大豆皂苷Ab的体外细胞毒性和免疫刺激活性研究

发布时间：2018-09-14 14:25

【摘要】：大豆皂苷(Soyasaponin)主要分布于大豆等豆科植物中,属于结构复杂的五环三萜类齐墩果烷型皂苷。大豆皂苷具有多种生理活性,在医药、保健品等领域的应用具有重要意义和广阔前景。大豆皂苷种类较多,其中大豆皂苷Ab是大豆皂苷复合物提纯和分离后所得的主要皂苷单体。目前对于大豆皂苷的生物活性研究多集中在大豆皂苷复合物上,对于其单体的研究报道较少。本文采用从大豆胚芽粉中提纯得到的大豆皂苷Ab为研究对象,通过体外实验研究了大豆皂苷对细胞的毒性作用、免疫刺激活性并初步探讨了相关分子信号机制。溶血性实验结果表明,与皂树皮皂苷相比,大豆皂苷Ab的溶血性极弱。大豆皂苷Ab浓度在10 mg/mL以内几乎无溶血性,当大豆皂苷Ab浓度达到20 mg/mL时溶血率仅约为7%,而对照组的皂树皮皂苷在20μg/mL时已经使红细胞完全溶血。此外,MTT实验结果表明,大豆皂苷Ab对RAW264.7细胞几乎没有毒性。在大豆皂苷Ab浓度为1~20μg/mL时,RAW264.7细胞存活率接近100%;而皂树皮皂苷在浓度为5μg/mL时,相应的细胞存活率仅50%左右,当皂树皮皂苷浓度增加至20μg/mL时,细胞存活率仅为6%左右。以上结果表明,大豆皂苷Ab的体外细胞毒性极低。ELISA实验结果表明,大豆皂苷Ab(0~200μg/mL)促进RAW264.7细胞释放细胞因子TNF-α和IL-1β,并且释放水平随大豆皂苷Ab浓度的增大而提高;双荧光素酶实验结果表明,大豆皂苷Ab可激活NF-κB转录因子。这些结果表明,大豆皂苷具有一定的免疫刺激活性。Western blot实验结果表明,将特异性沉默TLR4表达的siRNA转染RAW264.7细胞后,有效抑制了TLR4的表达,其抑制效率大于60%,并且转染了TLR4 siRNA组细胞在用大豆皂苷Ab作用后所释放的TNF-α、IL-1β水平显著低于未转染TLR4 siRNA组,NF-κB信号分子的活性也显著低于未转染siRNA2对照组。这些结果显示,TLR4可能在大豆皂苷Ab引起的RAW264.7细胞释放细胞因子TNF-α和IL-1β及NF-κB信号转导通路过程中发挥了重要功能。
[Abstract]:Soybean saponins (Soyasaponin) are mainly distributed in soybean and other legumes and belong to pentacyclic triterpenoid oleanolic saponins. Soybean saponins have a variety of physiological activities and have important significance and broad prospects in medicine, health products and other fields. There are many kinds of soybean saponins, among which daidzein Ab is the main saponins monomer after purification and separation of soybean saponins complex. At present, the bioactivity of soybean saponins is mainly focused on the soybean saponins complex, but there are few reports on the monomers of soybean saponins. In this paper, daidzein Ab extracted from soybean germ powder was used to study the cytotoxicity and immunostimulatory activity of soybean saponin in vitro. The hemolytic results showed that the hemolysis of daidzein Ab was very weak compared with that of saponin. The hemolysis rate of soybean saponin Ab was only about 7 when the concentration of Ab reached 20 mg/mL, while that of the control group had been completely hemolytic at 20 渭 g/mL. In addition, daidzein Ab showed little toxicity to RAW264.7 cells. The survival rate of RAW264.7 cells was close to 100 when the concentration of soja saponin Ab was 1 ~ 20 渭 g/mL, but the cell survival rate was only about 50% when the concentration of saponin was 5 渭 g/mL, and the cell survival rate was only 6% when the concentration of saponin increased to 20 渭 g/mL. The results showed that the cytotoxicity of daidzein Ab in vitro was very low. Elisa results showed that daidzein Ab (0 ~ 200 渭 g/mL) promoted the release of cytokines TNF- 伪 and IL-1 尾 from RAW264.7 cells, and the release level increased with the increase of Ab concentration. The results of double luciferase assay showed that daidzein Ab could activate NF- 魏 B transcription factor. These results indicated that daidzein had a certain immunostimulatory activity. Western blot assay showed that the expression of TLR4 was effectively inhibited by transfection of siRNA specifically silencing the expression of TLR4 into RAW264.7 cells. The inhibitory efficiency was more than 60%, and the level of TNF- 伪 -kappa 尾 released by the transfected TLR4 siRNA group was significantly lower than that of the untransfected TLR4 siRNA group. The activity of NF- 魏 B signaling molecules in the transfected TLR4 siRNA group was significantly lower than that in the untransfected siRNA2 group. These results suggest that TLR4 may play an important role in the release of cytokines TNF- 伪, IL-1 尾 and NF- 魏 B by RAW264.7 cells induced by daidzein Ab.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R151;TS201.2

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