【作者】 王莉；

【导师】 陈正行；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2009， 博士

【摘要】 米糠是稻米加工业的主要副产品,是我国丰富而可再生的多糖资源。尽管我国米糠产量居世界之首,但由于我国米糠合理利用的基础理论研究尚处于较低水平,限制了我国米糠潜在价值的进一步精深利用。本论文在系统研究具有抗肿瘤活性米糠多糖RBPS2a提取、分离纯化、结构特征的基础上,通过硫酸酯化改性得到了抗肿瘤活性更显著的硫酸酯化米糠多糖SRBPS2a,并对其体内外抗肿瘤活性机理以及诱导肿瘤细胞凋亡的分子机制进行了较为深入的研究。具有抗肿瘤活性的硫酸酯化米糠多糖国内外还鲜有报道,酯化修饰米糠多糖抑制肿瘤的作用机理和诱导肿瘤细胞凋亡的分子机制国内外也尚属空白,本课题的研究,对于提高米糠多糖的生物利用率,丰富硫酸酯化修饰活性多糖基础理论,都具有非常重要的现实意义和学术价值。运用响应面(RSM)和神经网络(ANNs)分析方法,探讨了米糠多糖超声波提取优化工艺。利用响应面设计研究了超声波功率、超声时间、提取温度和料液比对米糠多糖得率的影响。结果表明,提取温度(X1)与超声功率(X2),二次项中提取温度(X1×X1)以及提取温度与超声功率的交互作用(X1×X2)对响应值米糠多糖的得率存在显著的相关性。首次采用RBF神经网络与QPSO优化算法结合的方法,模拟与优化米糠多糖的超声波提取过程,其最优工艺条件为:提取温度82.5℃、超声波功率335W、提取时间37.5min和料液比18:1。所建立的RBF-QPSO算法对具有多变量、时变性和非线性特征的米糠多糖超声波提取过程的模拟和优化结果优于RSM。以体外抑制肿瘤细胞增殖活性为筛选导向,采用Q-Sepharose big beads离子色谱和Sepharose CL-6B凝胶色谱,分离纯化得到一种具有抗肿瘤活性的多糖组分RBPS2a。高效凝胶色谱(HPGPC)法测得其呈单一峰,重均分子量9×105 Da。RBPS2a的单糖分析、部分酸水解、甲基化分析、红外、核磁共振等分析结果表明,单糖组成为阿拉伯糖、木糖、葡萄糖和半乳糖,摩尔比率4:2:1:4;具有一条以β→(1,3)位键合的吡喃半乳糖主链,有两个分支残基,分别是α-D-木糖-(1→4)-α-D-阿拉伯糖-(1→残基和α-D-葡萄糖-(1→4)-a-D-阿拉伯糖-(1→残基。以肿瘤体外增殖抑制为指标,采用氯磺酸-吡啶法,研究米糠多糖的硫酸酯化,优化得到米糠多糖硫酸酯化修饰的最佳条件为:氯磺酸和吡啶的比例1:4,反应温度70℃,反应时间2 h。采用该工艺得到了取代度1.29,分子量3.5×105 Da,具有显著体外抗肿瘤活性的米糠多糖硫酸酯化衍生物SRBPS2a。通过对比傅立叶红外光谱和13C核磁共振谱图,酯化前后结构的分析结果证明,米糠多糖RBPS2a经硫酸酯化修饰后,主链结构保持β-(1→3)-D-甘露糖不变,侧链被切除,硫酸基团的取代发生在C-2和C-4位上,C-6氧化形成糖醛酸甲酯。硫酸酯化米糠多糖较酯化前的米糠多糖显示出较高的体外抗肿瘤活性,与其酯化后结构的改变有密切关系。通过建立小鼠抑制乳腺癌EMT-6模型,研究了SRBPS2a的体内抗肿瘤作用机理。结果表明,SRBPS2a在高(75 mg/kg?d)、中(50 mg/kg?d)剂量下,均具有较为显著的抑瘤作用,其抑瘤率分别达到55.95%和44.05%。与阳性对照组5-FU相比较,SRBPS2a组小鼠的状态良好且脾指数和胸腺指数均有所提高。HE染色结果显示,SRBPS2a中剂量组和高剂量组均能使EMT-6肿瘤细胞成团索状实性排列或弥散状分布,并发生肿瘤细胞大片坏死,坏死区可见凋亡细胞;免疫组化技术进一步显示,SRBPS2a能诱导肿瘤组织中Bax基因蛋白表达,同时也能显著下调Bcl-2基因蛋白的表达。SRBPS2a体外对HepG-2肿瘤细胞凋亡机制的研究表明,SRBPS2a作用后其细胞形态有明显差异。光镜下,细胞贴壁形态异常,细胞数减少;电镜观察显示,随着SRBPS2a浓度的增加,细胞表面微绒毛逐渐消失,细胞膜皱缩,外凸形成小泡;荧光电镜观察发现,高浓度样品作用下细胞核变小、皱缩,可见致密强荧光。PI单染色流式细胞仪检测发现,SRBPS2a作用后的细胞被阻滞在G2/M期,细胞出现较大的凋亡峰(23.4%),说明SRBPS2a能有效地诱导肿瘤细胞凋亡。以荧光染料PI和Rh123双重染色样作用的细胞,PI (-)和Rh123(-)细胞显著增加,推测SRBPS2a可能在不影响细胞膜完整性的情况下,诱导HepG-2细胞线粒体膜?Ψm下降;采用流式细胞仪检测结果表明,SRBPS2a作用48 h后通过上调HepG-2细胞中促凋亡因子Bax、下调抑凋亡因子Bcl-2,以及上调线粒体依赖的Caspase-3,激活凋亡信号通路,诱导细胞凋亡,从而达到抗肿瘤的作用。更多还原

