六味地黄丸诱导负载胃癌干细胞抗原的DC分化及其对CD4+CD25+调节性T细胞作用的研究

In the early research, we found that tonifying kidney medicine have the similar effect of cytokine to mobilize DC in vivo and improves the survival rate of patients when using tonifying kidney method combined with Dendritic Cell(DC) for treating cancer. Based on we further researched stem cell characteristics of gastric cancer stem cell. It is urgent to solve the next problems how to optimize concentrations of tonifying kidney medicine inducing DC differentiation and target DC killing gastric cancer stem cell..The root of cancer recurrent and metastatic is tumor stem cell remaining in vivo. The immunotherapy of DC for cancer is clinical significant cure. The main problems influence on DC cure for tumor are the insufficient quantity and low immunogenicity of DC in vivo. Therefore ,the tonifying kidney method inducing DC differentiation and then load with gastric cancer stem cell antigen would solve these problems..DC originates from hematopoietic stem cells which correlates with "kidney storing essence". Tonifying kidney method inducing DC differentiation complies with "Defensive Qi rooting in kidney" theory.Therefore, we design that optimize and screen different concentrations of Liuwei Dihuang pill to inducing DC differentiation and analyse the changing quantity of DC and observe the activity of DC loaded with gastric cancer stem cell antigen activating CD83+ and inhibiting CD4+CD25+ regulatory T cells.The project would verify that tonifying kidney method promoting DC differentiation and DC loaded with gastric cancer stem cell antigen play a important role on treatment of gastric cancer.

前期，我们在补肾法配合树突状细胞（DC）治疗肿瘤的研究发现，补肾药物具有类似细胞因子作用，可增强动员DC作用，提高患者生存率。在此基础上，继续研究胃癌干细胞的"干性"特征提示，如何优化补肾方药浓度进而诱导DC分化及DC靶向杀伤胃癌干细胞将是要解决的问题。.体内残存的肿瘤干细胞是肿瘤复发转移的根本原因。DC免疫治疗作为肿瘤治疗的重要方法，因体内DC数量不足和肿瘤免疫源性较低而影响疗效。故提出补肾法诱导负载胃癌干细胞抗原的DC分化，将可能解决DC数量不足和免疫源性较低的问题。.DC源于造血干细胞，而干细胞又和"肾藏精"相关，并且补肾法诱导DC分化与"卫气根于肾"理论相符。由此设计，优化不同浓度的六味地黄丸诱导DC分化，检测DC数量变化，观察负载胃癌干细胞抗原的DC激活CD83+，并抑制CD4+CD25+调节性T细胞活性的作用，以验证补肾法促进DC分化及DC负载胃癌干细胞抗原对肿瘤的杀伤作用。

目的：探讨六味地黄丸对树突状细胞增殖的影响及负载胃癌干细胞抗原的树突细胞对胃癌的靶向杀伤作用及其机制。.方法： 制备六味地黄丸含药血清，优化最佳给药浓度和作用时间，检测六味地黄丸对树突状细胞（Dendritic cells，DC ）增殖的影响,观察不同浓度和时间六味地黄丸作用下的细胞增殖变化；分离胃癌干细胞，用反复冻融法制备胃癌干细胞抗原，检测负载胃癌干细胞抗原的DC对胃癌的靶向杀伤及其CD4+CD25+调节性T细胞对抗肿瘤的免疫抑制。 .结果：1.与空白对照组比较，成熟型DC含药血清显著促进DC细胞增殖,且这种增殖在含药浓度15%，作用时间48h时最为显著。其IL—12分泌量显著提高（P<0.05）。六味地黄丸含药血清作用48小时，DC吸光光度值显著升高( P ＜ 0.05），DC对异基因T淋巴细胞具有增殖刺激作用，且随着细胞数量的增加作用增强（P<0.05），呈量效关系。2、经胃癌干细胞抗原刺激的DC细胞，DC表面成熟标志CD83和CD86 表达明显增加，CD83和CD86表达率为80.4%（P<0.01）；MTT检测结果显示：胃癌干细胞抗原组、胃癌细胞抗原组、DC-CIK组、DC组对胃癌细胞的杀伤率分别为（80.6±0.8）、（72.3.±0.6）、（58.4±0.2）和（49.7±0.8）%，（P<0.01）。3、ALDH1方法分选肿瘤干细胞发现，胃癌干细胞具有自我更新的生物学特性，胃癌干细胞克隆形成数量和形成率高于对照组，胃癌干细胞裸鼠皮下移植具有高致瘤性；4、DC吸光光度值升高对CD4+T细胞的增殖能力有明显调节作用；降低CD4+T细胞中CD4+CD25+调节性T细胞的数量，可同步升高CD4+CD25-效应性T细胞。.结论：.1、六味地黄丸具有诱导树突状细胞增殖的作用；.2、ALDH可作为分选胃癌干细胞的标志物，.3、胃癌干细胞可作为抗原负载DC，负载胃癌干细胞抗原的DC具有靶向增强免疫杀伤的作用。