人参皂苷Rg1对树突状细胞效应与分子机制的研究
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摘要
目的
人参是传统的补药,具有延长寿命和增强人体抗病能力的作用,可有效地抗应激、抗疲劳、抗肿瘤以及抗氧化等作用。人参皂苷是其主要有效成分,其中以人参皂苷Rg1含量较高。对人参的药物基因组学和调节阴阳平衡研究表明,人参皂苷Rgl具有抗肿瘤作用和类固醇激素样作用。树突状细胞(DC)是功能最强的专职抗原提呈细胞,它可捕获肿瘤细胞分泌的可溶性抗原,以MHCⅠ类分子或Ⅱ类分子限制的方式提呈给CD8+T细胞或CD4+T细胞,使之活化并发挥抗瘤效应。其中CD4+T细胞还参与激活B细胞、巨噬细胞、自然杀伤细胞(NK)和细胞毒淋巴细胞(CTL),协同发挥抗肿瘤作用。人参皂苷Rg1作为人参的主要成分,可提高小鼠的非特异性免疫功能,但其机制尚不清楚,是否能通过影响DC功能而提高免疫应答有待进一步研究。基因芯片在研究药物对机体免疫系统的调节,寻找药物靶向方面具有优越性,本课题运用功能基因芯片技术,研究与DC功能相关的基因,以期找到人参皂苷Rg1作用于DC的相关分子,明确人参皂苷Rg1发挥免疫调节作用的信号通路,为中医药“扶正祛邪”治疗原则以及人参的双向免疫调节作用提供实验依据,也为人参皂苷Rg1作为临床抗肿瘤新中成药的组成成份提供明确的生物学基础和实验依据。
方法
1、实验一针对人参皂苷Rg1是否影响卵清蛋白(OVA)免疫小鼠特异性免疫应答能力进行研究。以OVA为抗原,混合人参皂苷Rgl或氢氧化铝(Al(OH)3)佐剂,颈部皮下免疫BALB/c小鼠,间隔2周,免疫三次。分为生理盐水对照组(NS组)、人参皂苷Rgl对照组(Rg1组)、铝盐对照组(A1组)、OVA组、OVA+Al组和OVA+Rg1组共六组。免疫三次后第10天,采用MTT比色法测小鼠脾淋巴细胞的增殖能力、用间接ELISA法分别测总血清IgG、IgG1、IgG2a、IgG2b变化以及血清白细胞介素-4(IL-4)、γ干扰素(IFN-γ)的分泌水平等指标的变化,探讨人参皂苷Rgl对BALB/c小鼠免疫调节作用的影响。
2、实验二以体外培养小鼠骨髓来源树突状细胞模型为研究对象,以粒细胞及巨噬细胞集落刺激因子(GM-CSF)和IL-4刺激骨髓造血干细胞,从而使之朝树突状细胞方向分化。待培养至第7天收集细胞,加入不同药物刺激24小时,分为培养基对照组(Ⅰ组)、人参皂苷Rg1组(Ⅱ组)、脂多糖(LPS)组(Ⅲ组)和人参皂苷Rg1+LPS组(Ⅳ组)共四组。培养过程使用相差显微镜观察DC的形态变化,并在培养结束时采用流式细胞术检测各组DCs标志表面分子MHC-Ⅱ、CD40和CD86的表达情况,探讨人参皂苷Rgl能否能促进树突状细胞的成熟以及人参皂苷Rg1作用于DC的可能机制。
3、实验三中,对于树突状细胞吞噬抗原能力的检测,收集培养6天的骨髓造血干细胞(即未成熟DCs),加入不同浓度的人参皂苷Rg1刺激24小时后,加入FITC-OVA避光孵育1小时,最终冰上终止反应5分钟,流式细胞仪检测树突状细胞吞噬FITC-OVA的荧光强度。对于DCs处理提呈抗原能力的检测,收集培养6天的骨髓造血干细胞(即未成熟DCs),加入不同浓度的人参皂苷Rg1刺激细胞24小时后,用SIINFEKL肽段刺激,再加入1μg/ml的LPS刺激16小时后和B3Z T细胞孵育,通过检测上清中IL-2的水平判断致敏后的树突状细胞对B3Z T细胞的活化能力。细胞因子的检测分组和实验二相同,加入药物刺激后为检测IL-12分泌水平的孵育时间为24小时,而为检测IL-4和IL-10分泌水平的孵育时间为48小时,利用间接ELISA法检测树突状细胞培养上清中细胞因子IL-12、IL-4、IL-10水平。进一步揭示人参皂苷Rgl免疫调节作用的机制。
4、实验四分组和实验二相同,以GM-CSF和IL-4刺激骨髓造血干细胞,从而使之朝树突状细胞方向分化。待培养至第7天收集细胞,加入不同药物刺激24小时,分为培养基对照组、人参皂苷Rg1组、LPS组和人参皂苷Rgl+LPS组共四组。培养结束后,抽提树突状细胞总RNA,利用树突状和抗原呈递细胞基因芯片对细胞功能相关基因进行检测,以筛选人参皂苷Rgl作用于DC的差异表达基因,为进一步寻找药物作用靶点提供了线索。
5、实验五通过分析总结实验四的结果找出感兴趣的目的基因,以GAPDH为管家基因,提取细胞RNA,以等量RNA为模板,以各基因特异引物,采用半定量RT-PCR检测EGFR和p44 mRNA表达水平；不同药物和DC孵育后,裂解细胞提取蛋白质,总蛋白跑SDS-PAGE后转膜,采用Western blot法分析EGFR和ERK1/2蛋白表达水平从而进一步证实人参皂苷Rg1是否影响EGFR-p44 MAPK途径对DC产生调控作用。
结果
1、用人参皂苷Rg1、铝盐佐剂以及卵清蛋白(OVA)免疫各组小鼠,体重并无明显差异。OVA特异性总IgG抗体水平的OD值,按照从小到大的顺序依次是：OVA<(OVA+Rg1)<(OVA+A1),其中(OVA+Rg1)组明显高于OVA组(p<0.01);(OVA+Rg1)组IgG1、IgG2a、IgG2b、IgG3抗体水平明显高于OVA组(p<0.05);(Al+OVA)组IgG1、IgG3.IgG2b抗体具有较高水平,IgG2a低水平(p>0.05).OVA组与NS组相比,IgG1和IgG3具有较高水平；此外,(OVA+Rg1)组免疫小鼠脾细胞对OVA抗原刺激的增殖反应在各组中最高；ELISA结果显示(OVA+Rg1)组脾细胞分泌的IFN-γ含量最高,且(OVA+Rg1)组和OVA组存在明显差别(p=0.03)；而(OVA+Al)组与OVA组相比分泌更高水平IL-4(p=0.01)。
2、体外培养BM-DC 7d后,相差显微镜下可见典型形态的树突状细胞,收集总DC数超过85%。40lg/ml的人参皂苷Rgl处理不成熟BM-DC 24小时后,表面分子I-A/I-E、CD40和CD86变化不显著；加入LPS后,DC表面的I-A/I-E、CD40和CD86分子均显著升高(p<0.05,与对照组相比)；人参皂苷Rgl和LPS同时加入DC时,细胞表面的I-A/I-E分子表达显著性升高(p<0.05,与LPS组相比),而CD40和CD86升高不明显。
