【作者】 金守杰；

【导师】 宋扬；

【作者基本信息】 青岛大学 ， 营养与食品卫生学， 2012， 硕士

【摘要】 目的：通过建立诱发性大鼠肝癌动物模型,考察刺参酸性粘多糖(SJAMP)在诱发大鼠肝肿瘤过程中的作用,并从免疫功能角度研究其对肝肿瘤发生的影响。方法：将50只wistar大鼠随机分为正常对照组(A组)、模型组(B组)、SAJMP低剂量组(17.5mg/Kg,C组),SJAMP中剂量组(35mg/Kg,D组),SJAMP高剂量组(70mg/Kg,E组)。除正常对照组(A组)外,其余各组灌胃2‰DEN生理盐水溶液以诱发肝肿瘤,SJAMP干预组(C组、D组、E组)同时灌胃给予SJAMP,饲养期间每周至少称重一次,直至第16周；第16周用水合氯醛腹腔麻醉大鼠,腹主动脉取血,检测ALT、AST等肝功指标；无菌取脾、胸腺,立即称重,计算脾指数和胸腺指数；分别计数肝脏上有一条直径大于3mm和5mm的瘤结节,并测量最大瘤结节的长径和短径,计算抑瘤率；贴壁法分离纯化Mφ,用中性红吞噬实验检测脾巨噬细胞吞噬功能；MTT法检测脾巨噬细胞的杀伤功能；MTT法检测自然杀伤细胞活性；ELISA法检测血清中TNF-α和IL-2的水平；流式细胞术分析大鼠外周血T淋巴细胞亚群。结果：①与正常对照组相比,诱癌实验组大鼠体重增长较慢,至实验第16周时,E组和B组大鼠体重有统计学差异(P<0.05)；②诱癌实验组ALT、AST和GGT均升高(P<0.05),其中模型组升高最为显著,随干预剂量增加,SJAMP干预组以上指标呈逐渐下降趋势,且与模型组比较有统计学意义(P<0.01)；③模型组胸腺指数与正常组组相比有所降低(P<0.05),但脾指数有所升高(P<0.05),与模型组相比,SJAMP干预组脾指数和胸腺指数明显升高(P<0.05),并呈现出剂量-效应关系；④SJAMP可显著抑制肝肿瘤的发生,减少癌结节的发生数量,SJAMP干预组大鼠大于3mm和大于5mm的结节数明显少于模型组(P<0.05)；最大结节的平均体积明显小于模型组(P<0.01)；⑤模型组和SJAMP干预组巨噬细胞吞噬能力较正常对照组均增(P<0.05),SJAMP干预组增加更为明显,其中D组和E组与C组相比较,有统计学意义(P<0.01)；⑥与正常对照组相比,模型组巨噬细胞的杀伤能力降低(P<0.01),SJAMP干预组巨噬细胞的杀伤能力较模型组增加(P<0.01)；⑦模型组自然杀伤细胞活性较正常对照组明显降低(P<0.01), SJAMP干预可以提高诱癌大鼠的自然杀伤细胞活性(P<0.05)；⑧模型组和SJAMP干预组的血清TNF-α水平高于正常组(P<0.01),提示诱癌各组大鼠均可能存在肝脏损伤,SJAMP干预组中D组和E组与模型组相比较,差别均具有统计学意义(P<0.01)；⑨模型组大鼠血清IL-2水平明显降低于正常组(P<0.01),SJAMP干预组的IL-2水平较模型组升高(P<0.01)；⑩模型组CD3+、CD4+T淋巴细胞和CD4+/CD8+比值比正常组均明显降低(P<0.05),而SJAMP干预组随干预剂量的增加,CD3+、CD4+、CD8+T淋巴细胞和CD4+/CD8+比值逐渐增高。结论：SJAMP不能完全阻断肝癌的发生过程,但对肝癌的发生有明显的抑制作用,其机制可能是通过刺激免疫器官组织增生,保护正常肝细胞,增强机体的巨噬细胞、自然杀伤细胞和T淋巴细胞的免疫能力,调节具有抗肿瘤效应的TNF-α、IL-2等重要细胞因子来实现的。更多还原

