基于“有故无殒”理论评价雷公藤甲素对荷瘤小鼠的肝毒性反应

发布时间：2018-01-05 15:16

本文关键词：基于“有故无殒”理论评价雷公藤甲素对荷瘤小鼠的肝毒性反应　出处：《北京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的根据中医理论"有故无殒"思想,以CT26细胞及荷瘤小鼠为研究对象,在评价雷公藤甲素抗肿瘤作用的同时,比较观察不同剂量雷公藤甲素对正常生理状态及荷瘤病理状态小鼠的肝毒性反应,为雷公藤的临床应用及深度开发提供更多的资料。研究方法离体实验:雷公藤甲素(50nM)孵育CT26细胞12小时、24小时,经MTS法检测细胞的活力、计算增殖抑制率;用流式细胞术检测雷公藤甲素(25nM和50nM)诱导CT26细胞凋亡的作用。整体实验:以雄性BALB/C小鼠为研究对象,将2×106个CT26细胞注入小鼠右前肢下的皮下部位,建立荷瘤小鼠模型。采用随机分组的方法将实验动物分为假手术组、肿瘤模型组、雷公藤甲素治疗组(0.4mg/kg),每组10只,腹腔注射连续给药21天后,取材,用以观察雷公藤甲素抗CT26结肠癌的药效。观测指标包括:小鼠体重、肿瘤生长情况及行为学变化,测量肿瘤的长度和宽度。通过体重及肿瘤监测、肿瘤组织冰冻切片TUNEL染色及免疫组织化学法检测肿瘤组织冰冻切片内Caspase-3的表达情况等方法观察其抗肿瘤作用。对正常小鼠及荷瘤小鼠在相同条件下暴露低剂量(0.1mg/kg)、中剂量(0.2mg/kg)、高剂量(0.4mg/kg)的雷公藤甲素,腹腔注射连续给药25天后,取材,通过大体观察、血清生化检测(ALT、AST)、肝组织匀浆液检测(MDA、SOD、GSH-PX)、组织病理学观察、iNOS、Nrf2蛋白表达分析等方法对比不同剂量雷公藤甲素对正常生理状态及荷瘤病理状态下小鼠的肝毒性反应。研究结果(1)雷公藤甲素可显著抑制CT26的生长增殖并诱导其细胞凋亡。(2)0.4mg/kg雷公藤甲素腹腔注射给药,雷公藤甲素可显著抑制荷瘤小鼠皮下肿瘤的生长,小鼠行为学及体重出现显著改变,肿瘤组织内出现明显的细胞凋亡。(3)对于丙氨酸氨基转移酶(ALT),随着雷公藤甲素药物剂量的增加,正常组及肿瘤组的ALT均增加,在高剂量时,正常组ALT高于肿瘤组;对于天门冬氨酸氨基转移酶(AST),随着雷公藤甲素药物剂量的增加,正常组及肿瘤组的AST变化不明显,肿瘤组的AST值明显高于正常组。(4)肝组织匀浆检测结果表明:对于丙二醛(MDA),随着雷公藤甲素药物剂量的增加,正常组及肿瘤组的MDA含量均增加,在低剂量及中剂量时,正常组要高于肿瘤组;对于超氧化物歧化酶(SOD),随着雷公藤甲素药物剂量的增加,正常组及肿瘤组的SOD活力均降低,正常组与肿瘤组间比较无统计学差异;对于谷胱甘肽过氧化物酶(GSH-PX),随着雷公藤甲素药物剂量的增加,正常组及肿瘤组的GSH-PX活力均降低,在低剂量及高剂量时,肿瘤组要高于正常组。(5)肝组织HE染色结果表明:随着雷公藤甲素药物剂量的增加,肝脏组织细胞病变越明显,正常组的肝脏组织损伤程度明显重于肿瘤组。(6)iNOS免疫组化显色结果表明:随着雷公藤甲素药物剂量的增加,iNOS在肝脏组织中的表达随之增加,在低剂量及中剂量时,正常组与肿瘤组之间iNOS的表达没有显著性差异;在高剂量时,正常组的iNOS的阳性表达要明显高于肿瘤组。(7)Nrf 2免疫组化及Western Blot结果表明:随着雷公藤甲素药物剂量的增加,Nrf 2蛋白在肝脏组织中的表达随之增加,在低剂量时,正常组与肿瘤组之间Nrf2的表达量没有显著性差异,在中剂量及高剂量时,正常组Nrf2的表达量要明显高于肿瘤组。研究结论1.雷公藤甲素具有显著的抗肿瘤作用,其初步机制与诱导细胞凋亡有关;2.雷公藤甲素在病理状态下小鼠的肝毒性反应比正常生理状态下的肝毒性反应要轻;3.雷公藤甲素的肝毒性发生机制与诱导氧化应激有关;4.高剂量的雷公藤甲素对的正常组及肿瘤组小鼠均可产生明显的肝毒性反应,提示雷公藤甲素用量超出一定范围后,无论机体处于生理状态还是病理状态,其肝毒性反应程度均很严重。
[Abstract]:The purpose of the study according to the theory of TCM "so no artist" thought, using CT26 cells and tumor bearing mice as the research object, in the evaluation of triptolide antitumor effect at the same time, comparative observation of liver toxicity of different dose of triptolide on normal physiological state and pathological state of tumor bearing mice, provide more information for clinical application and the depth of the development of Tripterygium. Research methods in vitro: triptolide (50nM) CT26 cells were incubated for 12 hours, 24 hours, were detected by MTS activity, calculation of inhibition rate; flow cytometry was used to detect the effects of triptolide (25nM and 50nM) induced CT26 cell apoptosis. The whole experiment in male BALB/C mice as the research object, the 2 x 106 CT26 cells were injected into the subcutaneous site of mice under the right forelimb, establishing tumor bearing mice model. Using the method of randomized experimental animal tumor divided into sham operation group, model group, Lei The triptolide treatment group (0.4mg/kg), 10 rats in each group, intraperitoneal injection after 21 days of medication, were used to observe the effect of triptolide against CT26 colon cancer. The observation index included: mice weight, tumor growth and behavior changes, measure the length and width. The tumor weight and tumor monitoring the frozen tumor tissue, detection of tumor tissue sections with TUNEL staining and immunohistochemistry of frozen sections in the expression of Caspase-3 was observed. The anti-tumor effect of normal mice and tumor bearing mice in the same conditions exposed to low dose (0.1mg/kg), middle dose (0.2mg/kg), high dosage (0.4mg/kg) triptolide. Intraperitoneally administered continuously for 25 