3、人参皂苷Rg1可以在体外促进未成熟DC分泌IL-12,和LPS同时作用时,有协同增强作用；此外人参皂苷Rgl还能促进未成熟树突状细胞分泌IL-4和IL-10；人参皂苷Rg1可以显著增强未成熟树突状细胞吞噬FITC-OVA抗原,并且促使成熟树突状细胞处理抗原肽,并提呈给T细胞使之活化分泌IL-2。
4、树突状细胞RNA提取A260/A280的比值在2.0左右,变性琼脂糖凝胶电泳RNA条带清晰,28s:18s rRNA条带亮度接近2：1；经终浓度为40μg/ml的人参皂苷Rg1和/或1μg/ml LPS处理24小时后,人参皂苷Rgl组与对照组相比较(Ⅱ/Ⅰ)上调≥2倍基因23个,下调≥2倍基因45个；LPS组与对照组相比较(Ⅲ/Ⅰ)上调≥2倍基因15个下调≥2倍基因47个；人参皂苷Rg1+LPS组与人参皂苷Rg1组相比(Ⅳ/Ⅱ)上调≥2倍基因35个,下调≥2倍基因15个；人参皂苷Rg1+LPS组与LPS组相比(Ⅳ/Ⅲ)上调≥2倍基因37个,下调≥2倍基因10个,在差异表达基因中,给予人参皂苷Rg1后,Erbb2和Ifi44基因在DCs呈现较高转录水平。
5、从基因芯片结果中找出感兴趣的差异表达基因Erbb2和Ifi44,进一步利用半定量RT-PCR和Western blot法检测Rgl组、LPS组以及Rg1+LPS组分别处理DC后的EGFR.p44基因的转录水平及蛋白质表达水平。其中Rgl组与对照组相比,两条基因的结果条带清晰,EGFR.p44基因转录水平明显增加；Western blot法分析EGFR和p44蛋白分泌水平均上调。
结论
1、人参皂苷Rg1能增强卵清蛋白免疫小鼠的特异性免疫应答。
2、人参皂苷Rg1不能促进未成熟树突状细胞的成熟,但能促进成熟DCs高表达I-A/I-E分子；人参皂苷Rg1能增强未成熟DCs的抗原吞噬能力；人参皂苷Rgl能促进成熟DCs的抗原提呈能力。
3、人参皂苷Rg1对DCs功能基因的调控涉及到DC的迁移、抗原吞噬、处理和提呈、分泌细胞因子等各个环节,尤其是能通过EGFR-MAPK途径调控下游细胞因子IL-10的产生,从而影响T细胞的极化状态调节免疫应答。
综上所述,尽管人参皂苷Rgl不能促进未成熟树突状细胞的成熟,但能增强小鼠树突状细胞的吞噬抗原、处理呈递抗原的能力并影响其产生细胞因子。上述作用可能涉及到多个靶点,通过本实验研究可以明确人参皂苷Rg1能够上调EGFR-MAPK途径调控DC功能而调节下游细胞因子IL-10的产生,从而调控免疫应答。这种作用是否是直接作用抑或是间接作用,需要进一步实验证明,下一步拟利用生物传感器或iRNA干扰技术进一步明确人参皂苷Rg1是否直接作用于EGFR分子。除此之外,我们的实验结果表明人参皂苷Rgl能辅助增强小鼠特异性免疫应答能力。
本研究的实验结果证明了人参皂苷Rg1具有双向的免疫调节作用,探讨了人参皂苷Rgl作用于树突状细胞的分子机制及其发生作用的信号通路,为中医药“扶正祛邪’治疗原则以及人参的双向免疫调节作用提供实验依据,并为中医药调节免疫功能研究提供一个新的思路和方法,最终为将来人参皂苷Rg1作为临床抗肿瘤新中成药的组成成份提供明确的生物学基础和实验依据。
In Chinese medicine, ginseng has long been used as a general tonic to promote longevity and enhance bodily functions. It has also been claimed to be effective in combating stress, fatigue, oxidants and cancer.Ginsenoside Rgl is one of ginseng's active ingredients and has anti-tumor, angiomodulating and steroid-like activities through investigating its pharmacogenomics from the perspective of the Yin/Yang therory. Though celluar immunity mediated by T cells、NK cells and macrophages is predominant in tumor immunity, humoral immunity plays an important role in the virus-induced tumors. However, dendritic cells (DC), which is the most powerful professional antigen presenting cells, can capture soluble antigens secreted by tumor cells, present them to be recongnized by CD4+T cells by the MHCⅠor MHCⅡmolecules restricted manner and finally activate CD8+T or CD4+T cells which play anti-tumor effect. In addition, CD4+T cells also involved in activation of B cells, macrophages, NK cells and CTL, synergistic antitumor effects. It is reported that ginsenoside Rgl as the main component of ginsenoside improved the non-specific immune function in mice, however, wheather this enhancement is mediated by regulating the functions of DCs remains unknown. Gene chip technology has advantages in studying the regulation of the immune system and finding out the key molecules on the immune cells influence by drugs. This study, is focused on the