SKAP1通过调控ICAM-1对宫颈癌模型小鼠肿瘤生长、转移及免疫调节机制研究

Study on the mechanism of SKAP1 on tumor growth,metastasis and immune regulation in cervical cancer model mice by regulating ICAM-1

目的 探讨SKAP1通过调控细胞间黏附分子-1(ICAM-1)对宫颈癌模型小鼠肿瘤生长、转移及免疫调节的机制。方法 培养人宫颈癌细胞He La,构建SKAP1-mi RNA慢病毒干扰质粒和阴性对照Neg-mi RNA空白慢病毒干扰质粒,分别感染He La细胞,随机分为对照组(细胞未做处理)、空质粒组(Neg-mi RNA组)和SKAP1基因沉默组(SKAP1-mi RNA组),每组10只。将稳定表达的3组细胞配制成单细胞悬液分别接种于对应组BALB/c裸鼠右前腋窝中部外侧皮下,记录各组肿瘤生长情况,30 d后采用活体荧光成像技术检测肿瘤转移情况。处死裸鼠,取肿瘤组织,采用RealTime PCR法检测各组裸鼠肿瘤组织SKAP1、ICAM-1 m RNA相对表达量,免疫组化法检测SKAP1、ICAM-1蛋白表达情况,Western blot法检测SKAP1、ICAM蛋白相对表达量,流式细胞术检测免疫细胞CD3+、CD4+、CD8+水平,ELISA法检测Th1和Th2细胞因子水平。结果 细胞接种后饲养期间内,对照组和空质粒组裸鼠各死亡1只,SKAP1基因沉默组死亡2只。各组瘤体体积均随时间延长而增大(P<0.05);与对照组、空质粒组比较,SKAP1基因沉默组各时间段瘤体体积减小、肿瘤细胞转移面积减小、荧光信号强度降低、肿瘤组织SKAP1和ICAM-1 m RNA及其蛋白相对表达量均降低,差异有统计学意义(P<0.05);与对照组、空质粒组比较,SKAP1基因沉默组肿瘤组织免疫细胞CD3+、CD4+、CD4+/CD8+及Th1细胞因子IL-2、IFN-γ水平均升高,CD8+及Th2细胞因子IL-4、IL-10水平均降低(P<0.05)。结论 沉默SKAP1基因可以抑制He La细胞宫颈癌裸鼠移植瘤的生长及转移,逆转小鼠Th1向Th2漂移,恢复Th1表达优势,这一作用可能是通过下调ICAM-1表达实现的。

Objective To investigate the mechanism of SKAP1 on the tumor growth,metastasis and immune regulation in cervical cancer model mice by regulating the intercellular adhesion molecule-1 (ICAM-1).Methods The human cervical cancer cells He La were cultured,and the SKAP1-mi RNA lentiviral interference plasmid and negative control Neg-mi RNA blank lentiviral interference plasmid were constructed and infected with He La cells respectively.They were randomly divided into a control group(untreated cells),an empty plasmid group (Neg-mi RNA group) and a SKAP1 gene silencing group (SKAP1-mi RNA group),with 10 mice in each group.The three groups of stably expressed cells were prepared into a single cell suspension and inoculated subcutaneously in the middle and lateral sides of the right anterior axilla of the BALB/c nude mice in the corresponding groups,and the tumor growth in each group was recorded.After 30 days,the tumor metastasis was detected by in vivo fluorescence imaging technology.The nude mice were sacrificed,and the tumor tissues were collected.The relative m RNA expression levels of SKAP1 and ICAM-1 in the tumor tissues of nude mice in each group were detected by Real-Time PCR,the protein expression levels of SKAP1 and ICAM-1 were detected by immunohistochemistry,the relative protein expression levels of SKAP1 and ICAM were detected by Western blot,the levels of CD3+,CD4+,and CD8+in the immune cells were detected by flow cytometry,and the levels of Th1 and Th2 cytokines were detected by ELISA.Results The tumor volumes in the three groups were increased with time(P<0.05).Compared with the control group and the empty plasmid group,the tumor cell metastasis area,the fluorescence signal intensity,and the relative m RNA and protein expression levels of SKAP1 and ICAM-1 in the tumor tissues of the SKAP1 gene silencing group were signifi cantly decreased (P<0.05).Compared with the control group and the empty plasmid group,the levels of CD3+,CD4+,CD4+/CD8+and Th1 cytokines IL-2 and IFN-γ in the tumor tissue immune cells in the SKAP1 gene silencing group were increased,while the levels of CD8+and Th2 cytokines IL-4 and IL-10 were decreased (P<0.05).Conclusion The silencing SKAP1 gene can inhibit the growth and metastasis of He La cervical cancer xenografts in nude mice,reverse the shift of Th1 to Th2 in them,and restore the dominance of Th1 expression,which may be achieved by down-regulating the expression of ICAM-1.