调气消积汤对Lewis肺癌小鼠抑瘤作用的实验研究

【作者】 梁靓靓；

【导师】 蔡玉文；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2010， 博士

【摘要】 目的:肺癌是我国常见的恶性肿瘤之一,严重威胁人类的健康与生命。针对肺癌的致病特点,我们根据多年的临床实践,筛选有效药物组方而成的调气消积汤具有调畅气机、清热解毒、软坚散结、活血化瘀的功效,在治疗中重视升降理论的应用,注重人体气机运动的调畅,是临床治疗肺癌的有效方剂。本课题以中医“气机升降”理论为指导,以小鼠Lewis肺癌移植瘤型作为体内试验研究对象,从生存质量、肿瘤抑制率、免疫功能、移植瘤细胞凋亡以及血管生成等方面探讨调气消积汤抗肺癌的作用及机制,从而为临床应用调气消积汤治疗肺癌提供科学理论依据。材料和方法:将48只近交系C57BL/6小鼠常规接种Lewis肺癌,5天后选取荷瘤成功的小鼠随机分四组:荷瘤对照组(MG)、调气消积汤组(TG)、顺铂组(DG)、综合组(调气消积汤+顺铂组)(ZG),每组10只;健康的10只小鼠组成空白对照组(CG)。每天分别予饮用水、调气消积汤各0.4ml灌胃12天;隔日一次腹腔注射生理盐水、顺铂0.13ml共3天。停药后24小时取材。观察各组小鼠日常生活状况和肿瘤生长情况,用H.E.染色法通过光镜观察肿瘤组织病理形态改变。观察调气消积汤对脾脏指数的影响,应用形态学方法计数小鼠外周血T淋巴细胞转化率,MTT比色法检测小鼠脾T淋巴细胞的增殖情况,用Western Blot法检测肿瘤坏死因子-α(TNF-α)的表达。应用电镜技术与TUNEL法检测调气消积汤对Lewis肺癌小鼠肿瘤细胞凋亡的影响,用Western Blot法检测突变型P53、Bcl-2基因蛋白的表达。应用Western Blotting法检测Lewis肺癌小鼠肿瘤组织的微血管密度(MVD)、血管内皮生长因子(VEGF)和基质金属蛋白酶-9(MMP-9)蛋白表达的影响。结果:1.从体重、进食量、小鼠的反应性来看,调气消积汤能改善Lewis肺癌小鼠一般状况,五分钟自主活动及站立次数增加,与荷瘤对照组比差异有统计学意义(P<0.01),与空白组比无明显差异(P>0.05);其中顺铂组基本丧失站立能力,表现最差。2.光镜下观察,各实验组与荷瘤对照组比较,肿瘤细胞体积变小,异型细胞较少,细胞核致密,染色质浓缩边集,病理性核分裂较少;间质可见较多的淋巴细胞等炎性细胞浸润,毛细血管内肿瘤细胞少见,周围坏死灶面积增大。3.调气消积汤组、顺铂组和综合组的瘤重均低于荷瘤对照组,差异显著(P<0.01),三组的抑瘤率分别为29.37%、71.23%和75.19%,说明调气消积汤具有抑制肿瘤生长的作用,与顺铂合用后抑瘤作用明显增强。4.荷瘤对照组的脾脏指数明显升高,是空白对照组的111.54%,差异显著(P<0.01);调气消积汤可使荷瘤小鼠的脾脏指数降低并趋向正常,与空白组比较无统计学意义(P>0.05);顺铂组和综合组的脾脏指数明显降低,分别是空白对照组的36.55%和54.62%。5.从小鼠外周血T淋巴细胞转化率及脾T淋巴细胞增殖活性来看,荷瘤对照组明显低于空白对照组,且二者相比较,差异显著(P<0.01或P<0.05);而调气消积汤组比荷瘤对照组明显升高,差异显著(P<0.01),与空白对照组相比差异无统计学意义(P>0.05);顺铂组最低,与空白对照组及荷瘤对照组相比差异显著(P<0.01)。说明提调气消积汤能提高荷瘤小鼠的免疫功能。6.Western Blotting法结果显示,顺铂组及综合组均可升高Lewis肺癌移植瘤组织中TNF-α蛋白的表达,与荷瘤对照组比较有显著性差异(P<0.01);而调气消积汤组与荷瘤对照组比较无明显差异(P>0.05)。7.电镜下观察,可见细胞凋亡的特异性标志——凋亡小体形成。8.TUNEL原位法检测结果显示,荷瘤对照组凋亡形态的细胞较少见,绿色荧光强度低;各治疗组与荷瘤对照组相比凋亡细胞明显增多,绿色荧光强度高;尤以综合组增多最为明显。说明调气消积汤与顺铂合用可增强促肺癌细胞凋亡的能力。9.Western Blotting法结果显示,调气消积汤组、顺铂组及综合组均可降低突变型p53蛋白的表达,其灰度值与β-actin的比值分别为:(0.752±0.006)、(0.723±0.008)、(0.721±0.011),与荷瘤对照组(0.777±0.008)相比,差异有统计学意义;其中综合组的突变型p53蛋白表达最低。各实验组Bcl-2蛋白的表达情况与突变型p53相同。10.Western Blotting法结果表明,各治疗组的MVD、VEGF和MMP-9蛋白的表达均低于荷瘤对照组,差异具有统计学意义(P<0.05或P<0.01);调气消积汤组MVD、VEGF和MMP-9蛋白的表达与顺铂组相比,差异无统计学意义(P>0.05)。结论:1.调气消积汤可以改善荷瘤小鼠的一般生活状况,对Lewis肺癌有抑制作用,与化疗合用后抑瘤效果明显增强。2.调气消积汤对Lewis肺癌小鼠的免疫器官(脾)有一定的保护作用;可以通过提高荷瘤小鼠外周血T淋巴细胞转化率、脾T淋巴细胞增殖活性,来增强荷瘤小鼠的免疫功能;对肿瘤组织中TNF-α蛋白的表达无明显促进和抑制作用。3.调气消积汤能促进Lewis肺癌小鼠肿瘤细胞凋亡,与化疗药物合用能增强促肺癌细胞凋亡的能力;下调凋亡相关基因突变型p53、Bcl-2的表达水平,可能是调气消积汤发挥抑瘤作用的分子机制之一。4.调气消积汤能降低Lewis肺癌移植瘤的微血管密度(MVD);并通过下调移植瘤中血管内皮生长因子(VEGF)及基质金属蛋白酶-9(MMP-9)的表达,来发挥抑制肿瘤血管形成、抗肿瘤的作用。更多还原

【Abstract】 Purpose:Lung cancer is one of the common malignant tumors in China, it threat human health and life seriously. For the lung cancer characteristics, based on years of clinical practice, we have to use Tiaoqixiaoji Decoction in the clinical treatment of lung cancer to achieve a good effect.For“air-lift”theory of Traditional Chinese medicine as a guide, for Lewis lung cancer of mice as a study, we discuss the possible mechanism of Tiaoqixiaoji Decoction in mice bearing Lewis lung cancer cells, at general life status、the inhibition ratio of tumor、immunologic function、apoptosis and tumor angiogenesis