influence which ginsenoside Rgl has on the specific immune responses in BALB/c mice as well as the key molecules on DCs which ginsenoside Rgl targets by gene chip technology, in order to find out the target signal passway of ginsenoside Rgl on DCs and provide the experimental evidence for the therapeutic principles of strenghthening body resistance and eliminating evil in TCM, the two-direction regulation of ginseng as well as ginsenoside Rg1 as the main composition of new anti-tumor Chinese medicines.
Method
1. At first, the influence of ginsenoside Rg1 on immune responses immunized by ovalbumin (OVA) in BALB/c mice was investigated.50μg OVA mixed ginsenoside Rgl or aluminum hydroxide (Al (OH) 3) adjuvant, were given to immunize BALB/c mice three times at a two-week interval.10 days after three immunizations, lymphocyte proliferation, specific total IgG and IgG subclass, cytokines IL-4 and IFN-y secreted by splenocytes from culture supernatants were measured respectively by MTT assay and ELISA.
2. The influence of ginsenoside Rg1 on the surface molecules of bone marrow derived dendritic cells. After culturing bone marrow derived cells stimulated by Granulocyte macrophage colony stimulating factor (GM-CSF) and Interleukin-4 for 7 days, bone marrow derived dendritic cells (BM-DCs) were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours. Finally, the expression of the surface molecule I-A /I-E, CD40 and CD86 were detected by flow cytometry in order to investigate whether ginsenoside Rg1 could promote immature DCs to mature.
3. In this study, the impacts of ginsenoside Rgl on the secretion of cytokines, phagocytosis and antigen-presenting of murine bone marrow dendritic cells were analyzed. After culturing bone marrow derived cells stimulated by GM-CSF and Interleukin-4 for 7 days, BM-DCs were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours or 48 hours. Then culture supernatant were collected and the sectretion of cytokines IL-12, IL-4, IL-10 levels by an indirect ELISA assay. In addition, after BM-DCs were treated by ginsenoside Rgl and then FITC-OVA for 1 hour, the phagocytosis was detected by flow cytometry. Finally, the influence of ginsenoside Rg1 on antige-presenting of BM-DCs were detected by IL-2 secreted from active B3Z T cells.
4. The influence of ginsenoside Rg1 on the expression of functional gene from mouse bone marrow-derived DC was analyzed by gene chip technique. After culturing bone marrow derived cells stimulated by GM-CSF and Interleukin-4 for 7 days, BM-DCs were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours and then the total RNA was extracted. Oligo dendritic &antigen presenting cell gene chips were utilized to find out the difference genes.
