摘要
树突状细胞（DC）是人体最重要的抗原递呈细胞，它具有摄取、加工、处理、递呈抗原并启动免疫应答的作用。人参是常用的补益类中药，人参皂苷Rg1是人参中主要有效成分之一。药理研究表明，Rg1具有提高免疫功能及抗衰老等作用，口服Rg1后，Rg1经人肠道内细菌代谢为一极性更小的化合物Rh1, 并在血中可同时检测到Rg1及Rh1的存在。本研究通过观察人参皂苷Rg1及其肠内菌代谢产物Rh1对DC功能的影响，在细胞、分子水平阐述人参皂苷Rg1、Rh1提高免疫力的作用机制。
1．从人外周血中分离DC前体细胞，用国产细胞因子IL-4、GM-CSF及TNFα共同诱导7天以后，经光镜和电镜下的形态特征、S-100蛋白免疫组化染色及混合淋巴细胞反应鉴定。结果DC成熟率达95-99%。
2.分离DC前体细胞及人脐静脉内皮细胞（HUVEC），用Rg1、Rh1分别处理DC前体细胞及HUVEC，用虎红染色法观察DC前体细胞与HUVEC的粘附。结果发现:(1)用Rg1、Rh1处理HUVEC 2h后，DC前体细胞与其粘附24h, 不论有无TNFα的刺激，Rg1 100(g/ml及Rh1各剂量组均可增强DC前体细胞与HUVEC的粘附(P<0.001)，且Rh1 1、10(g/ml比同剂量的Rg1作用更强（P<0.001）；粘附48h后，在无刺激剂存在下，Rh1各剂量组及Rg1均可明显促进DC前体细胞与HUVEC的粘附，其作用亦强于同剂量的Rg1（P<0.001）;在TNFα刺激下，与TNFα对照组相比， Rh1 10、100(g/ml可显著提高粘附（P<0.05-0.01），且其作用强于同剂量的Rg1(P<0.01)。（2）用Rg1、Rh1处理DC前体细胞后，DC前体细胞与HUVEC粘附24h, 无论有无TNFα的刺激，Rg1、Rh1均能显著增强DC前体细胞与HUVEC的粘附（P<0.001）; 粘附48h后，仅Rg1 10(g/ml及Rh1 100(g/ml可促进DC前体细胞的粘附（P<0.05-0.01）;在TNFα作用下， Rh1各剂量组均显著促进DC前体细胞与HUVEC的粘附（P<0.01-0.001），但Rg1 10、100(g/ml却表现出对粘附的抑制作用（P<0.001）。
3．用MTT法检测Rg1 及Rh1对DC刺激T淋巴细胞增殖作用的影响。结果表明： T：DC 为10:1及25:1时，与对照组相比，Rg1的高浓度(100(g/ml)可明显促进T细胞的增殖；T:DC为10：1时，Rh1 10(g/ml抑制T细胞的增殖, 而在T:DC为25:1时则表现为促增殖作用（P<0.05）。提示在适宜的T:DC比及适当的浓度下，Rh1与Rg1的作用一致，均能促进DC对T细胞增殖的刺激作用。
4．用中性红摄入比色法观察DC-LPAK的杀伤活性。结果发现： Rg1及Rh1均能促进DC-LPAK对乳头瘤细胞的杀伤能力；在对L929细胞的杀伤实验中, 在效靶比为10：1时，各组均未观察到差异；在效靶比为5：1时，与乳头瘤+LPAK对照组相比， Rg1各剂量组及Rh1 10ug/ml均能显著提高LPAK的杀伤能力（P<0.05~0.01）; 与乳头瘤+LPAK+DC对照组相比，Rg1各剂量组可显著提高LPAK细胞的杀伤能力（P<0.01~0.001），且随剂量的降低作用减弱。结果提示Rg1与Rh1在适宜的浓度及效靶比时，可促进DC刺激的LPAK细胞杀伤肿瘤细胞的能力。
5．用免疫组化图像分析法检测成熟DC表面分子的表达。结果表明，Rg1和Rh1可同时增强成熟DC表达HLA-DR、CD25、CD44、 CD54、CD11c分子，Rh1 的作用比Rg1强，对E-seletctin没有明显影响。
6．用ELISA法检测Rg1及Rh1对DC分泌IL-12 P40蛋白的影响，结果发现，Rg1、Rh1 各剂量均可促进成熟DC 分泌IL-12 P40；用RT-PCR法检测Rg1及Rh1对DC IL-12 mRNA表达的影响，Rg1 100、1(g/ml及Rh1 100(g/ml均可显著促进DC IL-12 P40 mRNA的表达，Rg1 100μg/ml促进功能更为显著。说明Rg1及Rh1对DC细胞分泌IL-12 P40的促进作用是通过增加IL-12 P40 mRNA转录而实现的。
上述结果提示： Rg1及其代谢产物 Rh1可通过促进前体DC与血管内皮细胞的粘附，而促进DC前体细胞的移行和发育成熟，并通过促进DC表面粘附分子，共刺激分子的表达及IL-12 mRNA的转录和蛋白的翻译，刺激T细胞的增殖，及增强LPAK细胞的杀伤活力。说明被吸收入血的Rg1和Rh1共同作用于DC，通过对DC移行、成熟、功能的上调作用而促进免疫应答的启动，增强机体的免疫功能。
Dendritic cell (DC) plays important roles not only in taking antigen but also in activating T cell. It is believed that DC is a pivotal Antigen Presenting Cell (APC) in the immune system. In this dissertation, we compared the effects of Ginsenoside Rg1 and its metabolite, Ginsenoside Rh1, on dendritic cell, then elucidated the immuno-regulating mechanism of Ginsenoside Rg1.
