【作者】 申海涛；

【导师】 张祥宏； 王凤荣；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2004， 硕士

【摘要】 目的：黄芪(astragalus membranaceus AS)是豆科多年生草本植物的根部，根据产地不同分为蒙古黄芪、膜荚黄芪（东北黄芪）。具有补气升阳、益卫固表、托毒生肌、利水退肿的功效，是中医临床最常用的补益药之一。药理学研究表明，黄芪主要含有甙类、多糖、黄酮、氨基酸、及少量微量元素等。多年来临床应用黄芪已被证明具有利尿、降压、保护机体造血功能以及提高机体免疫力等功效。近年来许多科研工作者探讨了黄芪的抑瘤作用及其机制。有研究认为黄芪具有肿瘤抑制作用，但也有研究认为黄芪具有促进肿瘤的生长的作用。目前研究工作或侧重于体外条件下对肿瘤细胞的影响，或通过对荷瘤动物瘤体大小、重量观察进行抑瘤效果的探讨，综合系统的研究工作尚很少。从整体水平上综合系统地探讨黄芪对肿瘤影响的研究至今国内外未见有报道。本研究拟采用多种实验方法从ASA对肿瘤重量和体积、形态学变化、肿瘤细胞增殖与凋亡以及肿瘤组织淋巴细胞浸润、荷瘤动物免疫机能等方面综合分析灌胃不同剂量黄芪提取物（ASA）对H22荷瘤昆明小鼠的影响。旨在从整体水平上客观评价和权衡应用黄芪治疗对荷瘤动物的利弊得失，进一步探讨黄芪对肿瘤的作用及机理，以期为黄芪应用于肿瘤的临床治疗提供理论依据。方法：1 动物模型的建立和实验分组：将50只雄性<WP=4>18-20g昆明小鼠右腋下接种健康腹水型H22瘤株，随机分5组，对照组和实验组。实验组分4组，按小剂量0.03125mg/g，中剂量0.125mg/g，大剂量 0.5mg/g，最大剂量2mg/g给小鼠灌胃黄芪提取物。对照组灌胃等量生理盐水。连续灌胃10天，停止灌胃后次日脱颈处死动物，分别进行抑瘤效果和对免疫机能影响的观察。实验重复一次，方法同前。2 肿瘤组织指标观察和检测：A:肿瘤生长抑制情况：取瘤体，称重，量体积。B:瘤体常规取材分两部分，一部分10%甲醛固定，常规石蜡切片制备。切片HE染色，置光镜下观察，高倍镜下计数肿瘤组织浸润的淋巴细胞；以HPIA-1000高清晰度彩色病理图文分析系统计算整个组织切片中坏死区域所占比例。免疫组织化学染色，S-P法检测PCNA蛋白表达。C:另一部分70%酒精固定，待制备单细胞悬液，FCM检测细胞凋亡率、增殖指数、Bcl-2、Bax蛋白表达。3 CD4+、CD8+细胞标记率检测：取脾固定于70%酒精中，待制备单细胞悬液，FCM检测CD4+、CD8+细胞标记率。4小鼠腹腔巨噬细胞吞噬鸡红细胞试验：连续灌胃10天后，每只小鼠腹腔注射10%的鸡红细胞悬液1ml，30分钟后脱颈处死，仰位固定，腹腔注入D-HankS液2ml，吸出腹腔洗液立刻滴入载玻片上。温育、固定后，姬姆萨染色，高倍镜下计算吞噬率、吞噬指数。结果：1 动物抑瘤实验结果: 第一次实验中，各实验组的平均瘤重、体积小于对照组，无显著性差异（P?0.05）。第二次实验中，各实验组平均瘤重、体积与对照组比较仍无显著性差异（P?0.05）。2 形态学观察：肿瘤组织中可见大量出血、坏死区及炎细胞、淋巴细胞浸润区域。大剂量组、最大<WP=5>剂量组平均一个高倍视野淋巴细胞浸润数分别为51.76±0.43、73.94±10.20，多于对照组35.65±5.37(P<0.05)。中剂量组、小剂量组与对照组比较无显著性差异（P?0.05）。HPIA-1000高清晰度彩色病理图文分析系统分析各实验组坏死面积比例与对照组比较无显著性差异（P?0.05）。3 黄芪对肿瘤细胞增殖的影响：免疫组织化学检测表明，PCNA蛋白阳性染色呈黄棕色，位于细胞核，各实验组PCNA蛋白均呈阳性表达，平均阳性率>50%，与对照组比较无显著性差异（P?0.05）。FCM检测各实验组肿瘤细胞增殖指数与对照组比较无显著性差异（P?0.05）。4 FCM检测黄芪对肿瘤细胞凋亡的影响：除小剂量组外，其余各组肿瘤细胞凋亡率分别是11.1±0.87%、10.83±0.87%、7.35±0.77%，明显高于对照组3.7±0.59%（P<0.01）。黄芪中剂量（0.125mg/g）组肿瘤细胞凋亡率最大。5 FCM检测黄芪对Bcl-2 、Bax蛋白阳性表达率的影响：各实验组Bcl-2阳性表达率分别是5.33±0.18%、4.96±0.12%、4.72±0.29%、4.80±0.43%，低于对照组6.54±0.70%（P<0.01）。除小剂量组外，各实验组Bax阳性表达率分别为6.32±0.36%、6.01±1.28%、5.94±0.15%，高于对照组4.92±0.25%（P<0.01）。各实验组Bcl2/Bax蛋白比值分别为1.12±0.11、0.79±0.04、0.81±0.14、0.81±0.05，低于对照组1.33±0.09（P<0.01）。6 黄芪对荷瘤小鼠免疫功能的影响：腹腔冲洗液滴片姬姆萨染色后，高倍镜下观察可见巨噬细胞中吞噬核椭圆如枣核的鸡红细胞.各实验组腹腔巨噬细胞吞噬率和吞噬指数与对照组比较，其均数明显高于对照组，差异有显著性（P<0.001）。FCM检测黄芪各实验组 CD4+、CD8+细胞标记率：各实验组脾脏CD4+ T淋巴细胞标记率分<WP=6>别为33.90±0.89%、33.98±2.00%、36.77±1.07%、38.20±1.35%，大于对照组31.75±0.90%（P<0.05）。各实验组脾脏CD8+ T淋巴细胞标记率与对照组比较无显著性差异（P?0.05）。各实验组CD4+/CD8+的比值分别为1.33±0.10、1.35±0.10、1.39±0.02、1.38±0.04，大于对照组1.20±0.08（P<0.05）。结论：1灌胃不同剂量黄芪提取物对肿瘤组织重量和体积没有影响。2形态学观察灌胃黄芪提取物可以增加肿瘤组织?更多还原