5.With GAPDH as the housekeeping gene, was extracted RNA, the same amount of RNA as template, the gene specific primers, using semi-quantitative RT-PCR and semi-quantitative detection of p44 mRNA expression levels EGFR; DC lysis cell extracts after incubation of protein, total protein run SDS-PAGE after the transfer film by Western bolt analysis EGFR and p44 protein secretion, and whether the aforementioned cytokine ELISA assay consistent with the results, thus confirming whether ginsenoside Rg1 by EGFR-p44 MAPK pathway on the regulation of DC generation.
Result
1. Among the different groups, body weights did not differ significantly. OD values of OVA-specific IgG antibodies responses are ranging in descending order:OVA<(OVA+ Rgl)<(OVA+Al). Among them, those from (OVA+Rg1) group were significantly higher than those from OVA group (p<0.01). In addition, IgG1, IgG2a, IgG2b, IgG3 antibody levels from(OVA+Rg1) group were significantly higher than those from OVA group (p<0.05); IgG1, IgG3, IgG2b levels from (Al+OVA) group were obviously higher while its IgG2a responses didn't increase significantly compared with the NS group. What's more, IgG1 and IgG3 responses from OVA-immunized alone group were evidently higher than those from NS group. In cellular immunity, the stimulation index of splenocytes from (OVA+Rg1) group was significantly enhanced compared with that from OVA group. Finally, the ELISA results showed that IFN-γlevels from (OVA+Rg1) group were the highest, and significant difference existed between that from (OVA+Rgl) group and OVA group (p=0.03) while (OVA+Al) groups secreted significantly higher levels of IL-4 compared with OVA group.
2. After cultivating bone marrow derived hematopoietic stem cells for 7 days, more than 85% of the dendritic cells were collected. The surface molecule I-A/I-E, CD40 and CD86 on DCs did not change significantly after DCs were treated with ginsenoside Rgl at a concentration of 40μg/ml. However, after adding LPS, those were significantly higher (p <0.05, compared with the media control group). In addition, the cell surface I-A/I-E molecules on DCs from ginsenoside Rgl plus LPS group was significantly increased in (p <0.05, compared with LPS group) while CD40 and CD86 remained unchanged.
3. Ginsenoside Rgl not only promoted DC in vitro to secrete higher levels of IL-12 but also strengthened this promotion if added with LPS simultaneously. Secondly, ginsenoside Rgl promoted immature dendritic cells to secrete IL-4 and IL-10 molecules. What's more, ginsenoside Rgl had an obvious influence on the phagocytosis of FITC-OVA antigen for immature DCs.Finally, ginsenoside Rgl promoted mature DCs to process and present peptides which activated B3Z T cells to secrete obviously higher levels of IL-2.
4. A260/A280 ratios were about 2.0 for RNA extraction from dendritic cells. In denaturing agarose gel electrophoresis, bands were clear and 28s rRNA band intensity is nearly twice as much as that for 18s rRNA.There were 23 genes whose ratios of Rgl group to media group (Ⅱ/Ⅰ) were above 2, and 45 genes below 0.5. As for those of LPS group to media group (Ⅲ/Ⅰ),there were 15 genes above 2 and 47 below 0.5. In addition, there were 35 above 2 and 15 below 0.5 for the ratios of Rgl+LPS to Rgl (Ⅳ/Ⅱ) while there were 37 above 2 and 10 below 0.5 for (Ⅳ/Ⅲ). Among those differently expressing genes, Erbb2 and Ifi44 showed obviously expressed in DCs stimulated by ginsenoside Rg1.
5. The results of semi-quantitative RT-PCR and Western Blot indicated that in DCs stimulated by ginsenoside Rgl, both EGFR and p44 which is also called ERK2, assumed obviously higher transcription and expression levels, compared with the media group.
Conclusion
1. Ginsenoside Rgl can enhance the specific immune responses in OVA-immunized mice.
2. Though ginsenoside Rgl can't promote the maturation of imDCs, it can up-reguate the expression of I-A/I-E molecules in mDCs. In addition, it can strengthen the phagocytosis of imDCs. Finally, it may enhance the antigen-presenting capability of mDCs.
3. The differently expressing genes in DCs stimulated by ginsenoside Rgl are involved in multiple processing including the migration, phagocytosis, antigen-presenting and secreting cytokines in DCs. It is worthwhile that ginsenoside Rgl may up-regulate the EGFR-MAPK passway in order to secret IL-10 which influences the Th polarization and regulate the immune response.
In conclusion, though ginsenoside Rgl can't promote the maturation of imDCs, it can strenghthen the photocytosis, antigen-presenting and the secretion of cytokines in murine DCs. These might be involed in multiple targets. Microarray and Western blot results indicated that ginsenoside Rgl might up-regulate the EGFR-MAPK passway to aid in secreting cytokines, which regulates the immune responses. However, whether the effect is direct or not need to be proved by further experiments such as biosensors or iRNA technology. What's more, ginsenoside Rgl enhances the specific immunity in OVA-immunized mice.
This study proves that ginsenoside Rg1 has two-diection immune-regulation and up-regulate the EGFR-MAPK passway, which provides the experimental evidence for the therapeutic principles of strenghthening the Vital and Dispenlling the Pathogen in TCM, the two-direction regulation of ginseng as well as ginsenoside Rgl as the main composition of new anti-tumor Chinese medicines.
