文献类型：学位论文

中文题名：翡翠贻贝糖胺聚糖抗肿瘤活性及其免疫调节作用的研究

英文题名：Research of Anti-tumor Activity and Immunomodulatory Effect of Glycosaminoglycan from Perna Viridis

作者：李瑞[1];

机构：[1]广东海洋大学;

导师：吴红棉;广东海洋大学

授予学位：硕士

语种：中文

中文关键词：翡翠贻贝;糖胺聚糖;提取条件优化;抗肿瘤活性;免疫调节作用

外文关键词：Perna viridis; glycosaminoglycan; optimum condition of extraction; anti-tumor activity; regulating immune function

中文摘要：目的:以翡翠贻贝全脏器为原料,优化翡翠贻贝糖胺聚糖粗品/(PVG/)的提取工艺条件,经初步纯化后对其抗肿瘤活性进行研究,并探讨其在免疫调节作用方面的抗肿瘤机理。 方法:1.采用二次回归正交旋转组合设计研究料液比、加酶量、酶解时间3个因素对糖胺聚糖粗品得率与糖胺聚糖含量乘积的影响,从而确定翡翠贻贝糖胺聚糖粗品/(PVG/)提取的最佳工艺条件。2.将粗品溶解后,调整溶液pH值,使大部分蛋白沉淀后,冷冻干燥得精制品/(PVG-1/),DEAE-52-纤维素纯化PVG-1得到翡翠贻贝糖胺聚糖纯化品/(PVG-1a/)3.采用最大耐受剂量实验对PVG-1的急性毒性进行检验。4.MTT法检测PVG-1和PVG-1a对体外培养的K562慢性髓性白血病细胞、CNE-2Z人鼻咽癌细胞和S180骨肉瘤细胞的抑制作用,并探讨PVG-1和PVG-1a对5-Fu的增敏作用;在体外实验基础上,体内实验验证PVG-1对S180移植瘤的抑制作用。5.采用计算免疫器官指数法检测PVG-1对正常小鼠、免疫抑制小鼠及荷瘤小鼠的脾指数、胸腺指数的影响;检测PVG、PVG-1、PVG-1a体外对小鼠腹腔巨噬细胞吞噬中性红及产生一氧化氮/(NO/)能力的影响;通过脾细胞增殖实验检测PVG、PVG-1、PVG-1a体外对小鼠脾淋巴细胞增殖的影响;通过脾淋巴细胞与K562肿瘤细胞的共培养,体外检测PVG、PVG-1、PVG-1a对自然杀伤细胞/(NK细胞/)活性的影响。 结果:1.获得了翡翠贻贝糖胺聚糖提取的最佳工艺组合:液料比2.30:1、加酶量0.94/%、酶解时间3.46h,模型预测值为25.50/(×10~/(-4/)/),重复验证实验结果与预测值的标准误差为1.10/%,低于5/%。重复验证实验糖胺聚糖粗品平均得率为0.99/%,糖胺聚糖平均含量为26.04/%。2.经过初步精制和离子交换柱层析纯化,翡翠贻贝糖胺聚糖的含量逐步提高,精制品PVG-1中糖胺聚糖含量为45.6/%;经柱层析后的纯化品中糖胺聚糖含量为59.4/%。3.最大耐受量实验结果显示,翡翠贻贝糖胺聚糖精制品PVG-1对小鼠的经口急性毒性剂量大于15.0 g·kg~/(-1/)。4./(1/)PVG-1对K562慢性髓性白血病细胞、CNE-2Z人鼻咽癌细胞的生长有抑制作用;PVG-1和PVG-1a对体外培养的S180骨肉瘤细胞的生长有显著抑制作用;PVG-1、PVG-1a与5-Fu合用可以增敏5-Fu的抗肿瘤药效。/(2/)PVG-1对小鼠体内S180移植瘤的生长具有显著抑制作用,200 mg·kg~/(-1/)的PVG-1的抑制率达到60.6/%;CTX与PVG-1合用组的抑制率达到78.8/%,显著提高了CTX的抑瘤效果。5./(1/)PVG-1能够提高正常小鼠脾指数和胸腺指数,200mg//kg剂量PVG-1对正常小鼠脾指数的提高最明显,100mg//kg剂量PVG-1对对正常小鼠胸腺指数的提高最明显; PVG-1能够修复由CTX引起的小鼠免疫低下状况。/(2/)PVG-1各剂量组都能够提高荷瘤小鼠的脾指数和胸腺指数,在所选实验剂量范围内表现出良好的剂量效应关系;PVG-1中剂量/(100 mg//kg/)能修复由于CTX治疗肿瘤的同时导致的荷瘤小鼠免疫低下。/(3/)PVG、PVG-1和PVG-1a能显著增强小鼠腹腔巨噬细胞吞噬中性红和产生NO的能力; PVG、PVG-1和PVG-1a各剂量组均能显著促进小鼠脾淋巴细胞增殖,样品相同剂量条件下,PVG-1a的刺激指数高于PVG-1,PVG-1高于PVG;200μg//ml的PVG-1a与ConA协同刺激脾淋巴细胞增殖的效果最好,刺激指数达到1.63。/(4/)PVG、PVG-1和PVG-1a的四个剂量作用的NK细胞能够增强NK细胞的杀伤活性;PVG-1a组作用的NK细胞在总体上杀伤活性好于PVG、PVG-1。 结论:通过响应面分析得到翡翠贻贝糖胺聚糖提取的最佳工艺条件;翡翠贻贝糖胺聚糖对K562、CNE-2Z和S180肿瘤细胞有抑制作用,能增敏5-Fu和CTX的抗肿瘤药效;翡翠贻贝糖胺聚糖具有调节免疫功能,其体内抗肿瘤作用机理之一可能是调节机体免疫力。