【Abstract】 Rice bran is the important by-product of the rice milling industry, it was considered as the rich and reproducible resource. Although the yield of rice bran in our country was primacy in the world, the low-level of study on rice bran has limit the intensively utilization potential value of rice bran. In this paper, we studied the isolation, purification and structural feature of RBPS2a, the sulfated rice bran polysaccharide SRBPS2a showed the potent antitumor activities, therefore, the mechanism of antitumor activity on SRBPS2a both in vitro and in vivo and the the molecular mechanism on apoptosis of tumor cells were also investigated. There is limited reported on mechanism of antitumor activity and apoptosis of tumor cells of sulfated rice bran polysaccharide. The present study could improve the utilize value and enrich the basic theory of rice bran polysaccharide.In order to maximize the yield of rice bran polysaccharide, response surface methodology (RSM) and RBF neural network were employed to optimize the ultrasound-assisted extraction condition. The RSM model showed that the yield of rice bran polysaccharide was related to temperature(X1), ultrasonic power(X2), interactions of temperature(X1×X1) and quadratic of temperature and ultrasonic power(X1×X2).According to the RBF- QPSO, the optimum extraction conditions were temperature of 82.5℃, ultrasonic power of 335 W, extraction time of 37.5 min and Water/solid ratio of 18:1. RBF-QPSO was better than RSM in ultrasonic-assisted extraction of rice bran polysaccharide.Based on the bioactivity-directed test, a novel heteropolysaccharide RBPS2a with anti-tumor activity was obtained from rice bran by hot water extraction, ethanol precipitation, and purified by Sepharose CL-6B gel chromatography after Q-Sepharose big beads chromatography. RBPS2a was eluted as a single symmetrical narrow peak on high-performance gel-permeation chromatography (HPGPC) and the average molecular weight was 9×105 Da. Gas chromatography of absolute acid hydrolysate of RBPS2a suggested that it was composed of arabinose, xylose, glucose and galactose with a molar ratio of 4:2:1:4. The Fourier-transform infrared spectra (FT-IR) and 1H, 13C NMR spectroscopy analysis revealed that RBPS2a had a backbone consisting ofβ- (1→3)- linked D- galacopyranosyl residues substitured at O-2 with glycosyl residues composed ofα- D- xylose–(1→4)-α- D- arabinose (1→andα- D- glucose–(1→4)-α- D-arabinose (1→linked residues.Sulfated rice bran polysaccharides (SRBPS), were prepared by chlorosulfonic acid-pyridine (CSA-Pyr) method, according to the antitumor activities test. The optimum modification conditions were reaction temperature of 70℃, the ratio of chlorosulfonic acid to pyridine of 1:4 and the reaction time of 2h. Under this condition, sulfated derivatives SRBPS2a exhibit relatively strong antitumor activity in vitro. The average molecular weight of SRBPS2a was 3.5×105 Da and the degree of sulfation (DS) was 1.29. The Fourier-transform infrared spectra (FT-IR) and 13C NMR spectroscopy analysis revealed that SRBPS2a was mainly consist ofβ- (1→3)- D- galacopyranosyl residues and the sulfate substitution site is on C-2 and C-4, the side chains were cut off during the sulfated reaction. Furthermore, SRBPS2a exhibited evident growth inhibition on Mouse mammary tumor EMT-6 cells both in vitro and in vivo.The antitumor activity and its mechanism of SRBPS2a were examined and evaluated with the mice transplanted EMT-6 tumor in vivo. At the dosage of 75 mg/kg.d and 50 mg/kg.d, SRBPS2a had strong antitumor activity with 55.95% and 44.05%. Compared with the 5-FU group, SRBPS2a could increase both the thymus index and the spleen index. The histopathology of tumors from the various groups indicated that the tumor cells of untreated mice grew vigorously, however the tumor cells from the different SRBPS2a treated groups had clear nucleus pycnosis and necrosis areas in different degree. The expression of tumor gene Bax and Bcl-2 in tumor tissues were also detected with immuno-histochemistry. After the expression between the experimental and control groups, there was obvious difference in the quantities of Bax and Bcl-2 genes expression between the experimental and control group, which indicated that SRBPS2a could control growth of tumor and accelerate the tumor cell apoptosis by influencing the expression related tumor genes.HepG-2 cell line was used to investigate the mechanism of SRBPS2a in vitro. Results showed that the tumor cells treated with SRBPS2a for 48 h showed significant morphological changes, such as cell shrinkage, membrane blebbing and volume reduction. Scanning electron microscope (SEM) photos showed that the villus of the HepG-2 cell was disappeared at higher concentration and the surface of membrane was smooth and shrinkage to form apoptotic bodies. Moreover, typical apoptotic morphological features (volume reduction, chromatin condensation, nuclear fragmentation) were also observed by fluorescent microscopy (Hoechst 33342). Flow cytometric analysis showed that the HepG-2 cell cycle was arrested in the G2/M phase, and the subdiploid peak of DNA characteristic of apoptotic was observed, and the apoptosis rate was about 23.4%. Further investigation results showed that the apoptotic machinery of HepG-2 induced by SRBPS2a was associated with a decrease in Bcl-2/Bax ratio, drop in mitochondrial trans-membrane potential, and upgrade the protein expression of caspase-3 by immunocytochemical staining.更多还原