摘要
目的 探讨溶质载体8号家族中的2号成员基因（SLC8A2）对胶质瘤细胞U87的迁移侵袭能力和裸鼠移植瘤生长的影响及其机制.方法 构建稳定表达SLC8A2基因的U87-SLC8A2细胞株（实验组）和稳定表达GFP的U87-NC细胞株（阴性对照组）.Transwell小室及预铺50μl Matrigel基质胶的Transwell小室检测SLC8A2对U87细胞侵袭和迁移能力的影响,逆转录-聚合酶链反应（RT-PCR）检测转染该基因后肿瘤细胞中基质金属蛋白酶（MMP）-2、MMP-3、MMP-7、MMP-9及MT1-MMP转录水平的变化,明胶酶谱法检测分泌至细胞培养基中MMP-2酶活性的变化；裸鼠皮下移植瘤实验（5只/组）检测SLC8A2对U87细胞致瘤性的影响.结果 U87-NC和U87-SLC8A2穿过Transwell小室基膜的细胞数分别为（279.70±17.50）个及（4.30±0.58）个,而穿过预铺Matrigel基质胶的Transwell小室基膜的细胞数分别为（274.0±21.3）个和（12.7±1.2）个,差异有统计学意义（P＜0.01）；与U87-NC比较,U87-SLC8A2细胞中MMP-2、MMP-3、MMP-7、MMP-9、MTl-MMP的转录水平分别下降了（55.4±3.2）％、（78.7±5.3）％、（79.7±4.6）％、（53.9±3.2）％及（70.9±4.4）％（P＜0.01）,且U87-SLC8A2细胞分泌的MMP-2酶活性较U87-NC下降了（93.2±5.1）％（P＜0.01）；接种U87-SLC8A2细胞的裸鼠全都不致瘤,而接种对照组细胞的裸鼠全部致瘤成功.结论 SLC8A2可下调MMP的活性而抑制胶质瘤细胞U87的侵袭迁移能力,并能显著抑制U87细胞在裸鼠体内的生长及增殖.
Objective To explore the effect of solute carrier family 8,member 2 （SLC8A2） on invasion,migration and in vivo xenograft growth of glioma cell line U87,and the potential mechanisms.Methods The lentivirus plasmid containing SLC8A2 （Lenti-SIC8A2-IRES-EGFP） and negative control （Lenti-EGFP） were transfected into U87 cells.The cells stably transfected with SLC8A2 and negative control called U87-SLC8A2 and U87-NC respectively were constructed.Real-time quantitative polymerase chain reaction （Real-time PCR） and western blot were carried out to detect the expression of SLC8A2 in transfected cells.The cell invasion and migration ability were detected by Transwell assay （50 μl Matrigel matrix per well were used in invasion assay）.The mRNA expression levels of matrix metalloproteinase （MMP）-2,MMP-3,MMP-7,MMP-9 and MT1-MMP were detected by reverse transcriptase-PCR （RT-PCR）.The enzymatic activity of MMP-2 secreted into culture medium was determined by Gelatin Zymography assay.The nude mouse xenograft model （five mice per group） was used to examine the effect of SLC8A2 on the growth of glioma cell line U87 in vivo.Results Compared to the control cells,the ability of invasion and migration were markedly inhibited in the cells transfected SLC8A2.The cell number of U87-NC and U87-SLC8A2 moving through the upper chambers of the transwell inserts were （279.7 ±17.5） and （4.3 ± 0.58） respectively in invasion assay （P ＜ 0.01）,and that were （274 ± 21.3） and （12.7 ± 1.2） respectively in migration assay （P ＜ 0.01）.The mRNA levels of MMP-2,MMP-3,MMP-7,MMP-9 and MT1-MMP,as well as the enzymatic activity of MMP-2 were downregulated by （55.4 ±3.2）％,（78.7±5.3）％,（79.7±4.6）％,（53.9±3.2）％,（70.9±4.4）％ and （93.2±5.1）％ in U87-SLC8A2 cells compared with U87-NC cells （P ＜ 0.01）.No mouse developed tumor in U87-SLC8A2 cell group,while all five mice developed tumors in U87-NC and U87 group.Conclusion The invasion,migration and in vivo xenogr
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第9期1796-1799,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81072058）
国家教育部回国基金资助项目（K512300412）
江苏省自然科学基金资助项目（BK2010230）
