化浊解毒固本方联合5-氟尿嘧啶对H22荷瘤小鼠VEGF、MMP-9表达的影响

发布时间：2018-04-30 13:42

本文选题：原发性肝癌 + 5-氟尿嘧啶　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:本研究以癌株传代的方式建立H22荷瘤小鼠模型,通过观察化浊解毒固本方、5-氟尿嘧啶(5-Fluorouridine,5Fu)以及化浊解毒固本方联合5-氟尿嘧啶等不同治疗干预对H22荷瘤小鼠生存状态、瘤体大小、免疫情况、病理变化以及相关肿瘤指标的影响,评价联合用药的抗癌效果,从肝癌的治疗机理入手,探讨中医药在肝癌的中西医结合治疗中所发挥的作用,为原发性肝癌的中西医结合治疗提供实验依据。方法:通过癌株传代法将稀释后的肿瘤细胞注射于小鼠右腋皮下,建立H22肝癌小鼠模型。将已成功生长肿瘤的40只小鼠随机分为4组,每组10只,包括模型组(M)、化浊解毒固本方组(Z)、5-氟尿嘧啶组(X)以及化浊解毒固本方联合5-氟尿嘧啶组(L)。造模成功后给予相应药物进行干预,其中中药组以化浊解毒固本方配方冲剂0.2ml/d剂量灌胃治疗;西药组以5-氟尿嘧啶20mg/kg/d腹腔注射治疗;联合用药组上午给予化浊解毒固本方灌胃治疗,下午予5-氟尿嘧啶腹腔注射治疗;模型组以0.4ml/d生理盐水进行灌胃维持。连续给药12天,定期观察各组小鼠的毛色光泽、进食情况、精神状态、体重变化以及死亡情况等,于末次治疗后次日,颈椎脱臼法处死小鼠,并钝性剥离小鼠肿瘤组织、脾脏组织、胸腺组织,分别计量称重,计算各治疗组的抑瘤率、脾指数、胸腺指数并进行统计分析,对肿瘤组织进行甲醛固定、病理切片,采用免疫组化法(Immunohistochmeistry,IHC)检测VEGF、MMP-9蛋白的表达,并对苏木素-伊红(Hematoxyli/eosin,HE)染色后的肿瘤组织进行病理观察。结果:1小鼠的一般状况各实验组小鼠治疗后生存状态都明显不如治疗前,尤其模型组,小鼠精神萎靡,毛色枯糙,食欲不振,体型瘦削,体重无明显增长,右腋下肿物明显肿大可见。与西药组相比,中药组和联合用药组在精神状态、食欲情况和体重增长等方面都占明显优势。2小鼠的肿瘤生长情况在抑瘤方面,联合用药组抑瘤率最高,西药组抑瘤率高于中药组。平均瘤重方面,联合用药组瘤重最小,其次是西药组、中药组、模型组。三个治疗组瘤重均小于模型组,差异有统计学意义(p0.05);其中,联合用药组瘤重小于西药组和中药组,差异显著(p0.05);西药组瘤重小于中药组,差异有统计学意义(p0.05)。3小鼠脾指数情况中药组小鼠脾指数最高,明显高于其它各组,差异具有统计学意义(p0.05);联合用药组次之,明显高于西药组和模型组,差异明显(p0.05);西药组小鼠脾指数较之模型组,差异无统计学意义(p0.05)。4小鼠胸腺指数情况与模型组相比,各治疗组荷瘤小鼠胸腺指数均显著升高,差异有统计学意义(p0.05);肝癌小鼠各治疗组间相比,中药组、联合用药组小鼠胸腺指数明显高于西药组,差异明显(p0.05);联合用药组胸腺指数与中药组相比,差异无统计学意义(p0.05)。5小鼠肿瘤组织中vegf的表达各治疗组肝癌小鼠肿瘤组织中vegf含量明显低于模型组,差异有统计学意义(p0.05);联合用药组肿瘤组织中vegf含量显著低于中药组和西药组,差异有统计学意义(p0.05);西药组肿瘤组织中vegf含量低于中药组,差异有统计学意义(p0.05)。6小鼠肿瘤组织中mmp-9的表达肝癌小鼠各组间比较,肿瘤组织中mmp-9蛋白表达量有显著差异,与模型组相比,各治疗组组织mmp-9蛋白表达量均明显降低,差异具有统计学意义(p0.05);其中联合用药组mmp-9表达量最低,明显低于中药组和西药组,差异显著(p0.05);西药组组织mmp-9低于中药组,差异有统计学意义(p0.05)。7小鼠肿瘤组病理变化情况模型组肿瘤组织生长旺盛,肿瘤细胞特异性强,核染颜色很深,嗜碱性很强,镜下血管丰富;中药组肿瘤组织生长缓慢,大量肿瘤细胞出现变性裂解,组织出现大面积坏死,细胞质细胞核嗜酸性增强;西药组肿瘤组织生长不旺盛,明显出现片状肿瘤坏死区和空泡,细胞异型性降低,细胞核呈现弱酸性;联合用药组肿瘤组织生长不明显,部分肿瘤细胞发生坏死溶解,细胞质细胞核嗜酸性很强,镜下血管较模型组明显减少。,结论:1化浊解毒固本方能够增强5-氟尿嘧啶的抑瘤作用,提高肿瘤抑制率,其作用机理可能与抑制肿瘤血管生成有关。2化浊解毒固本方能够明显改善5-氟尿嘧啶对H22肝癌小鼠的免疫抑制作用,增强肝癌小鼠的免疫功能,提高肝癌小鼠的生存质量。
[Abstract]:Objective: to establish a H22 tumor bearing mouse model in this study, the survival state of the tumor bearing mice, the size of the tumor, the immune situation, the pathological changes and the related tumor indexes were observed by observing the different treatments of the H22, 5- fluorouracil (5-Fluorouridine, 5Fu) and the combined treatment of the turbid and detoxifying decoction. To evaluate the anti-cancer effect of combined use of the drug, from the treatment mechanism of liver cancer, to explore the role of traditional Chinese medicine in the combination of traditional Chinese and Western medicine in the treatment of liver cancer, and to provide experimental basis for the combination of traditional Chinese and Western medicine for primary liver cancer. 40 mice with successful tumor growth were divided into 4 groups randomly, with 10 rats in each group, including model group (M), turbidity detoxification solid recipe group (Z), 5- fluorouracil group (X) and combined 5- fluorouracil group (L). The dose of 0.2ml/d was treated with dose of gavage; the western medicine group was treated by intraperitoneal injection of 5- fluorouracil 20mg/kg/d; the combined drug group was given the gavage in the morning, and the 5- fluorouracil was administered intraperitoneally in the afternoon. The model group was administered with 0.4ml/d physiological saline for 12 days, and the hair color luster was observed regularly in each group. Eating condition, mental state, body weight change and death condition, the next day after the last treatment, the mice were killed by dislocated cervical vertebra, and the tumor tissue, spleen tissue and thymus tissue of the mice were weighed blunt, and the tumor suppressor rate, the spleen index, the thymus index and the statistical analysis were calculated. Immunohistochmeistry (IHC) was used to detect the expression of VEGF, MMP-9 protein, and the pathological observation of the tumor tissue after hematoxylin eosin (Hematoxyli/eosin, HE) staining. Results: the general condition of 1 mice was obviously inferior to that of the mice after treatment, especially in the model group, and in mice. The mental state, appetite and body weight growth of the Chinese medicine group and the combination group are all obviously superior to the tumor growth of.2 mice, and the combination group has the highest tumor suppressor rate, and the combination