1. Separating pregnior DC from PBMC, then inducing these cells with IL-4, GM-CSF and TNFa. 7 days later, identifying mature DC by the combination of microscope, electro-microscope and immunohistochemistry staining with S-100 protein. The results showed that the mature rate of DC is 95-99%。
2. The adhesion rate between pregnior DC and HUVEC was evaluated after treatment with Rg1 and Rh1 separately. (1) After 2h treatment with Rg1 and Rh1, HUVEC was cultured with pregnior DC. The results after 24 hours co- cultivation showed that Rg1 100(g/ml and all dose of Rh1 could enhance the adhesion rate between pregnior DC and HUVEC with and without stimulation of TNFa, moreover the effect of Rh1 1,10(g/ml is more significant than the same concentration of Rg1 (P<0.001). the results after 48h co-cultivation showed that all dose of Rh1 could promote the adhesion rate in the lack of stimulator and the effect is more significant than the same concentration of Rg1 (P<0.001); with TNFαas stimulator, Rh1 10,100(g/ml could enhance significantly the adhesion rate（P<0.05-0.01）and the effect is more significant than the same concentration of Rg1 (P<0.001).(2) After 2h treatment with Rg1 and Rh1, pregnior DC was cultured with HUVEC. The results After 24h co-cultivation showed that both Rg1 and Rh1 could enhance significantly the adhesion rate between pregnior DC and HUVEC (P<0.001) with and without the stimulation of TNFα; The results after 48h co-cultivation showed that only Rg1 10(g/ml and Rh1 100(g/ml could improve the adhesion rate (P<0.05-0.01). With TNFαas stimulator, all dose of Rh1 could improve the adhesion rate （P<0.01-0.001）, however Rg1 10,100(g/ml showed obvious inhibitory effect on adhesion (P<0.001).
3. The effect Rg1 and Rh1 on the proliferation of T cell stimulated by DC was detected using MTT method. The result demonstrated that Rh1 10(g/ml play inhibitory and stimulating effect on the proliferation of T cell when the ratio between T cell and DC was 10：1 and 25:1 separately（P<0.05）.Both Rg1 and Rh1 have no effect on the proliferation of T cell when the ratio of TC and DC is 50:1.
4. The cytotoxicity activity of DC-LPAK was detected using neutral red phagocytosis assay. The results showed that both Rg1 and Rh1 could enhance the cytotoxicity activity of DC-LPAK on papilla tumor cell line; When the E:T was 5:1, all dose of Rg1 could enhance the cytotoxicity of DC-LPAK on L929 cell line（P<0.01；0.05）,. However only 10μg/ml Rh1 can improve the cytotoxicity of DC-LPAK (P<0.05).
5. The immunohistochemistry results showed that both Rg1 and Rh1 could stimulate the expression of surface molecules of DC, such as HLA-DR，CD25, HLA-DR, CD44, CD54 and CD11c. However they have no significant effect on the expression of E-seletctin.
6. The effect of Rg1 and Rh1 on the expression and transcription lever of IL-12 P40 was detected using ELISA and RT-PCR.. The result showed that all dose of Rg1 and Rh1 could enhance the expression of IL-12 P40. Rg1 100, 1(g/ml and Rh1 100(g/ml could improve the transcription lever the IL-12 P40 mRNA and the effect of Rg1 100μg/ml is more significant.
The results demonstrated that both Rg1 and its metabolite Rh1 could enhance the migration of precursor DC, they can improve the proliferation of T cell and enhance the cytotoxity activity of LPAK by improving the expression of adhesion and co-stimulating moleculars on the surface of mature DC and the transcription and translation of IL-12 . The result showed that the immune effects of Rg1 was proformed by Rg1 and Rh1 which were absorbed into blood.
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