摘要
多糖具有广泛的生物活性，其肿瘤防治作用引起了大家的关注。目前，全球至少有超过30种多糖在临床进行抗肿瘤、抗艾滋病及治疗糖尿病试验。在日本已有香菇多糖，在我国则有牛膝多糖、云芝多糖、灵芝多糖和猪苓多糖被批准用于临床。本实验室研究表明，低分子苹果多糖对防治结/直肠癌，具有潜在的开发应用价值。然而，天然多糖类药物研发存在以下突出问题：（1）聚糖多以高分子形式存在，分子量范围跨度大，均一性差，结构不清楚，难于质量控制。（2）药物活性重复性差，难于在分子水平阐明其作用机制。（3）大分子多糖在体内很难进行跨膜转运，体内过程不明。（4）提取分离技术与质量控制滞后，现有的提取工艺难以获得结构均一的多糖。低分子化、均一化是实现天然糖类药物质量可控的重要策略。
目的：
建立苹果寡糖的制备、分离纯化、结构鉴定的新方法，观察苹果寡糖的抗肿瘤作用及其机制，为开发具有自主知识产权的苹果寡糖类药物奠定基础。
方法：
1．采用水提醇沉的方法，从苹果渣中分离得到苹果粗多糖，去除蛋白和色素，再经透析分段、DEAE-52和Sepharose CL-4B凝胶柱层析分离得到两个组分AP-1和AP-2B。对苹果多糖和纯化后的两种多糖进行糖含量、分子量和UV、IR、旋光度以及单糖组成表征。
2．采用碱解和酶解相结合的方法，制备苹果寡糖，采用阴离子交换树脂对寡糖混合物进行分离，得到OS-1、OS-2、OS-3、OS-4和OS-5。采用HPLC柱前衍生化法、MS和NMR对OS-5结构进行分析表征。
3．MTT法检测OS-1、OS-2、OS-3、OS-4、OS-5和AP-1对6株肿瘤细胞及人正常上皮细胞HIEC活性的影响，筛选出抗肿瘤效果最好的寡糖进行体外抗肿瘤机制研究。采用OS-5处理HT29细胞，通过流式细胞仪检测HT29细胞凋亡率和细胞周期变化情况，透射电镜法观察细胞形态学的变化；蛋白印迹法检测凋亡和周期相关蛋白的变化情况，同时采用real time PCR法检测相关蛋白mRNA的表达，探讨OS-5抗肿瘤作用机制。
4．复制致炎剂/致癌剂诱导的ICR小鼠结肠癌模型和裸鼠HT29细胞移植瘤模型，进行实验治疗学观察。通过病理组织学方法，观察结肠癌发生情况；通过ELISA和Western Blot检测方法，观察结肠癌小鼠血清TNF-α和Galectin-3含量变化和凋亡相关蛋白的变化。采用肿瘤体积和生存时间等指标，观察OS-5对移植瘤小鼠肿瘤的抑制效应。
结果：
1．经HPSEC分析可知，苹果多糖是包含有三个不同分子量组分的混合多糖，组分1和组分2为均一性多糖，分子量分别为10kD和1900kD。单糖组成分析结果显示，苹果多糖的单糖组成为鼠李糖、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖，摩尔比为1.00:17.67:8.50:4.01:2.03。组分1单糖组成为鼠李糖、半乳糖醛酸和葡萄糖，摩尔比为1.20:10.00:5.25；组分2单糖组成为鼠李糖、半乳糖醛酸、葡萄糖和半乳糖，摩尔比为1.00:20.30:8.50:8.03。
2．HPSEC分析结果表明，OS-5纯度为99.21%，OS-5是由GalA组合而成，分子量为898，其结构是由（1→4）-β和（1→6）-α-D-GalA线性连接形成的半乳糖醛酸寡聚糖，具体结构为α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-β-D-GalAp-(1→4)-α-D-GalAp。
3．HIEC细胞经9μg/mL OS-5处理36h，其增殖率为37.3±1.8%。OS-5对A549、H1299、SMMC7721、HCT116和SW480细胞的活性抑制作用较小。给药36h，9μg/mLOS-5对H1299、SW480细胞活性抑制率分别为24.99±0.6%、23.4±1.4%，对A549细胞和HCT116细胞的抑制率均低于10%。不同浓度OS-5作用于SMMC7721细胞24h后，细胞抑制率均为25%，不呈现浓度依赖性。OS-5对HT29细胞活性抑制作用比较突出，且呈现时间和浓度依赖性。给予HT29细胞9μg/mLOS-5处理36h后，抑制率为52.6±1.8%。电镜观察发现，10μmol/L OS-5处理后的HT29细胞呈现典型凋亡形态。流式细胞术结果表明，10μmol/L OS-5处理后的HT29细胞凋亡率为45.9%（P<0.01），S期细胞占60.86±3.44%，与正常对照组细胞（未用OS-5处理，S期细胞占24.64±1.29%）具有显著性差异（P<0.01），提示OS-5可诱导HT29细胞发生凋亡和S期阻滞。经OS-5处理后的HT29细胞，Bax蛋白表达量明显升高，同时抗凋亡蛋白Bcl-2和Bcl-xl的表达显著降低。OS-5能够显著降低HT29细胞中Cdk2和cyclin B1蛋白的表达，同时增加cyclin A1蛋白表达量。
4．DMH/DSS诱发的结肠癌小鼠给予OS-5后免疫器官指数和结肠长度有所改善，胸腺指数比正常组和模型组显著增高（P<0.05）；HE染色结果显示，OS-5可抑制结肠组织癌细胞的浸润生长现象；OS-5治疗组动物血清中TNF-α水平显著升高（P<0.01），血清中Galectin-3水平比模型组降低，但无显著性差异；Western Blot显示OS-5治疗组结肠黏膜中Bax表达量升高，Bcl-2和Bcl-xl表达降低，模型组黏膜中Bax表达量显著降低（P<0.01）。裸鼠移植瘤实验表明，OS-5对HT29细胞移植瘤具有明显的抑制作用。第35d时，5mg/kg、10mg/kg和20mg/kg OS-5给药组肿瘤体积分别为：529.84±44.01mm~3、443.81±38.94mm~3和358.96±28.68mm~3，与模型组肿瘤体积998.61±30.79mm~3相比，各给药组肿瘤体积明显减小（P<0.05）。模型组裸鼠中位生存时间为38.5d，10mg/kg和20mg/kg OS-5给药组裸鼠中位生存时间分别为39d和47d。