【Abstract】 Objective: Astragalus membranaceus(AS) is one of the most commonly used traditional Chinese herbal medicine, which possesses “Buqishengyang, Yiweigubiao, Tudushengji, Lishuituizhong” function. Modern pharmacological studies showed AS contains glycoside, polysaccharide, chromocor, amino acid, and microelement. Clinical usage suggested that AS is effective in many fields such as enhancement of immunological function, protection of hemopoiesis, etc.Recently, many studies on the anti-cancer effects of AS have been carried out. The results were quite controversial. Some studies showed that AS could inhibit the growth of tumors. Yet, there were some studies showed that AS may accelerate the growth of tumors. Up to now, the studies were carried out either on the improvement of immuno-function in vivo, or on the effects of tumor cells in vitro, or on the weight and volume of tumors in experimental animals. Few studies involving both tumor inhibiting and immunological function has been seen. There is no report of works on the systemic evaluation of effects of AS on tumor apoptosis, growth and immune system of the animal with tumor in whole animal level. <WP=8>To explore the systemic effects of ASA on H22 in mice and to objectively evaluate the putative advantages and disadvantage of AS on the animals with tumor, the following studies were carried out.Methods: 1 Animals and treatment：Fifty male Kunming mice, 18-20g in body weight were first inoculated with H22. The mice were then randomly divided into control and four ASA treatment groups. The mice in ASA treatment groups were treated by gavage with ASA at the dosage of 0.031 mg/g, 0.125mg/g, 0.5 mg/g and 2mg/g body weight respectively. The mice in control group were treated accordingly with normal saline. The experiment was repeated once. 2 Evaluation of tumor inhibiting effects：The mice were killed after 10 days of treatment. The mean weight and volume of the tumors were first measured and then the tumor tissues were divided into two parts. One part was fixed in 10% formaldehyde for the preparation for morphological observation and immunohistochemical staining of PCNA. The other was fixed in 70% ethanol for preparation of single cell suspension for flow cytometric analysis of apoptosis, proliferation and the expression of Bcl-2 and Bax. 3 Splenic CD4+ and CD8+ lymphocyte analysis：At the same time, splenic tissues from experimental mice were fixed in 70% ethanol and prepared for single cell suspension for the analysis of the fraction and ratio of CD4+ and CD8+ lymphocytes. 4 Peritoneal macrophages phagocytosis assay：Peritoneal <WP=9>macrophages phagocytosis of chicken RBC were carried out and the rate of phagocytosis and the index of phagocytosis were calculated under light microscope with Giemsa stain.Results: 1 The results of tumor inhibiting effects：In the first experiment, the mean weight and volume of the tumor in experimental groups were slightly lower than that in control group, but no statistic significance could be found（P?0.05）. In the repeat experiment, there were no difference in both tumor weight and volume among the groups（P?0.05）.2 Morphological observation: Areas of necrosis and hemorrhage and infiltration of lymphocytes could be seen in all the tumor tissue sections. The number of infiltrated lymphocytes in the 0.5mg/g and 2mg/g groups were 51.76±0.43, 73.94±10.20，which were significantly higher than that in control group (35.65±5.37, P<0.05).The HPIA-1000 analysis system revealed that there were no difference in the ratio of areas of necrosis among different groups （P?0.05）. 3 The effects of AS on the proliferation: Positive PCNA nuclear staining cells could be seen in all groups by immunohistochemical staining. No difference in the positive expression rate of PCNA among the experimental groups and control group were found. FCM analysis showed that no significant differences in PI were seen between experimental groups and control group（P?0.05）.4 The effects of AS on the apoptosis: FCM analysis revealed that the apoptosis rat更多还原