group has the highest tumor suppressor rate. The tumor weight of the western medicine group was higher than that of the traditional Chinese medicine group. The tumor weight of the combined drug group was the smallest, followed by the western medicine group, the traditional Chinese medicine group and the model group. The tumor weight of the three treatment groups was less than the model group, the difference was statistically significant (P0.05); the tumor weight of the combined drug group was less than the western medicine group and the traditional Chinese medicine group, the difference was significant (P0.05); the tumor weight of the western medicine group was less than the Chinese traditional medicine group. The difference was statistically significant (P0.05).3 mice spleen index, the spleen index of the Chinese medicine group was the highest, obviously higher than the other groups, the difference was statistically significant (P0.05); the combination of drug group was significantly higher than the western medicine group and the model group, the difference was obvious (P0.05); the difference of spleen index in the western medicine group was less than that of the model group (P0.05).4 small Compared with the model group, the thymus index of the mice was significantly higher than that in the model group, and the difference was statistically significant (P0.05). The thymus index of the mice in the Chinese medicine group and the combination group was significantly higher than that in the western medicine group (P0.05), and the thymus index of the combined drug group had no difference compared with the Chinese medicine group. The expression of VEGF in the tumor tissue of.5 mice was significantly lower than that in the model group. The difference was statistically significant (P0.05), and the content of VEGF in the tumor tissue of the combined drug group was significantly lower than that of the traditional Chinese medicine group and the western medicine group (P0.05); VEGF contained in the tumor tissue of the western medicine group contained VEGF content (P0.05). The difference was significantly lower than the Chinese medicine group. The difference was statistically significant (P0.05) in the tumor tissue of.6 mice, the expression of MMP-9 protein in the tumor tissues was significantly different. Compared with the model group, the expression of MMP-9 protein in each group was significantly decreased, and the difference was statistically significant (P0.05), and the combination group mmp- was statistically significant (P0.05), and mmp- in the combination group mmp- was statistically significant (mmp-). The expression of 9 was the lowest, obviously lower than that of the traditional Chinese medicine group and the western medicine group (P0.05). The MMP-9 in the western medicine group was lower than the traditional Chinese medicine group. The difference was statistically significant (P0.05) the pathological changes in the tumor group of.7 mice were strong, the tumor cells were strong, the color of the nuclear dye was very strong, the blood vessels were rich under the microscope, and the swelling of the traditional Chinese medicine group was swollen. The tumor tissue grew slowly, a large number of tumor cells appeared denatured and cracked, tissue appeared large area necrosis and cytoplasm nucleus eosinophilia increased, and the tumor tissue in the western medicine group was not strong, the tumor necrosis area and vacuoles appeared obviously, the cell isomorphism decreased and the nucleus showed weak acidity, and the tumor tissue of the combined drug group was not obvious, part of the tumor tissue was not obvious. The tumor cells were necrotic and dissolved, cytoplasmic cell nuclear eosinophilia was very strong, and the blood vessels under the microscope decreased obviously. Conclusion: 1 the anti tumor effect of 5- fluorouracil can be enhanced and the tumor suppressor rate is enhanced. The mechanism of the action may be related to the inhibition of the angiogenesis of the tumor angiogenesis with the inhibition of the tumor angiogenesis in the 5- fluoruria. The immunosuppressive effect of pyrimidine on H22 hepatoma mice can enhance the immune function of liver cancer mice and improve the quality of life of liver cancer mice.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

【参考文献】