【作者】 田庆锷；

【导师】 李焕德；

【作者基本信息】 中南大学 ， 药理学， 2013， 博士

【摘要】 目的：探讨黄芪多糖(Astragalus polysaccharide APS)体内外是否具有化疗增敏作用及其可能机制。方法：荷H22小鼠肝癌细胞株皮下移植瘤小鼠经腹腔给以APS及ADM,观察其体内对移植瘤生长的影响及对昆明小鼠主要脏器的毒性。用ELISA方法检测血清中IL-1a、IL-2、IL-6、TNF-α、IL-10等细胞因子的表达；用Western blot和荧光定量PCR检测肿瘤组织中的P-GP和MDR1mRNA表达。体外用MTT法检测APS单独和协同化疗药对H22/ADM敏感性；用Western blot和荧光定量PCR检测H22/ADM耐药细胞株的P-GP和MDR1mRNA表达；用R-123摄取实验研究不同浓度APS对H22/ADM细胞P-GP功能的影响。结果：各实验组KM小鼠皮下接种部位成瘤率100%,荷H22小鼠肝癌细胞株皮下移植瘤模型成功；体内实验表明,在剂量50mg/kg～200mg/kg范围内APS能减轻ADM所导致的KM小鼠体重下降,但对肝、心、肾、肺重量及其脏器系数无明显影响。在剂量100mg/kg～200mg/kg范围内APS能减轻由AMD导致的胸腺减小,在剂量50mg/kg～200mg/kg范围内APS能减轻由AMD导致的脾脏减小。在剂量50mg/kg～200mg/kg范围内APS联用ADM对荷瘤小鼠肿瘤生长有较强的抑制作用；在剂量50mg/kg～200mg/kg范围内,含APS组IL-1a、IL-2、IL-6、TNF-α等因子表达增加,而IL-10因子表达减少,与ADM组比较均有显著性差异(P<0.05),APS具有辅助ADM抗肿瘤作用机制之一可能与APS增加IL-1a、IL-2、IL-6、TNF-α等因子表达,而减少IL-10因子产生有关；在APS剂量50mg/kg～200mg/kg范围内MDR1mRNA和P-GP的表达随着APS剂量增加而减小,呈浓度依赖性。与ADM或ADM+RFP组比较,ADM+APS (200mg/kg)组MDR1mRNA和P-GP的表达明显减小(P<0.05)。APS能下调MDR1mRNA水平,减少P-GP表达,因此可能增加化疗药物在肿瘤细胞内的浓度,这也许是APS增强ADM的化疗敏感性,辅助ADM抗肿瘤作用的另一机制。体外研究发现,在终浓度0.8-500mg/mL范围,APS对H22/ADM耐药细胞株无抗肿瘤作用,但可增强5-Fu、 DDP、VP-16或CTX等常用化疗药物对H22/ADM耐药细胞株的化疗敏感性；APS可以使R-123潴留增加,对P-GP外排功能表现为抑制作用；APS下调MDR1mRNA作用随APS浓度增加而作用增强,呈浓度依赖性,APS各组MDR1mRNA表达随干预时间的延长而减少,均具有时间依赖性；在同一时间内,APS下调P-GP表达随APS浓度增加而增强,呈浓度依赖性,同一浓度的APS下调P-GP表达随干预时间的延长而增强(72h>48h>24h),呈时间依赖性。对于H22/ADM耐药细胞株,APS下调MDR1mRNA和P-GP的表达,抑制P-GP外排功能可能是APS增强耐药细胞株H22/ADM的化疗敏感性的机制之一。结论：APS具有辅助抗肿瘤作用,其机制可能与APS增加IL-1a、 IL-2、IL-6、TNF-α等因子表达,而减少IL-10因子产生,以及降低P-GP外排功能,下调P-GP和MDR1mRNA表达有关。更多还原

【Abstract】 Objective:To investigate the adjunct anticancer role of Astragalus polysaccharides in H22tumor-bearing mice and H22/ADM cell lines.Methods:H22tumor-bearing mice were treated by APS and ADM intraperitoneally, after ten days of treatment, blood samples were collected from mouse eyes and serum was harvested by centrifugation. Mice were sacrificed, and the whole body, tumor, spleen, and thymus were weighed immediately. The rate of tumor inhibition and organ indexes were calculated. The expression levels of cytokines in sera and the expression levels of P-glycoprotein (P-GP) and multidrug resistance (MDR)1mRNA in tumor tissues were detected using enzyme-linked immunosorbent assay, Western blotting, and quantitative myeloid-derived suppressor cells reverse transcription-polymerase chain reaction, respectively. H22/ADM cell lines were treated with different concentrations of APS and/or the most common chemotherapy drugs, such as Cyclophosphamid, Adriamycin,5-Fluorouracil, Cisplatin, Etoposide, and Vincristine. Chemotherapeutic drug sensitivity, P-glycoprotein function and expression, and MDR1mRNA expression were detected using MTT assay, flow cytometry, Western blotting, and quantitative RT-PCR.Results:The tumor inhibition rates in the treatment groups: Adriamycin (ADM)+Astragalus polysaccharides (APS)(50mg/kg), ADM+APS (100mg/kg), and ADM+APS (200mg/kg) were significantly higher than in the ADM group. The spleen indexes of the above groups were also significantly higher than in the ADM group, and the thymus indexes of the ADM+APS (100mg/kg), ADM+APS (200mg/kg) groups were significantly higher than in the ADM group. APS was found to exert a synergistic anti-tumor effect with Adriamycin and to alleviate the decrease in the sizes of the spleen and thymus induced by AMD. The expression of interleukin-1alpha (IL-la), interleukin-2(IL-2), interleukin-6(IL-6), and tumor necrosis factor-a (TNF-a) were significantly higher in the ADM+APS (50mg/kg), ADM+APS (100mg/kg) and ADM+APS (200mg/kg) groups than in the ADM group; and interleukin-10(IL-10) was significantly lower in the above groups than in the ADM group. Results show that APS could increase IL-la, IL-2, IL-6, and TNF-a expression and decrease IL-10levels. Compared to the ADM group, APS treatment at a dose concentration of50-200mg/kg could down regulate MDRl mRNA expression in a concentration-dependent manner. The expression level of P-GP was significantly lower in the ADM+APS (200mg/kg) group than in the ADM group.When used alone, APS had no anti-tumor activity in H22/ADM cells in vitro. However, it can increase the cytotoxicity of certain chemotherapy drugs, such as Cyclophosphamid, Adriamycin,5-Fluorouracil, Cisplatin, Etoposide, and Vincristine, in H22/ADM cells. It acts in a dose-dependent manner. Compared to a blank control group, APS increased intracellular Rhodamine-123retention and decreased P-glycoprotein efflux function in a dose-dependent manner. These factors were assessed24h,48h, and72h after administration. APS down regulated P-glycoprotein and MDR1mRNA expression in a concentration-dependent manner within a final range of0.8-500mg/L and in a time-dependent manner from24-72h.Conclusion:The adjunct anticancer mechanism of astragalus polysaccharide in vivo and in vitro may be related to its ability to increase expression of IL-la, IL-2, IL-6, and TNF-a, decrease IL-10, and down regulate MDR1mRNA and P-GP expression levels.更多还原