结论：
1．创立了可控性降解获得苹果寡糖的方法，得到了OS-1、OS-2、OS-3、OS-4和OS-5五个组分。
2．经分析鉴定，OS-5的结构为α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-β-D-alAp-(1→4)-α-D-GalAp，未见相同的结构报道。
3．体外实验发现，OS-5对HT29细胞的活性有明显抑制作用，其作用可能与上调Bax同时降低Bcl-2和Bcl-xl表达诱导细胞凋亡，以及通过降低Cdk2和cyclin B1蛋白的表达同时增加cyclin A1蛋白表达使细胞周期阻滞于S期有关。
4．OS-5对DMH/DSS诱发的小鼠结肠癌具有明显的治疗作用，并可显著抑制裸鼠HT29细胞移植瘤的生长，具有进一步开发前景。
The natural polysaccharide possesses a variety of biological activities. At present,more than30polysaccharides are in clinical trials for treatment of cancer, HIV infectionand diabete. Some polysaccharides have been approved to be used in clinic, such aslentinan in Japan, achyranthan, coriolus versicolor polysaccharide and ganoderma lucidumpolysaccharide in China. Our research shows that the low molecular apple polysaccharideinhibited the cancerization of colitis by influencing the expression of TLR-4and Gal-3.This reveals the potential application of apple polysaccharide in treatment colon/rectumcancer. However, there are some problems in the R＆D of natural polysaccharides:⑴Natural polysaccharides exist as the macromolecule forms, it has broad molecular weight range, poor homogeneity. It is hard to control the quality of polysaccharide.⑵It isdifficulty to elucidate the action mechanism of polysaccharide at the molecular level.⑶Itis very difficulty for natural macromolecular polysaccharide to transport in the body, andits pharmacokinetic characteristics in vivo is unclear.⑷It is difficult to extracthomogeneous polysaccharide. So, decrease the molecular weight and homogenization isan important strategy to realize the quality control of natural polysaccharide.
Objective:
⑴to establish a new method to separate, purify and identify the appleoligosaccharides;⑵to investigate the antitumor activities and its mechanism of appleoligosaccharide and lay a foundation for the development of apple oligosaccharide.
Methods:
1. The procedure of water extract-alcohol precipitation was used to isolate applepolysaccharide from apple pomace. Then the protein, pigment and small molecularsubstances were removed. The apple polysaccharide component1(AP-1) and component2(AP-2B) were obtained by dialysis, ion exchange chromatography and gel columnchromatography. The physiochemical properties were determined.
2. Alkali and enzymic hydrolysis were coherently used to preparation appleoligosaccharides. The monosaccharide and oligosaccharides were obtained by using anioncolumn chromatography. The structure of OS-5was investigated by MS and NMR.