【Abstract】 Objective Through the establishment of rat liver cancer model, the role of Stichopus japonicus acid mucopolysaccharide(SJAMP) in the development of hepatocellular carcinoma induced by diethylnitrosamine(DEN) was explored in rats and the possible immunological mechanisms was determined.Methods50wistar rats were randomly divided into five groups:normal control group(group A), model group(group B), SAJMP low dose group (17.5mg/Kg, group C), SJAMP moderate dose group (35mg/Kg, group D), SJAMP high dose group (70mg/Kg, group E) by weight. In addition to normal control group, all the rats were given orally2%o DEN saline solution to the16th week to induce hepatocelluar carcinoma, meanwhile they were intervened with different doses of SJAMP during the process of carcinogenesis. All the rats were weighed once a week during the breeding, and at the end of the16th week, all the rats were anaesthetized by using chloral hydrate, blood samples were drew from the abdominal aorta; spleen and thymus were excised under sterile conditions and weighed, spleen index and thymus index were calculated. The number of the nodules more than3mm or5mm in diameter were counted, in addition, the size of the largest nodule were compared among groups. The tumor inhibitory rate was calculated. Macrophagocytes(Mf) were purified via attachment culture, macrophage phagocytosis was observed with phagocytosing neutral red method; spleen macrophage killing capability and NK(Natural killer) cell activity were measured by MTT method, the levels of serum TNF-a and IL-2were detected with enzyme linked immunosorbent assay(ELISA); T lymphocyte subsets in peripheral blood were analyzed by flow cytometry.Results①Compared with normal control group, the induced hepatic carcinoma rats gain weight slower, at the16th week, the weights of rats of group E was obviously increased when compared to model group B(P<0.05);②The serum ALT,AST,and GGT of the induced hepatic carcinoma rats, especially in model group, increased significantly when compared to those of the normal control group, with the increase of the SJAMP intervention dose, the above indicators decreased gradually, and there were significant differences compared with model group;③The thymus index of model group decreased (P<0.05), while the spleen index increased(P<0.05) when compared to normal group; with the implement of the SJMAP intervention, these two indicators incresed significantly(P<0.05), and a dose-response relationship has been presented;④ith the implement of the SJAMP intervention,The occurrence of liver tumors was significantly inhibited and the number of tumor nodules was obviously reduced, the number of nodules of SJAMP intervention group greater than3mm or5mm in diameter were significantly less than model group (P<0.05), and the average volume of the largest nodule was less than the model group (P<0.01);⑤The ability of phagocytosis of macrophagocytes of the model group and SJAMP intervention groups increased(P<0.05) when compared to normal group, SJAMP intervention groups increased more significantly, there were statistical differences among group D, group E and group C (P<0.01);⑥The killing capacity of macrophages of rats in model group reduced (P<0.01) compared to the normal group, however, the killing capacity of macrophages in the SJAMP intervention group increased when compared with model group(P<0.01);⑦ompared with normal group, natural killer cell activity decreased significantly (P<0.01), SJAMP intervention can enhance natural killer cell activity of the induced hepatic carcinoma rats(P<0.05);⑧Serum TNF-a level of rats in model group and SJAMP intervention group were higher than normal control group(P<0.01), suggesting that liver injuries have occurred in all the DEN induced hepatic carcinoma rats, there were statistical differences when compare group D and E with model group(P<0.01);⑨Serum IL-2level of model group was was significantly lower (P<0.01) than the normal control group, while the level of SJAMP intervention groups increased compared to model group(P<0.01);⑩D3+, CD4+T lymphocytes and CD4+/of CD8+ratio of model group were significantly lower than normal group(P<0.05), however, with the increase of the SJAMP intervention dose, CD3+CD4+, CD8+T lymphocytes and CD4+/CD8+ratio of the induced hepatic carcinoma rats were gradually increased.Conclusion Although the occurrence of liver cancer can not be completely blocked, but can be significantly inhibited by SJAMP. The possible mechanisms may be: SJAMP can stimulate the immune organs and tissues, protect the normal liver cells, cahance cell-mediated immunity ability of macrophage, natural killer and T lymphocytes, regulate cytokines which have anti-tumor effect,such as TNF-a and IL-2.更多还原