in this experiment, in order to provide a scientific theoretical basis for the treatment of lung cancer. Material and method:Inoculated the forty-eight C57BL/6 mice with Lewis lung cancer cells, after 5 days choosing successful tumor-bearing mice, then randomly divided into four groups: Model group(MG)、Tiaoqixiaoji Decoction group(BG)、DDP (Cisplatin) group(DG)and Tiaoqixiaoji Decoction-DDP combination group,10 mice in each group; other ten healthy mice were control group(CG).Different drug were given to five groups: Drinking water、Tiaoqixiaoji Decoction at 0.4ml once a day by gastrogavage, total 12 days;Normal sodium、DDP at 0.13ml every other day by intraperitoneal injection, total 3 days . Then to observe the daily life and the tumor growing status of mice; H.E.staining was used to observe the pathological changes of tumor tissue by optical microscope. To observe the effect of the Tiaoqixiaoji Decoction on the index of spleen and count the T lymphocyte transformation rate of the peripheral blood by morphology method; The T lymphocyte proliferation of spleen of all group mice were determined by MTT colorimetric determination; To detect the expression of tumor necrosis factor-α(TNF-α)by Western Blotting method. Determination of cell apoptosis of tumorous tissue were performed by electron microscope (EM)and Tunel method; The expression of mutant-type p53、Bcl-2 of tumor tissues were determined by Western Blotting method. To detect the expression of Microvascular density(MVD)、Matrix metalloproteinases-9(MMP-9) and Vascular endothelial growth factor(VEGF) by Western Blotting method.Results:1. From the body weight、food intake、mice reactivity point of view, the Tiaoqixiaoji Decoction could improve the general status of the Lewis tumor-bearing mice. The times of autonomic activities and standing within five minute were increased, there were statistical difference compared with the MG(P<0.01), and no significant difference between the TG & CG(P>0.05).2. Compared with the MG, there were some cell morphological changes of the TG、DG and ZG. Such as the volume of some tumor cells were smaller and the abnormal cells became less, the cell nuclei were tightening, the Chromatin were condensed in margin, and the pathological karyokinesis reduced obviously; Lymphocytes infiltrated in interstitial and seldom tumor cells can be seen in capillaries, surrounding necrosis area increased.3. The weight of tumors of the TG、DG and ZG were much lower than the MG,and there were significant difference compared with the MG(P<0.01), the inhibition ratio were 29.37%、71.23% and 75.19%. It shows that the Tiaoqixiaoji Decoction could inhibit growth of tumors and the inhibitory effect appears obviously when Tiaoqixiaoji Decoction combines with DDP.4. The spleen index of the MG were significantly increased to 111.54% of the CG’s, there were statistical difference between the TG & CG(P<0.01); and the spleen index of the TG were closed to normal value, there was no statistical difference compared with the CG(P>0.05); the DG