外文摘要：Objective: To determine the optimum conditions of extraction of rough glycosaminoglycan from Perna viridis and after preliminary purification research its anti-tumor activity and investigate anti-tumor mechanisms in the aspect of immunomodulatory effect. Methods: 1. The optimum conditions of extraction of glycosaminoglycan from Perna viridis were determined by quadratic regression orthogonal rotational combing design studying the effects of three independent variables: solid-liquid ratio, enzyme dosage and enzymolysis time on the product of yield of crude glycosaminoglycan and content of glycosaminoglycan. 2. PVG-1 was obtained via freeze drying the treated crude glycosaminoglycan by dissolving the crude glycosaminoglycan and adjusting pH of solution to precipitate most of protein; the PVG-1 was purified by DEAE-52-cellulose chromatography. 3. The acute toxicity of PVG-1 was determined by the method of Test of Maximum Tolerated Dosage.4. MTT colorimetric assay was carried out to evaluate anti-tumor effect of PVG-1 and PVG-1a on human nasopharyngeal carcinoma cell CNE-2Z、chronic myelogenous leukemia cell K562 and osteosarcoma cell S180 in Vitro. Meanwhile the sensitization effect of PVG-1 and PVG-1a to 5-Fu were measured. On the basis of experiment in Vitro, the anti-tumor activity was verified via the experiment of inhabitation of PVG-1 on transplanted tumor S180 in Vivo. 5. The immune organs indexes were calculated to test the effects of PVG-1 on spleen and thymus index of normal mice、immunosuppressive mice and tumor-bearing mice; The effects of PVG、PVG-1、PVG-1a on phagocytic activity and activity of producing Nitro Oxide of mice peritoneal macrophages and on proliferation of mice spleen lymphocytes and on the cytotoxic activity of NK cells were studied in Vitro also. Results: 1. The optimum conditions of extraction of glycosaminoglycan from Perna viridis were acquired: solid-liquid ratio 2.30:1, enzyme dosage 0.94/% and enzymolysis time 3.46h; Standard error of results of repeated verifing experiments and model prediction 25.50/(×10~/(-4/)/)was 1.10/%. 2. Content of glycosaminoglycan from Perna viridis was improved via preliminary refining PVG and one fraction PVG-1a was acquired through DEAE-52-cellulose chromatography. The content of glycosaminoglycan of PVG-1 and PVG-1a were 45.6/% and 59.4/% respectively. 3. Test of Maximum Tolerated Dose experiment showed that dose of PVG-1 >15.0 g·kg~/(-1/) in mouse acute oral toxicity test. 4. /(1/) PVG-1 exhibited inhibitory effect on K562 and CNE-2Z in Vitro. PVG-1 and PVG-1a showed obvious inhibitory effect on S180 in Vitro. PVG-1、PVG-1a could enhance anti-tumor effect of 5-Fu. /(2/) PVG-1 had obvious inhibitory effect on S180 in Vivo, with the inhibition rate of 60.6/% of 200mg·kg~/(-1/) PVG-1. The inhibition rate of group of CTX together with middle dose of PVG-1 reached 78.8/%. Middle dose of PVG-1 obviously improved the anti-tumor effect of CTX. 5. /(1/) PVG-1 could increase the spleen and thymus indexes of normal mice, and 200mg//kg、100 mg//kg PVG-1 could most obviously increase the spleen and thymus indexes of normal mice respectively. PVG-1 could repair low-immunity expression of immunosuppressive mice. /(2/) Each dosage groups of PVG-1 could increase the spleen and thymus indexes of tumor-bearing mice. Middle dose of PVG-1 /(100 mg//kg/) could repair low-immunity expression of tumor-bearing mice while treated by CTX. /(3/) PVG、PVG-1and PVG-1a could promote phagocytic activity and activity of producing Nitro Oxide of mice peritoneal macrophages and significantly promote proliferation of mice spleen lymphocytes in Vitro. /(4/) Each group of PVG、PVG-1and PVG-1a could promote cytotoxic activity of NK cells in Vitro. And the effect of groups of PVG-1a on cytotoxic activity of NK cells was better than PVG、PVG-1. Conclusion: The optimum conditions of extraction of GAG from Perna viridis were acquired. GAG from Perna viridis possessed the inhibitory effect on K562、CNE-2Z and S180, and improved the anti-tumor effect of 5-Fu and CTX. GAG from Perna viridis played a critical role in regulate immune function. One of the mechanisms of anti-tumor activity was it could regulate immune function.

年份：2010