调“神”干预肺癌转移延长小鼠生存期研究

Study on the mechanism of extending the survival time of lung cancer mice by regulating ‘spirit’

目的:构建血道转移模型,从神经-内分泌-免疫学角度探讨调“神”预防肺癌转移的作用及机制。方法:采用巨猫牌R3H仓鼠饲养笼(84 cm×48.5 cm×45 cm),辅以跑步轮、隧道、木制玩具、小木屋和筑巢材料,构建小鼠富集饲养环境;以普通饲养笼作为小鼠标准饲养环境进行对照。采用小鼠Lewis肺癌细胞在C57BL/6小鼠上分别建立皮下移植瘤模型和尾静脉注射肺转移模型,观察不同饲养环境对荷瘤小鼠肿瘤生长、肺转移和小鼠生存期的影响;采用流式细胞术检测小鼠外周血中的免疫细胞水平;采用ELISA检测小鼠血清中肾上腺素和去甲肾上腺素水平;采用HE染色和免疫组化技术检测小鼠肺脏病理情况及转移前微环境相关指标。结果:富集环境组荷瘤小鼠的肿瘤大小、瘤重、肺转移灶数目等均显著低于标准环境组(P<0.05,P<0.01),且富集环境组小鼠生存期显著长于标准环境组(P<0.01)。两组小鼠之间脾脏指数及胸腺指数无显著差异。小鼠外周血中的髓源性抑制细胞(MDSCs)比例在标准环境组中显著升高(P<0.01);而CD_(4)^(+)T细胞、CD_(8)^(+)T细胞、NK、Tregs、肾上腺素和去甲肾上腺素水平在两组之间无显著差异。小鼠肺脏免疫组化结果显示富集环境组S100A9的表达水平明显高于标准环境组(P<0.05);而CD_(3)^(+)T细胞、CD_(4)^(+)T细胞、S100A8、MMP9的表达无明显差异。结论:引入富集环境模型,模拟临床调“神”干预,可通过降低小鼠外周血中MDSCs水平,干预免疫逃逸的发生,从而达到抑制荷瘤小鼠肿瘤生长和肺转移,以及延长小鼠生存期的作用。

Objective: Building a hematogenous metastasis model to investigate the function and mechanism of regulating ‘spirit’ to prevent lung cancer metastasis from the perspective of nerve-endocrine-immunology. Methods: The mice in intervention group were fed in enrichment environment cages(84 cm×48.5 cm×45 cm), equipped with running wheels,tunnels, wooden toys, wood cabins and nesting materials. The mice in control group were fed in common cages. Subcutaneous transplantation tumor model and tail vein injection lung metastasis model was established were established by subcutaneous and tail vein injection of mouse Lewis lung cancer cells respectively with C57BL/6 mice to observe the effect of different feeding environments on tumor growth, metastasis and survival time of tumor model mice;Flow cytometry was used to detect the immune cell level in the peripheral blood of mice in different groups. ELISA kits were used to detect the levels of epinephrine and norepinephrine in mouse serum in different groups. HE staining and immunohistochemical techniques were used to detect the pathological condition of the lungs of mice and the relevant indexes of the microenvironment before metastasis. Results:The tumor size, tumor weight, and number of lung metastases of tumor model mice in the enriched environment group were significantly lower than those of the standard environment group(P<0.05, P<0.01), with a longer survival time of the mice in the enriched environment group than that of the standard environment group(P<0.01). There was no statistic difference in spleen index and thymus index between the two groups of mice. Peripheral blood test results of immune cells of mice showed that the MDSCs level in the standard environment group was significantly higher than that in the enriched environment group(P<0.01).While there was no statistic difference between the two groups in the levels of CD_(4)^(+)T cells, CD_(8)^(+)T cells, NK, Tregs, norepinephrine and norepinephrine. Immunohistochemical results showed that S100A9 in the enriched environment group was significantly higher than that of mice in the standard environment group, with statistical difference(P<0.05). The levels of CD_(3)^(+)T cells, CD_(4)^(+)T cells,S100A8 and MMP9 had no statistic different between the two groups. Conclusion: By applying enrichment environment culturing method to imitate clinical intervention of regulating spirit, regulating spirit can inhibit the tumor growth and tumor metastasis of tumor model mice, prolonging the survival of mice by lowering the concentration of MDSCs of mice and inhibiting the immune escape happens.