and ZG spleen index decreased significantly, they were 36.55% and 54.62% of the CG respectively.5. From the T lymphocyte transformation rate of the peripheral blood and the T lymphocyte proliferation of spleen of all group mice point of view, the MG was obviously decreased to the CG, and there were significant difference(P<0.01 or P<0.05); but the TG was higher than the MG(P<0.01),and there was no statistical difference compared with the CG(P>0.05); the DG was the lowest,and there were significant difference compared with the CG、MG. Say that the Tiaoqixiaoji Decoction can improve the immunologic function of tumor-bearing mice.6. The high expression of TNF-αwas showed in Lewis lung tumor tissue of the DG and ZG by Western Blotting method,there were significant difference compared with the MG(P<0.01); but there were no statistical difference between the TG and MG(P>0.05).7. Through the observation of EM, we found the specific signs of apoptosis-- apoptotic bodies.8. In TUNEL situ assay showed, the apoptotic cells of the MG were rare and the green fluorescence intensity is low; increased apoptotic cells could be seen in all treatment groups, the green fluorescence intensity is high; the ZG was highest. It shows that the Tiaoqixiaoji Decoction and DDP combined could enhance the ability to promote apoptosis in lung cancer cells.9. Western Blotting results showed, the TG、DG and ZG all could reduce the expression of mutant-type p53 of tumor tissue, the ratio of the grey levels toβ-actin were : (0.752±0.006)、(0.723±0.008)、(0.721±0.011),there were statistical difference compared with the MG(0.777±0.008) (P<0.05 or P<0.01); the expression of mutant-type p53 of the ZG was lowest.10.Western Blotting results showed, compared with the MG, the expression of MVD、VEGF and MMP-9 decreased obviously in all treatment groups, and there were obviously difference(P<0.05 or P<0.01); and there were no statistical difference between the TG and DG in the expression of MVD、VEGF and MMP-9(P>0.05).Conclusion:1. The Tiaoqixiaoji Decoction is effective in improving the general life status of tumor-bearing mice,and it can inhibit growth of the Lewis lung cancer. If combine with DDP, it can enhance the ability of anti-tumor.2. The Tiaoqixiaoji Decoction can protect the immune organ of Lewis lung cancer in mice; it can improve the immunologic function of tumor-bearing mice by increasing the T lymphocyte transformation rate of the peripheral blood and the T lymphocyte proliferation of spleen; and can not effect expression of TNF-αin tumor tissue.3. The Tiaoqixiaoji Decoction can induce the apoptosis of the Lewis lung cancer cells, if combine with DDP, it can enhance the ability to promote apoptosis; its activity of inhibiting the growth of tumor may be relative to decreasing the expression of mutant-type p53 and Bcl-2.4. The Tiaoqixiaoji Decoction can degrade the MVD of Lewis lung cancer; and also can reduce the expression of VEGF and MMP-9 in order to play the effect of inhibiting tumor angiogenesis, anti-tumor.更多还原