3. MTT assay was adopted to investigate the effects of apple polysaccharides andoligosaccharides on the cell vitality. Apoptotic cells were measured with an AnnexinV-FITC staining, and cell cycle was determined by flow cytometry. Morphologicalchanges were examined by electron microscopy. The proteins and mRNA associated withapoptosis and cell cycle were demonstrated with western blot and real time PCRrespectively.
4. The colorectal cancer model (CACC) and tumor bearing nude mice model wereused to evaluate the anti-tumor activity of OS-5in vivo. Colon cancer occurrence wasobserved by pathological histology method. TNF-α and Galectin-3levels in serum andapoptosis proteins were observed through ELISA and Western Blot. The antitumor effects of OS-5on tumor bearing nude mice were evaluated by tumor volume and the survivaltime on nude mice model.
Results:
1. AP-1and AP-2B were showed as a single sharp peak by using HPSEC, whichsuggested they were homogeneous polysaccharides. The monosaccharide composition ofapple polysaccharide was Rha, GalA, Glc, Gla, Arb in molar ratio of1.00:17.67:8.50:4.01:2.03. The molecular weight of AP-1was10kD, and the monosaccharide composition wasRha, GalA, Glc in the molar ratio of1.20:10.00:5.25. The molecular weight of AP-2B was1900kD, and the monosaccharide composition was Rha, GalA, Glc, Gal in the molar ratioof1.00:20.30:8.50:8.03.
2. The purity of OS-5was99.21%. OS-5was an oligogalacturonide, its molecularweight was898. The structure of OS-5was α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-β-D-alAp-(1→4)-α-D-GalAp.
3. The proliferation rate of HIEC cell was37.3±1.8%after it was treated with9μg/mL OS-5for36h. OS-5showed low inbibitional effect to A549, H1299, SMMC-7721,HCT116and SW480cells.36h after treatment, the inhibition rate on H1299, SW480cellswere24.99±0.6%,23.4±1.4%respectively. For A549cells and HCT116cells, theinhibition rate was less than10%. After SMMC7721cells were treated with differentconcentration of OS-5for24h, cells inhibition rate was about25%without anyconcentration-dependent relationship. The inhibition effect of OS-5on HT29cells wassignificant and showed time and concentration dependent manner. Flow cytometry showedthat OS-5could induce HT29cells apoptosis and S phase arrest. The apoptosis ratio ofHT29cells after treated with10μmol/L OS-5was45.9%(P<0.01). The percentages of Sphase was60.86±3.44%after treated with10μmol/L OS-5, and it was significantlyhigher than normol cells (P<0.01). Bax protein of HT29cells expression was significantlyhigher after it was treated with OS-5, while the expression of anti-apoptotic proteins Bcl-2and Bcl-xl was significantly lower than normol cells. OS-5significantly reduced theexpression of Cdk2and cyclin B1proteins in HT29cells, while increased the expressionof cyclin A1.
4. After treated by OS-5, the thymus index was significantly higher than the normalgroup and the model group (P<0.05) in colon cancer model. HE staining showed thatOS-5can reduce colon cancer cells invasion and growth. In OS-5treatment group, TNF-αlevel in serum was significantly higher than the model group (P<0.01), Galectin-3level inserum was lower than the model group, but there was no significant difference. WBshowed that the expression of Bax was significantly increased and expression of Bcl-2andBcl-xl decreased in OS-5treatment group. The expression of Bax significantly decreasedin model group (P<0.01). The experiment of nude mice showed that OS-5couldsignificantly inhibit the proliferation of transplanted tumors in dose-depenent manner. On35day, the tumor volume in5mg/kg dose group,10mg/kg dose group and20mg/kg dosegroup was529.84±44.01mm~3,443.81±38.94mm~3and358.96±28.68mm~3respectively.The median survival time of nude mice model group was38.5d, it was prolonged to39and47days in10mg/kg and20mg/kg group respectively.
Conlusion：
1. A controllable method to degrade apple monosaccharide was established, and OS-1,OS-2, OS-3, OS-4and OS-5were obtained.
2. The structure of OS-5is α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-α-D-GalAp-(1→6)-β-D-alAp-(1→4)-α-D-GalAp, which has not been reported.
3. OS-5showed effectively antitumor activities on HT29cells. The possiblemechanism is associated with overexpression of Bax and decreased the levels of of Bcl-2and Bcl-xl. Apple oligosaccharide induced cell cycle arrest in S phase, which correlatedwith the decreased expression of Cdk2and cyclin B1on HT29cells.
4. OS-5shows anti-tumor activities both on DMH/DSS-induced colon cancer modeland tumor bearing nude mice model, indicating OS-5has potential in colon cancertreatment and be worthy of further development.
引文
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