IL-12诱导肝癌微环境中NK细胞活化发挥抗肿瘤作用

IL-12 plays anti-tumor effect by inducing NK cell activation in hepatic carcinoma microenvironment

目的:探讨IL-12通过诱导肝癌微环境中NK细胞活化诱导抗肿瘤的效果。方法:NOD/SCID小鼠皮下注射肝癌HepG2细胞,成瘤后腹腔注射人外周血淋巴细胞(peripheral blood lymphocyte,PBL),建立HCC-huPBL荷瘤小鼠模型。将荷瘤小鼠随机分为IL-12组和PBS对照组,瘤内注射IL-12后,观测荷瘤小鼠瘤体积、体重、一般状况的变化,IL-12瘤内注射后第30天ELISA法检测荷瘤小鼠肝癌组织微环境中IL-12、INF-γ含量以及小鼠外周血中天门冬氨酸氨基转移酶(aspartate amin-otransferase,AST)及谷丙转氨酶(alanine aminotransferase,ALT)的含量,免疫组化法检测IL-12治疗后肝癌微环境中NK细胞活化性受体NKG2D、NKp44、NKp30、NKp46,以及抑制性受体KIR2DL3/CD158b、NKG2A/CD159a的表达。结果:第12、18、24、30天IL-12组荷瘤小鼠瘤体积均小于PBS组[(594.47±205.51)vs(832.10±187.49)mm3,(963.61±427.95)vs(1 350.87±468.23)mm3,(1 285.02±368.56)vs(1 975.49±655.54)mm3,(1 903.64±471.34)vs(2 568.77±784.68)mm3,均P<0.05]。IL-12组小鼠肝癌组织中IL-12与INF-γ的表达水平均明显高于PBS组[(2.96±1.02)vs(1.35±0.75)pg/ml,(12.26±4.11)vs(7.81±3.46)pg/ml,均P<0.05]。IL-12组与PBS组相比,血清ALT水平第7天显著升高[(73.85±10.71)vs(41.73±13.13)U/L;P<0.05],第14天达到高峰。IL-12组治疗后肝癌组织中NK细胞活化性受体NKG2D、NKp44、NKp30的表达较PBS组高(P<0.05),NKp46的表达未见明显升高;而NK细胞抑制性受体CD158b和CD159a表达较PBS组低(P<0.05)。结论:肝癌模型小鼠瘤体内IL-12注射可上调瘤组织内NK细胞活化性受体、IL-12、IFN-γ的表达,下调抑制性受体的表达,从而抑制小鼠模型中肿瘤的生长。

Objective：To explore the enhanced anti-tumor effect of IL-12 through inducing NK cell activition in hepatic carcinoma microenviroment. Methods： The hepatic cacinoma HepG2 cells were subcutaneously injected into NOD/SCID mice, and human peripheral blood lymphocytes （PBL） were introperitoneally injected after tumor formation to establish HCC-huPBL tumor-bearing mouse model. The tumor-bearing mice were randomized into IL-12 group and PBS control group. Mice were intratumoral injected with IL-12, and the changes of tumor volume and body weight as well as general conditions of tumor-bearing mice were observed. ELISA assay was performed to examine the expression levels of IL-12 and INF-γ in the microenvironment of hepatic carcinoma tissues in tumor-bearing mice, and the aspartate aminotransferase （AST） and alanine aminotransferase （ALT） levels in peripheral blood of mice 30 days after IL-12 intratumoral injection. Immunohistochemistry assay was used to analyze the expressions of NK-activating receptors： NKG2D, NKp44, NKp30, NKp46, and inhibitory NK receptors： KIR2DL3/CD158b and NKG2A/CD159a in hepatic carcinoma microenvironment after IL-12 treatment. Results： On day 12, 18, 24 and 30, the tumor volumes were smaller in the IL-12 group than those in the PBS group （／[594.47±205.51／] vs ／[832.10±187.49／] mm3, ／[963.61±427.95／] vs ／[1 35087±468.23／] mm3, ／[1 285.02±368.56／] vs ／[1 975.49±655.54／] mm3, ／[1 903.64±471.34／] vs ／[2 568.77±784.68／] mm3, P〈0.05）. The expression levels of IL-12 and IFN-γ in the IL-12 group were significantly higher than those in the PBS group （／[2.96±1.02／] vs ／[1.35±0.75／] pg/ml, ／[12.26±4.11／] vs ／[7.81±3.46／] pg/ml, P〈005）. The serum ALT level significantly increased in the IL-12 group compared to the PBS group on day 7 （／[7385±10.71／] vs ／[41.73±13.13／] U/L, P〈0.05）, and reached a peak at day 14. The expressions of NK-activating receptors NKG2D, NKp44 and NKp30 were statistically higher in the IL-12 group than those in the PBS group （P〈005）, the expression level of NKp46 showed no significant up-regulation, while the expression levels of NK inhibitory receptors CD158b and CD159a were decreased compared to the PBS group （P〈0.05）. Conclusion： IL-12 intratumoral injection can up-regulate the expressions of NK-activating receptors, IL-12 and IFN-γ, and down-regulate the NK inhibitory receptors in the hepatic carcinoma mouse model, therefore effectively inhibiting the tumor growth in mouse model.