人参是传统的补药,具有延长寿命和增强人体抗病能力的作用,可有效地抗应激、抗疲劳、抗肿瘤以及抗氧化等作用。人参皂苷是其主要有效成分,其中以人参皂苷Rg1含量较高。对人参的药物基因组学和调节阴阳平衡研究表明,人参皂苷Rgl具有抗肿瘤作用和类固醇激素样作用。树突状细胞(DC)是功能最强的专职抗原提呈细胞,它可捕获肿瘤细胞分泌的可溶性抗原,以MHCⅠ类分子或Ⅱ类分子限制的方式提呈给CD8+T细胞或CD4+T细胞,使之活化并发挥抗瘤效应。其中CD4+T细胞还参与激活B细胞、巨噬细胞、自然杀伤细胞(NK)和细胞毒淋巴细胞(CTL),协同发挥抗肿瘤作用。人参皂苷Rg1作为人参的主要成分,可提高小鼠的非特异性免疫功能,但其机制尚不清楚,是否能通过影响DC功能而提高免疫应答有待进一步研究。基因芯片在研究药物对机体免疫系统的调节,寻找药物靶向方面具有优越性,本课题运用功能基因芯片技术,研究与DC功能相关的基因,以期找到人参皂苷Rg1作用于DC的相关分子,明确人参皂苷Rg1发挥免疫调节作用的信号通路,为中医药“扶正祛邪”治疗原则以及人参的双向免疫调节作用提供实验依据,也为人参皂苷Rg1作为临床抗肿瘤新中成药的组成成份提供明确的生物学基础和实验依据。
方法
1、实验一针对人参皂苷Rg1是否影响卵清蛋白(OVA)免疫小鼠特异性免疫应答能力进行研究。以OVA为抗原,混合人参皂苷Rgl或氢氧化铝(Al(OH)3)佐剂,颈部皮下免疫BALB/c小鼠,间隔2周,免疫三次。分为生理盐水对照组(NS组)、人参皂苷Rgl对照组(Rg1组)、铝盐对照组(A1组)、OVA组、OVA+Al组和OVA+Rg1组共六组。免疫三次后第10天,采用MTT比色法测小鼠脾淋巴细胞的增殖能力、用间接ELISA法分别测总血清IgG、IgG1、IgG2a、IgG2b变化以及血清白细胞介素-4(IL-4)、γ干扰素(IFN-γ)的分泌水平等指标的变化,探讨人参皂苷Rgl对BALB/c小鼠免疫调节作用的影响。
2、实验二以体外培养小鼠骨髓来源树突状细胞模型为研究对象,以粒细胞及巨噬细胞集落刺激因子(GM-CSF)和IL-4刺激骨髓造血干细胞,从而使之朝树突状细胞方向分化。待培养至第7天收集细胞,加入不同药物刺激24小时,分为培养基对照组(Ⅰ组)、人参皂苷Rg1组(Ⅱ组)、脂多糖(LPS)组(Ⅲ组)和人参皂苷Rg1+LPS组(Ⅳ组)共四组。培养过程使用相差显微镜观察DC的形态变化,并在培养结束时采用流式细胞术检测各组DCs标志表面分子MHC-Ⅱ、CD40和CD86的表达情况,探讨人参皂苷Rgl能否能促进树突状细胞的成熟以及人参皂苷Rg1作用于DC的可能机制。
3、实验三中,对于树突状细胞吞噬抗原能力的检测,收集培养6天的骨髓造血干细胞(即未成熟DCs),加入不同浓度的人参皂苷Rg1刺激24小时后,加入FITC-OVA避光孵育1小时,最终冰上终止反应5分钟,流式细胞仪检测树突状细胞吞噬FITC-OVA的荧光强度。对于DCs处理提呈抗原能力的检测,收集培养6天的骨髓造血干细胞(即未成熟DCs),加入不同浓度的人参皂苷Rg1刺激细胞24小时后,用SIINFEKL肽段刺激,再加入1μg/ml的LPS刺激16小时后和B3Z T细胞孵育,通过检测上清中IL-2的水平判断致敏后的树突状细胞对B3Z T细胞的活化能力。细胞因子的检测分组和实验二相同,加入药物刺激后为检测IL-12分泌水平的孵育时间为24小时,而为检测IL-4和IL-10分泌水平的孵育时间为48小时,利用间接ELISA法检测树突状细胞培养上清中细胞因子IL-12、IL-4、IL-10水平。进一步揭示人参皂苷Rgl免疫调节作用的机制。
4、实验四分组和实验二相同,以GM-CSF和IL-4刺激骨髓造血干细胞,从而使之朝树突状细胞方向分化。待培养至第7天收集细胞,加入不同药物刺激24小时,分为培养基对照组、人参皂苷Rg1组、LPS组和人参皂苷Rgl+LPS组共四组。培养结束后,抽提树突状细胞总RNA,利用树突状和抗原呈递细胞基因芯片对细胞功能相关基因进行检测,以筛选人参皂苷Rgl作用于DC的差异表达基因,为进一步寻找药物作用靶点提供了线索。
5、实验五通过分析总结实验四的结果找出感兴趣的目的基因,以GAPDH为管家基因,提取细胞RNA,以等量RNA为模板,以各基因特异引物,采用半定量RT-PCR检测EGFR和p44 mRNA表达水平；不同药物和DC孵育后,裂解细胞提取蛋白质,总蛋白跑SDS-PAGE后转膜,采用Western blot法分析EGFR和ERK1/2蛋白表达水平从而进一步证实人参皂苷Rg1是否影响EGFR-p44 MAPK途径对DC产生调控作用。
结果
1、用人参皂苷Rg1、铝盐佐剂以及卵清蛋白(OVA)免疫各组小鼠,体重并无明显差异。OVA特异性总IgG抗体水平的OD值,按照从小到大的顺序依次是：OVA<(OVA+Rg1)<(OVA+A1),其中(OVA+Rg1)组明显高于OVA组(p<0.01);(OVA+Rg1)组IgG1、IgG2a、IgG2b、IgG3抗体水平明显高于OVA组(p<0.05);(Al+OVA)组IgG1、IgG3.IgG2b抗体具有较高水平,IgG2a低水平(p>0.05).OVA组与NS组相比,IgG1和IgG3具有较高水平；此外,(OVA+Rg1)组免疫小鼠脾细胞对OVA抗原刺激的增殖反应在各组中最高；ELISA结果显示(OVA+Rg1)组脾细胞分泌的IFN-γ含量最高,且(OVA+Rg1)组和OVA组存在明显差别(p=0.03)；而(OVA+Al)组与OVA组相比分泌更高水平IL-4(p=0.01)。
2、体外培养BM-DC 7d后,相差显微镜下可见典型形态的树突状细胞,收集总DC数超过85%。40lg/ml的人参皂苷Rgl处理不成熟BM-DC 24小时后,表面分子I-A/I-E、CD40和CD86变化不显著；加入LPS后,DC表面的I-A/I-E、CD40和CD86分子均显著升高(p<0.05,与对照组相比)；人参皂苷Rgl和LPS同时加入DC时,细胞表面的I-A/I-E分子表达显著性升高(p<0.05,与LPS组相比),而CD40和CD86升高不明显。
3、人参皂苷Rg1可以在体外促进未成熟DC分泌IL-12,和LPS同时作用时,有协同增强作用；此外人参皂苷Rgl还能促进未成熟树突状细胞分泌IL-4和IL-10；人参皂苷Rg1可以显著增强未成熟树突状细胞吞噬FITC-OVA抗原,并且促使成熟树突状细胞处理抗原肽,并提呈给T细胞使之活化分泌IL-2。
