骨桥蛋白RNA干扰对人U251胶质瘤细胞在裸鼠体内生长的影响

Effect of Osteopontin Silencing by Lentivirus-mediated Delivery of RNA Interference on the Growth of U251 Cells in vivo

研究下调骨桥蛋白(osteopontin,OPN)对人U251胶质瘤细胞在裸鼠体内生长的影响并探讨其对胶质瘤生长、侵袭的可能机制.应用RNA干扰技术,将OPN基因的慢病毒干扰载体LV-OPNshRNA感染U251细胞.将对照和试验组U251细胞分别接种裸鼠,建立裸鼠荷瘤模型.3周后测量肿瘤的体积、瘤重并做肿瘤组织病理切片分析;利用RT-PCR和免疫印迹法检测OPN、尿激酶型纤维蛋白酶原激活物(uPA)、基质金属蛋白酶(MMP-2、MMP-9)的mRNA和蛋白表达;免疫组化法检测肿瘤组织微血管密度和血管内皮生长因子(VEGF)表达情况.经OPN的RNA干扰后,能显著降低肿瘤组织OPN mRNA水平及蛋白表达,有效抑制肿瘤细胞生长及侵袭能力,肿瘤体积及重量的减小有统计学意义(P<0.05).感染组uPA、MMP-2和MMP-9的mRNA和蛋白表达明显减少,肿瘤组织的MVD值和VEGF的表达均显著降低.上述结果表明,抑制OPN的表达能明显抑制人U251胶质瘤细胞在裸鼠体内的生长和侵袭,OPN可能通过激活uPA、MMP-2和MMP-9等蛋白酶降解细胞外基质和促进肿瘤血管生成,参与胶质瘤的生长.

To observe the effect of osteopontin（OPN） silencing on the growth of human glioma U251 cells of nude mice in vivo and to investigate the mechanism of OPN gene on the proliferation and invasion of glioma U251 cell.LV-N-OPN/U251 cells transfected with blank lentivirus vector,LV-OPNshRNA/U251cells transfected with shRNA lentivirus vector osteopontin,and untransfected U251 cells were injected into the oxter of the nude mice,respectively to establish the xenograft.The excised tumor masses on the 21st day,the volume and the weight of tumors were checked.Histopathological changes were observed.The mRNA and protein expression of OPN,urokinase type plasminogen activator（uPA）,matrix metalloproteinase（MMP-2 and MMP-9） were measured by RT-PCR and immunoblotting.The microvessel density（MVD） and vascular endothelial growth factor（VEGF）were detected by immunohistochemistry.OPN interference RNA inhibited OPN expression,cell proliferation and invasion,both volume and weight of tumor were decreased obviously.In addition,the expression of uPA,MMP-2 and MMP-9 were significantly decreased,the expression of MVD and VEGF were also significantly lower.The results indicated that OPN might increase the abilities of invasion of glioma by degrade extracellular matrix to activate uPA,MMP-2,MMP-9 and to promote angiogenesis of tumor.