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为阐明冬凌草甲素(ORI)抗小鼠H22肝癌中的T细胞免疫应答机制,本研究利用C57/BL6小鼠H22肝癌皮下移植瘤模型,随机分为荷瘤对照组(PBS组)、ORI高、中、低剂量处理组(75、50、25mg·kg~(-1)·d~(-1))、阳性对照组(5-Fu组)和空白组(NC组),连续腹腔注射给药12d后处死。以瘤重、抑瘤率观察ORI对移植瘤生长的影响;用乳酸脱氢酶释放法检测对小鼠H22细胞杀伤活性;流式细胞术检测小鼠脾脏细胞中CD4~+T细胞分泌IL-17、IL-2、TNF-α、IFN-γ等细胞因子能力,并检测CD8~+T和CD4~+T细胞表面PD-1的表达。结果显示:低、中、高剂量ORI处理组和荷瘤对照组比较均有显著性缩小(P<0.05);ORI能明显提高荷瘤小鼠脾脏细胞对H22细胞的杀伤活性(P<0.05);抑制CD4~+T细胞中细胞因子的分泌,尤其对IL-17和IL-2的抑制最为明显,对TNF-α和IFN-γ抑制在ORI高浓度情况下发挥作用;同时,ORI处理组中CD8~+T细胞的PD-1表达降低,而CD4~+T细胞表面PD-1的表达呈现一定程度的上调。上述结果表明ORI对小鼠H22肿瘤具有明显的体内抑瘤作用,除了直接的杀伤作用外,ORI还可以通过提高CD8~+T的杀伤作用,降低CD4~+T细胞的炎症特性发挥抗肿瘤免疫应答功效,其中的可能机制是影响两种细胞表面PD-1的表达。因此,ORI还可通过协同T细胞抗肿瘤免疫应答机制促进对肿瘤的抑制作用。
In order to elucidate the mechanism of T cell immune response in oridonin H22 hepatocellular carcinoma treated by ORI, we used the H22 hepatocellular carcinoma subcutaneous xenograft model of C57 / BL6 mice to randomly divide them into the tumor-bearing control group (PBS group) ORI group (75, 50, 25 mg · kg -1 · d -1), positive control group (5-Fu group) and blank group (NC group) The rats were sacrificed 12 days after injection. The effect of ORI on tumor growth was observed by tumor weight and tumor inhibition rate. The killing activity of mouse H22 cells was detected by lactate dehydrogenase release assay. Flow cytometry was used to detect the secretion of IL- 17, IL-2, TNF-α, IFN-γ and other cytokines, and detect the expression of PD-1 on CD8 ~ + T and CD4 ~ + T cells. The results showed that compared with the tumor-bearing control group, ORI treatment group showed a significant reduction (P <0.05); ORI significantly increased the cytotoxicity of spleen cells to H22 cells (P <0.05) ; Inhibition of cytokine secretion in CD4 ~ + T cells, especially IL-17 and IL-2 inhibition of the most obvious, the inhibition of TNF-α and IFN-γ ORI at high concentrations play a role; the same time, ORI treatment group The expression of PD-1 in CD8 ~ + T cells decreased, while the expression of PD-1 on CD4 ~ + T cells showed a certain degree of up-regulation. The above results show that ORI has a significant antitumor activity in vivo against mouse H22 tumors. In addition to the direct killing effect, ORI can also exert anti-tumor immunity by increasing the cytotoxicity of CD8 + T and decreasing the inflammatory characteristics of CD4 ~ + T cells Response efficacy, one of the possible mechanisms is to affect the expression of PD-1 on both cell surfaces. Therefore, ORI can also promote tumor suppression through synergistic T cell anti-tumor immune response mechanism.