4、树突状细胞RNA提取A260/A280的比值在2.0左右,变性琼脂糖凝胶电泳RNA条带清晰,28s:18s rRNA条带亮度接近2：1；经终浓度为40μg/ml的人参皂苷Rg1和/或1μg/ml LPS处理24小时后,人参皂苷Rgl组与对照组相比较(Ⅱ/Ⅰ)上调≥2倍基因23个,下调≥2倍基因45个；LPS组与对照组相比较(Ⅲ/Ⅰ)上调≥2倍基因15个下调≥2倍基因47个；人参皂苷Rg1+LPS组与人参皂苷Rg1组相比(Ⅳ/Ⅱ)上调≥2倍基因35个,下调≥2倍基因15个；人参皂苷Rg1+LPS组与LPS组相比(Ⅳ/Ⅲ)上调≥2倍基因37个,下调≥2倍基因10个,在差异表达基因中,给予人参皂苷Rg1后,Erbb2和Ifi44基因在DCs呈现较高转录水平。
5、从基因芯片结果中找出感兴趣的差异表达基因Erbb2和Ifi44,进一步利用半定量RT-PCR和Western blot法检测Rgl组、LPS组以及Rg1+LPS组分别处理DC后的EGFR.p44基因的转录水平及蛋白质表达水平。其中Rgl组与对照组相比,两条基因的结果条带清晰,EGFR.p44基因转录水平明显增加；Western blot法分析EGFR和p44蛋白分泌水平均上调。
结论
1、人参皂苷Rg1能增强卵清蛋白免疫小鼠的特异性免疫应答。
2、人参皂苷Rg1不能促进未成熟树突状细胞的成熟,但能促进成熟DCs高表达I-A/I-E分子；人参皂苷Rg1能增强未成熟DCs的抗原吞噬能力；人参皂苷Rgl能促进成熟DCs的抗原提呈能力。
3、人参皂苷Rg1对DCs功能基因的调控涉及到DC的迁移、抗原吞噬、处理和提呈、分泌细胞因子等各个环节,尤其是能通过EGFR-MAPK途径调控下游细胞因子IL-10的产生,从而影响T细胞的极化状态调节免疫应答。
综上所述,尽管人参皂苷Rgl不能促进未成熟树突状细胞的成熟,但能增强小鼠树突状细胞的吞噬抗原、处理呈递抗原的能力并影响其产生细胞因子。上述作用可能涉及到多个靶点,通过本实验研究可以明确人参皂苷Rg1能够上调EGFR-MAPK途径调控DC功能而调节下游细胞因子IL-10的产生,从而调控免疫应答。这种作用是否是直接作用抑或是间接作用,需要进一步实验证明,下一步拟利用生物传感器或iRNA干扰技术进一步明确人参皂苷Rg1是否直接作用于EGFR分子。除此之外,我们的实验结果表明人参皂苷Rgl能辅助增强小鼠特异性免疫应答能力。
本研究的实验结果证明了人参皂苷Rg1具有双向的免疫调节作用,探讨了人参皂苷Rgl作用于树突状细胞的分子机制及其发生作用的信号通路,为中医药“扶正祛邪’治疗原则以及人参的双向免疫调节作用提供实验依据,并为中医药调节免疫功能研究提供一个新的思路和方法,最终为将来人参皂苷Rg1作为临床抗肿瘤新中成药的组成成份提供明确的生物学基础和实验依据。
In Chinese medicine, ginseng has long been used as a general tonic to promote longevity and enhance bodily functions. It has also been claimed to be effective in combating stress, fatigue, oxidants and cancer.Ginsenoside Rgl is one of ginseng's active ingredients and has anti-tumor, angiomodulating and steroid-like activities through investigating its pharmacogenomics from the perspective of the Yin/Yang therory. Though celluar immunity mediated by T cells、NK cells and macrophages is predominant in tumor immunity, humoral immunity plays an important role in the virus-induced tumors. However, dendritic cells (DC), which is the most powerful professional antigen presenting cells, can capture soluble antigens secreted by tumor cells, present them to be recongnized by CD4+T cells by the MHCⅠor MHCⅡmolecules restricted manner and finally activate CD8+T or CD4+T cells which play anti-tumor effect. In addition, CD4+T cells also involved in activation of B cells, macrophages, NK cells and CTL, synergistic antitumor effects. It is reported that ginsenoside Rgl as the main component of ginsenoside improved the non-specific immune function in mice, however, wheather this enhancement is mediated by regulating the functions of DCs remains unknown. Gene chip technology has advantages in studying the regulation of the immune system and finding out the key molecules on the immune cells influence by drugs. This study, is focused on the influence which ginsenoside Rgl has on the specific immune responses in BALB/c mice as well as the key molecules on DCs which ginsenoside Rgl targets by gene chip technology, in order to find out the target signal passway of ginsenoside Rgl on DCs and provide the experimental evidence for the therapeutic principles of strenghthening body resistance and eliminating evil in TCM, the two-direction regulation of ginseng as well as ginsenoside Rg1 as the main composition of new anti-tumor Chinese medicines.
