【摘要】：大豆皂苷(Soyasaponin)主要分布于大豆等豆科植物中,屬于結(jié)構(gòu)復(fù)雜的五環(huán)三萜類(lèi)齊墩果烷型皂苷。大豆皂苷具有多種生理活性,在醫(yī)藥、保健品等領(lǐng)域的應(yīng)用具有重要意義和廣闊前景。大豆皂苷種類(lèi)較多,其中大豆皂苷Ab是大豆皂苷復(fù)合物提純和分離后所得的主要皂苷單體。目前對(duì)于大豆皂苷的生物活性研究多集中在大豆皂苷復(fù)合物上,對(duì)于其單體的研究報(bào)道較少。本文采用從大豆胚芽粉中提純得到的大豆皂苷Ab為研究對(duì)象,通過(guò)體外實(shí)驗(yàn)研究了大豆皂苷對(duì)細(xì)胞的毒性作用、免疫刺激活性并初步探討了相關(guān)分子信號(hào)機(jī)制。溶血性實(shí)驗(yàn)結(jié)果表明,與皂樹(shù)皮皂苷相比,大豆皂苷Ab的溶血性極弱。大豆皂苷Ab濃度在10 mg/mL以?xún)?nèi)幾乎無(wú)溶血性,當(dāng)大豆皂苷Ab濃度達(dá)到20 mg/mL時(shí)溶血率僅約為7%,而對(duì)照組的皂樹(shù)皮皂苷在20μg/mL時(shí)已經(jīng)使紅細(xì)胞完全溶血。此外,MTT實(shí)驗(yàn)結(jié)果表明,大豆皂苷Ab對(duì)RAW264.7細(xì)胞幾乎沒(méi)有毒性。在大豆皂苷Ab濃度為1~20μg/mL時(shí),RAW264.7細(xì)胞存活率接近100%;而皂樹(shù)皮皂苷在濃度為5μg/mL時(shí),相應(yīng)的細(xì)胞存活率僅50%左右,當(dāng)皂樹(shù)皮皂苷濃度增加至20μg/mL時(shí),細(xì)胞存活率僅為6%左右。以上結(jié)果表明,大豆皂苷Ab的體外細(xì)胞毒性極低。ELISA實(shí)驗(yàn)結(jié)果表明,大豆皂苷Ab(0~200μg/mL)促進(jìn)RAW264.7細(xì)胞釋放細(xì)胞因子TNF-α和IL-1β,并且釋放水平隨大豆皂苷Ab濃度的增大而提高;雙熒光素酶實(shí)驗(yàn)結(jié)果表明,大豆皂苷Ab可激活NF-κB轉(zhuǎn)錄因子。這些結(jié)果表明,大豆皂苷具有一定的免疫刺激活性。Western blot實(shí)驗(yàn)結(jié)果表明,將特異性沉默TLR4表達(dá)的siRNA轉(zhuǎn)染RAW264.7細(xì)胞后,有效抑制了TLR4的表達(dá),其抑制效率大于60%,并且轉(zhuǎn)染了TLR4 siRNA組細(xì)胞在用大豆皂苷Ab作用后所釋放的TNF-α、IL-1β水平顯著低于未轉(zhuǎn)染TLR4 siRNA組,NF-κB信號(hào)分子的活性也顯著低于未轉(zhuǎn)染siRNA2對(duì)照組。這些結(jié)果顯示,TLR4可能在大豆皂苷Ab引起的RAW264.7細(xì)胞釋放細(xì)胞因子TNF-α和IL-1β及NF-κB信號(hào)轉(zhuǎn)導(dǎo)通路過(guò)程中發(fā)揮了重要功能。
[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- 偽 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- 偽 -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- 偽, IL-1 尾 and NF- 魏 B by RAW264.7 cells induced by daidzein Ab.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R151;TS201.2

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