Immune Killing Effect of Silencing TIPE2 Expression on T Lymphocytes Against Lung Adenocarcinoma Cells LA795
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摘要:目的
利用特异性小干扰RNA（small interfering RNA, siRNA）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siRNA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siRNA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siRNA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。
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关键词:
- 肿瘤坏死因子α诱导蛋白2 /
- 基因沉默 /
- T细胞 /
- 肺癌 /
- 免疫治疗
Abstract:ObjectiveTo silence the expression of tumor necrosis factor-α-induced protein-8-like 2 (TIPE2) gene of T-lymphocytes by specific small interfering RNA (siRNA) and investigate the cytotoxicity activity of transfected T lymphocytes against lung adenocarcinoma cells LA795 in vitro.
MethodsSpleen T lymphocytes of mice were separated by magnetic bead, and we screened and validated an effective siRNA to silence TIPE2 gene of T lymphocytes. After 48h transfection, the secretion of IFN-γ was measured by enzyme-linked immuno-sorbent assay (ELISA). At the end, 72h 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.
ResultsWe screened a siRNA 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 48h 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 72h 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.
ConclusionSilencing 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.
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Key words:
- TIPE2 /
- Gene silencing /
- T lymphocytes /
- Lung cancer /
- Immunotherapy
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图 1 荧光显微镜下的FAM-siRNA转染荧光 (×100)
Figure 1 Fluorescence observation for transfection of FAM-siRNA under fluorescence microscope (×100)
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图 3 不同siRNA分组TIPE2蛋白的表达情况
Figure 3 Expression of TIPE2 protein in different siRNA groups
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图 4 转染48 h后T细胞细胞因子IFN-γ的分泌水平比较
Figure 4 Comparison of IFN-γ secretion level in T cells 48h after transfection
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图 5 各组T细胞与小鼠肺腺癌LA795共同培养72 h后的肿瘤杀伤率
Figure 5 Killing rates of tumor cells 72h after T Cell and LA795 co-culture
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