Method
1. At first, the influence of ginsenoside Rg1 on immune responses immunized by ovalbumin (OVA) in BALB/c mice was investigated.50μg OVA mixed ginsenoside Rgl or aluminum hydroxide (Al (OH) 3) adjuvant, were given to immunize BALB/c mice three times at a two-week interval.10 days after three immunizations, lymphocyte proliferation, specific total IgG and IgG subclass, cytokines IL-4 and IFN-y secreted by splenocytes from culture supernatants were measured respectively by MTT assay and ELISA.
2. The influence of ginsenoside Rg1 on the surface molecules of bone marrow derived dendritic cells. After culturing bone marrow derived cells stimulated by Granulocyte macrophage colony stimulating factor (GM-CSF) and Interleukin-4 for 7 days, bone marrow derived dendritic cells (BM-DCs) were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours. Finally, the expression of the surface molecule I-A /I-E, CD40 and CD86 were detected by flow cytometry in order to investigate whether ginsenoside Rg1 could promote immature DCs to mature.
3. In this study, the impacts of ginsenoside Rgl on the secretion of cytokines, phagocytosis and antigen-presenting of murine bone marrow dendritic cells were analyzed. After culturing bone marrow derived cells stimulated by GM-CSF and Interleukin-4 for 7 days, BM-DCs were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours or 48 hours. Then culture supernatant were collected and the sectretion of cytokines IL-12, IL-4, IL-10 levels by an indirect ELISA assay. In addition, after BM-DCs were treated by ginsenoside Rgl and then FITC-OVA for 1 hour, the phagocytosis was detected by flow cytometry. Finally, the influence of ginsenoside Rg1 on antige-presenting of BM-DCs were detected by IL-2 secreted from active B3Z T cells.
4. The influence of ginsenoside Rg1 on the expression of functional gene from mouse bone marrow-derived DC was analyzed by gene chip technique. After culturing bone marrow derived cells stimulated by GM-CSF and Interleukin-4 for 7 days, BM-DCs were treated by different concentrations of ginsenoside Rg1 and LPS for 24 hours and then the total RNA was extracted. Oligo dendritic &antigen presenting cell gene chips were utilized to find out the difference genes.
5.With GAPDH as the housekeeping gene, was extracted RNA, the same amount of RNA as template, the gene specific primers, using semi-quantitative RT-PCR and semi-quantitative detection of p44 mRNA expression levels EGFR; DC lysis cell extracts after incubation of protein, total protein run SDS-PAGE after the transfer film by Western bolt analysis EGFR and p44 protein secretion, and whether the aforementioned cytokine ELISA assay consistent with the results, thus confirming whether ginsenoside Rg1 by EGFR-p44 MAPK pathway on the regulation of DC generation.
