢病毒48 h后FQ-PCR和Westemblot法检测结果显示SHIP基因在K562细胞中表达明显升高；K562/wtSHIP组细胞迁移指数为（15.8±1.4）％,明显低于K562/muSHIP组[（54.3±2.4）％]和转染空载体的细胞对照组[K562/猫免疫缺陷病（pFIV）组,（50.3±3.8）％]（P＜0.01）；K562/muSHIP组与K562/pFIV组差异无统计学意义（P＞0.05）；侵袭实验显示K562/wtSHIP组迁移到碳酸脂膜的细胞[（32±6）/视野]较K562/pFIV细胞[（78±13）/视野]和K562/muSHIP细胞[（83±16）/视野]明显减低.而K562/pFIV细胞与K562/muSHIP细胞比较,在侵袭能力上差异无统计学意义（P＞0.05）.K562/muSHIP细胞p-FAK和NF-KB表达明显高于K562/wtSHIP细胞.结论 SHIP基因碳末端PxxP结构域对于SHIP基因负调节功能有重要作用.此位点发生突变通过影响FAK蛋白磷酸化、NF-KB活化以及MMP-9的表达影响K562细胞的体外迁移和侵袭能力；SH IP基因碳末端PxxP结构域点突变可能为致病性突变,可能是白血病细胞中SHIP功能异常的原因之一.

英文摘要：

Objective To explore the effect of mutation in PxxP domain of SHIP on migration and invasion of leukemia cells and its mechanism. Methods The lentiviral vector mediated wild type SHIP （ wtSHIP） and mutant SHIP（muSHIP） plasmids were transfected into K562 cells through gene transfection techniques. Expression of SHIP at mRNA and protein level was detected by real-time PCR and Western blot, respectively. Transwell assay was used to analyze the difference between the migration and invasion ability of the K562/wtSHIP and the K562/muSHIP cells after transfection. Primary migration associated factor FAK, MMP and NF-κB were assayed by Western blot. Results After transfection, the SHIP expression in transfected K562 cells were significantly increased. Compared with the migration ability of K562/wtSHIP [ （15.8± 1.4）% ], that of K562/muSHIP cells [ （54.3 ±2.4）% ] increased greatly and almost at the same level of that of K562/pFIV [ （ 50. 3 ±3. 8 ） % ] （ P 〈 0. 01 ）. The invasion assay also showed that K562/wtSHIP [ （32±6）/HPI has a lower invasion ability than that of the K562/muSHIP group [（83±16）/HP] and K562/pFIV group [ （78± 13）/HP] （P 〈0.01 ）. Western blot analysis showed that the expression of p-FAK and NF-κB was up-regulated in K562/muSHIP group compared to that of the K562/wtSHIP group. Conclusions The results confirmed that mutation in PxxP domain of SHIP gene played an important role in negative regulating function of SHIP gene. The mutation affects the cell migration and invasion ability through increase in MMP-9 expression, FAK phosphorylation and NF-κB activation. It suggested that the mutation of PxxP domain in SHIP gene might be pathogenic, and be one of the reasons for SHIP abnormality in leukemia.