days, from the general observation of serum biochemical detection (ALT, AST), the detection of liver tissue homogenate (MDA, SOD, GSH-PX, iNOS), histopathological observation, the expression of Nrf2 protein in the methods of comparative analysis of different agents The amount of liver toxicity of triptolide on normal physiological and pathological conditions in tumor bearing mice. Results (1) the proliferation of triptolide could significantly inhibit CT26 and induce cell apoptosis. (2) 0.4mg/kg triptolide intraperitoneal injection, triptolide can significantly inhibit subcutaneous tumor of mice bearing the growth of mice behavior and body weight had significant change, significant apoptosis occurred in the tumor tissue. (3) to alanine aminotransferase (ALT), with the increase of triptolide dose, normal group and tumor group ALT were increased at high doses, ALT higher than the normal group in tumor group for; aspartate aminotransferase (AST), with the increase of triptolide dose, AST changes the normal group and tumor group was not obvious, the AST value of tumor group was significantly higher than the normal group. (4) the detection results of liver homogenate showed that for C Two aldehyde (MDA), with the increase of triptolide dose, the content of MDA in normal group and tumor group were increased in the low dose and middle dose, normal group is higher than that of tumor group; for superoxide dismutase (SOD), with the increase of triptolide dose, normal group and the tumor group SOD activity decreased, no significant difference between normal group and tumor group; for glutathione peroxidase (GSH-PX), with the increase of triptolide dose, normal group and tumor group GSH-PX activity decreased in the low dose and high dose group, the tumor is higher than that of the normal group. (5) liver tissue HE staining results showed that with the increase of triptolide dose, tissue cells of liver is more obvious, the degree of liver tissue injury in normal group was significantly heavier than tumor group. (6) iNOS immunohistochemical coloration. The results show that with triptolide drug dose The increased expression of iNOS in liver tissues increased in low dose and middle dose, there was no significant difference in expression of iNOS between normal group and tumor group; at high doses, the positive expression of iNOS in the normal group was higher than that of tumor group. (7) Nrf 2 immunohistochemistry and Western Blot the results showed that with the increase of triptolide dose, the expression of Nrf 2 protein in liver tissues increased at low doses, there is no significant difference between the expression of Nrf2 between normal group and tumor group, the middle dose and high dose, the expression of Nrf2 in the normal group was significantly higher than the tumor group. Conclusion 1. triptolide has significant anti-tumor effects, the mechanisms involved in the induction of apoptosis; liver toxicity of triptolide in 2. pathological conditions of mice are lighter than liver toxicity in normal condition; 3. Triptolide Liver toxicity mechanism and oxidative stress induced by about 4.; high dose of triptolide on the normal group and tumor group of mice liver can produce significant toxicity, suggesting that triptolide content beyond a certain range, regardless of the body in a physiological state or pathological conditions, the degree of liver toxicity was very serious.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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