Result
1. Among the different groups, body weights did not differ significantly. OD values of OVA-specific IgG antibodies responses are ranging in descending order:OVA<(OVA+ Rgl)<(OVA+Al). Among them, those from (OVA+Rg1) group were significantly higher than those from OVA group (p<0.01). In addition, IgG1, IgG2a, IgG2b, IgG3 antibody levels from(OVA+Rg1) group were significantly higher than those from OVA group (p<0.05); IgG1, IgG3, IgG2b levels from (Al+OVA) group were obviously higher while its IgG2a responses didn't increase significantly compared with the NS group. What's more, IgG1 and IgG3 responses from OVA-immunized alone group were evidently higher than those from NS group. In cellular immunity, the stimulation index of splenocytes from (OVA+Rg1) group was significantly enhanced compared with that from OVA group. Finally, the ELISA results showed that IFN-γlevels from (OVA+Rg1) group were the highest, and significant difference existed between that from (OVA+Rgl) group and OVA group (p=0.03) while (OVA+Al) groups secreted significantly higher levels of IL-4 compared with OVA group.
2. After cultivating bone marrow derived hematopoietic stem cells for 7 days, more than 85% of the dendritic cells were collected. The surface molecule I-A/I-E, CD40 and CD86 on DCs did not change significantly after DCs were treated with ginsenoside Rgl at a concentration of 40μg/ml. However, after adding LPS, those were significantly higher (p <0.05, compared with the media control group). In addition, the cell surface I-A/I-E molecules on DCs from ginsenoside Rgl plus LPS group was significantly increased in (p <0.05, compared with LPS group) while CD40 and CD86 remained unchanged.
3. Ginsenoside Rgl not only promoted DC in vitro to secrete higher levels of IL-12 but also strengthened this promotion if added with LPS simultaneously. Secondly, ginsenoside Rgl promoted immature dendritic cells to secrete IL-4 and IL-10 molecules. What's more, ginsenoside Rgl had an obvious influence on the phagocytosis of FITC-OVA antigen for immature DCs.Finally, ginsenoside Rgl promoted mature DCs to process and present peptides which activated B3Z T cells to secrete obviously higher levels of IL-2.
4. A260/A280 ratios were about 2.0 for RNA extraction from dendritic cells. In denaturing agarose gel electrophoresis, bands were clear and 28s rRNA band intensity is nearly twice as much as that for 18s rRNA.There were 23 genes whose ratios of Rgl group to media group (Ⅱ/Ⅰ) were above 2, and 45 genes below 0.5. As for those of LPS group to media group (Ⅲ/Ⅰ),there were 15 genes above 2 and 47 below 0.5. In addition, there were 35 above 2 and 15 below 0.5 for the ratios of Rgl+LPS to Rgl (Ⅳ/Ⅱ) while there were 37 above 2 and 10 below 0.5 for (Ⅳ/Ⅲ). Among those differently expressing genes, Erbb2 and Ifi44 showed obviously expressed in DCs stimulated by ginsenoside Rg1.
5. The results of semi-quantitative RT-PCR and Western Blot indicated that in DCs stimulated by ginsenoside Rgl, both EGFR and p44 which is also called ERK2, assumed obviously higher transcription and expression levels, compared with the media group.
Conclusion
1. Ginsenoside Rgl can enhance the specific immune responses in OVA-immunized mice.
2. Though ginsenoside Rgl can't promote the maturation of imDCs, it can up-reguate the expression of I-A/I-E molecules in mDCs. In addition, it can strengthen the phagocytosis of imDCs. Finally, it may enhance the antigen-presenting capability of mDCs.
3. The differently expressing genes in DCs stimulated by ginsenoside Rgl are involved in multiple processing including the migration, phagocytosis, antigen-presenting and secreting cytokines in DCs. It is worthwhile that ginsenoside Rgl may up-regulate the EGFR-MAPK passway in order to secret IL-10 which influences the Th polarization and regulate the immune response.
In conclusion, though ginsenoside Rgl can't promote the maturation of imDCs, it can strenghthen the photocytosis, antigen-presenting and the secretion of cytokines in murine DCs. These might be involed in multiple targets. Microarray and Western blot results indicated that ginsenoside Rgl might up-regulate the EGFR-MAPK passway to aid in secreting cytokines, which regulates the immune responses. However, whether the effect is direct or not need to be proved by further experiments such as biosensors or iRNA technology. What's more, ginsenoside Rgl enhances the specific immunity in OVA-immunized mice.
This study proves that ginsenoside Rg1 has two-diection immune-regulation and up-regulate the EGFR-MAPK passway, which provides the experimental evidence for the therapeutic principles of strenghthening the Vital and Dispenlling the Pathogen in TCM, the two-direction regulation of ginseng as well as ginsenoside Rgl as the main composition of new anti-tumor Chinese medicines.
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