Notch1 siRNA对小鼠恶性黑色素瘤细胞体内外增殖的抑制作用研究

Inhibitory effect of Notch1 siRNA on proliferation and tumor growth of malignant melanoma cells in vivo and in vitro

目的探讨靶向Notch1基因小干扰RNA(Notch1 siRNA,以下简称siNotch)对小鼠恶性黑色素瘤细胞体内外增殖的抑制作用。方法将siNotch转染小鼠恶性黑色素瘤细胞株B16F1,诱导RNA干扰,采用RT-PCR、Western blot、MTT法及细胞克隆试验体外检测细胞Notch1基因和蛋白表达及细胞增殖、克隆形成变化。随后分别将转染及对照细胞皮下接种于同基因C57BL/6小鼠(分别为转染组及对照组),在成瘤过程中瘤内注射相应siNotch1,记录肿瘤生长情况并绘制生长曲线,处死动物后取肿瘤比较各组肿瘤体积,并行HE及免疫组化染色观察肿瘤组织病理学变化和Notch1蛋白表达情况。结果 siNotch1转染B16F1细胞后,细胞增殖能力、克隆形成率及小鼠体内成瘤等均受到抑制(P<0.01),免疫组化显示转染组Notch1蛋白表达阳性细胞百分比明显低于对照组(P均<0.01)。结论 siNotch1可降低细胞Notch1的表达,从而抑制小鼠MM细胞的增殖与生长,可作为治疗恶性黑色素瘤的新方法。

Objective To observe the inhibit effeet of siRNA on Notch 1 in mouse malignant melanoma cells in vivo and vitro.Method We used siRNA targeting Notch1 gene to transfect melanoma cell line B16F1and induced RNA inter-ference.RT-PCR, Western blotting, MTT method and cell cloning assay in vitro were used to detect the expression of Notch1 gene and protein expression , cell proliferation and cell clone formation changes .Then we transfected or inoculated the control cells subcutaneously into syngeneic C 57BL/6 mice, and in the intratumoral process , the corresponding siRNA was injected .The tumor growth was recorded and the growth curve was drawn .After that , we executed the animal to get tumor and compared the tumor volume .Meanwhile, HE and immunohistochemical staining were used to observe the patho-logical changes of tumor tissue and the expression of Notch 1 protein.Results The cell proliferation , clone formation rate and tumor formation in mice were inhibited after Notch1 siRNA transfecting B16F1 cells （P〈0.01）, and immunohisto-chemistry showed that Notch1 protein positive cell percentage of the siNotch 1 transfected group was lower than that of con-trol group （all P〈0.01）.Conclusions Targeting Notch1 gene siRNA can inhibit the proliferation and growth of mouse melanoma cells by decreasing the expression of Notch 1, which can be used as a new method for the treatment of malignant melanoma.