摘要
目的利用特异性小干扰RNA(small interfering RNA,si RNA)技术沉默T细胞TIPE2基因表达,体外观察转染T细胞对肺腺癌细胞LA795的免疫杀伤作用。方法磁珠分选小鼠脾脏T细胞,筛选并验证能有效沉默T淋巴细胞TIPE2基因的si RNA序列,转染48 h后,利用酶联免疫吸附法(ELISA)比较各分组细胞上清液中IFN-γ分泌水平;转染72 h后,观察对比单纯T细胞(空白组)、阴性对照T细胞(阴性对照组)及转染T细胞(实验组)与小鼠肺腺癌细胞LA795共培养时肿瘤杀伤率。结果我们筛选出一条能有效抑制TIPE2基因表达的si RNA序列,细胞转染率79.63%,蛋白表达抑制率达到79%,转染48 h后,检测实验组、阴性对照组、空白组细胞因子IFN-γ分泌水平分别为(678.96±26.91)pg/ml、(401.69±13.67)pg/ml、(387.03±15.14)pg/ml(P<0.001);转染72 h后,与空白组及阴性对照组T细胞相比,在不同的效靶比水平(20:1、40:1、80:1),转染T细胞杀瘤活性均更高。当效靶比为80:1时,实验组肿瘤细胞杀伤率达到(47.91±3.25)%。结论利用特异性si RNA技术能有效沉默TIPE2基因,并促进抗肿瘤活性细胞因子IFN-γ分泌,增强T细胞对小鼠肺腺癌细胞LA795的体外免疫杀伤作用。
Objective To silence the expression of tumor necrosis factor-α-induced protein-8-like 2（TIPE2） gene of T-lymphocytes by specific small interfering RNA（si RNA） and investigate the cytotoxicity activity of transfected T lymphocytes against lung adenocarcinoma cells LA795 in vitro. Methods Spleen T lymphocytes of mice were separated by magnetic bead, and we screened and validated an effective si RNA to silence TIPE2 gene of T lymphocytes. After 48 h transfection, the secretion of IFN-γ was measured by enzyme-linked immuno-sorbent assay（ELISA）. At the end, 72 h after transfection, we compared the cytotoxicity activity changes against LA795 cells among transfected T lymphocytes（experimental group）, negative control and simplex T lymphocytes（blank group） in vitro. Results We screened a si RNA sequences to effectively inhibit the expression of TIPE2 gene, the transfection rate was 79.63%, and the inhibition rate for TIPE2 protein expression reached 79%. After 48 h transfection, the secretion level of IFN-γ in experimental group, negative control and blank group respectively were（678.96±26.91）,（401.69±13.67） and（387.03±15.14）pg/ml（P〈0.001）. After 72 h transfection, compared with negative control and blank group, the transfected T lymphocyte showed more efficient killing ability against LA795 cells in different level of effect/target（20：1, 40：1, 80：1）. The killing rate of experimental group reached（47.91±3.25）% in effect/target of 80：1. Conclusion Silencing TIPE2 could significantly promote the secretion level of IFN-γ of mice T lymphocyte, enhance the cytotoxicity activity of T lymphocyte against LA795 lung adenocarcinoma cells in vitro.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2016年第8期673-677,共5页
Cancer Research on Prevention and Treatment
