PDTC通过减少肌肉萎缩及脂肪降解缓解肿瘤恶病质

发布时间：2018-07-13 17:12

【摘要】：背景：肿瘤恶病质(cancer cachexia)是一种进行性骨骼肌消耗(伴随或不伴随脂肪消耗),并且不能通过营养支持完全逆转的功能性损伤疾病。其发生机制复杂,目前的治疗效果有限。研究抗肿瘤恶病质药物尚无统一标准和系统的体内外生物筛选评价模型,昂贵费时的体内筛选延缓了抗肿瘤恶病质药物的发现与研究,建立并优化具有代表性的体内外抗肿瘤恶病质生物评价模型,将为抗肿瘤恶病质的研究带来极大便利。NF-κB信号通路与肿瘤恶病质中肌肉萎缩和脂肪消耗密切相关,吡咯烷二硫代氨基甲酸盐(PDTC)能通过抑制肿瘤组织中NF-κB的表达缓解恶病质小鼠体重下降,但其对脂肪细胞和骨骼肌细胞的直接作用,缓解肿瘤恶病质的分子作用机制仍不明确,有待进一步的研究。目的：建立具有代表性的稳定可行的体内外肿瘤恶病质模型,用于抗肿瘤恶病质新药的研究与开发。利用建立的体内外模型评价PDTC对肿瘤恶病质的缓解作用,探究其对脂肪和骨骼肌的直接作用及可能的分子作用机制。方法：(1)采用MTT法、甘油检测试剂盒、免疫荧光法及Western Blot检测TNF-α对成熟脂肪细胞及肌管的影响,构建TNF-α诱导的肌萎缩及脂解模型；(2)C26细胞培液直接作用成熟脂肪细胞,或C26细胞培液与2% HS分化液1：1稀释后作用于肌管,分别采用油红O染色、甘油三脂GPO-POD酶法检测试剂盒、免疫荧光法及Western Blot检测C26细胞培液对成熟脂肪细胞及肌管的影响,构建C26细胞培液诱导的肌萎缩及脂解模型；(3)以C26细胞悬液接种SPF级BALB/c雄鼠腋下,监测小鼠体重、瘤体积及摄食量等,构建C26肿瘤恶病质小鼠模型；(4)MTT法或CCK8法检测PDTC对成熟脂肪细胞或成熟肌管细胞的增殖活力影响；(5)油红O染色法、甘油含量检测试剂盒及甘油三脂GPO-POD酶法检测试剂盒测定PDTC对TNF-α及C26细胞培液诱导的脂解的缓解作用,Western Blot检测NF-κB的活化、Perilipin变化及相关蛋白表达；(6)免疫荧光进行染色后,]mage J软件统计分化肌管直径检测：PDTC对TNF-α及C26细胞培液诱导的肌管萎缩的缓解作用,Western Blot检测NF-κB的活化状况、萎缩相关蛋白MuRF-1和Atrogin-1以及肌生成相关蛋白MyoD的表达情况；(7)监测C26荷瘤小鼠体重,腓肠肌及附睾脂肪组织的重量,生化检测仪测定血生化指标,HE染色观察腓肠肌及附睾脂肪组织的显微结构变化,评价PDTC对C26荷瘤小鼠肿瘤恶病质的缓解作用。结果：(1)甘油检测及MTT法显示TNF-α 50 ng/mL时甘油释放量最大且对脂肪细胞活性无影响,并且p-p65表达增加,Perilipin蛋白表达减少；Image J及免疫荧光实验发现TNF-α 100 ng/mL时肌管直径明显小于对照组,且p-p65表达上调,MHC表达下调；(2)甘油三脂检测及免疫印迹实验表明在C26细胞培液刺激下,脂肪细胞中甘油三脂含量显著下降,p-p65表达增加,Perilipin表达下调；Image J测量发现肌管直径减小,且p-p65表达增加,MHC表达减少；(3)C26荷瘤模型组在实验第12天左右出现恶病质表型,瘤体积持续增大,体重及腓肠肌重量下降：(4)MTT法测得PDTC (0-100 pM)对成熟脂肪细胞作用24 h及48 h,对细胞活性无影响；CCK8法显示当PDTC浓度大于50州时对成熟肌管作用48 h,对肌管细胞活性有一定影响：(5)脂解模型中,PDTC 5μM时即可降低TNF-α诱导的甘油释放,100μM时极大的缓解了脂解情况,Perilipin的表达随PDTC浓度的增加逐渐增加；PDTC 10 μM时脂肪细胞内甘油三脂含量显著高于C26细胞培液组,且随PDTC浓度的增大,脂肪细胞甘油三脂量逐渐增多,p-p65及p-HSL表达随PDTC浓度的增加逐渐减少,脂滴围蛋白(Perilipin)的表达则随PDTC浓度的增加逐渐增加；(6)肌萎缩模型中,PDTC在25μM及50μM都能显著改善TNF-α及C26细胞培液引起的肌管萎缩,p-p65、E3泛素链接酶MuRF-1及Atrogin-1表达随PDTC浓度的增加逐渐减少,MyoD及MI-IC表达呈剂量依赖性增加；(7)小鼠肿瘤恶病质模型中,PDTC剂量依赖性的缓解恶病质小鼠体重、腓肠肌及附睾脂肪组织重量的下降,改善血中TG、GLU、ALB含量及甘油释放量。结论：成功建立了由TNF-α及C26细胞培液诱导的体外脂解模型、肌萎缩模型及体内C26小鼠恶病质模型,为肿瘤恶病质药物研究提供了一个便捷系统的平台；PDTC能通过降低NF-κB活性,抑制酯酶及泛素蛋白酶体系统的激活,改善肌生成途径,从而很好缓解由TNF-α及C26培液诱导的成熟脂肪细胞的脂解及成熟肌管的萎缩程度,也能较好的延缓肿瘤恶病质小鼠体重、腓肠肌及附睾脂肪重量的下降,表现出显著的缓解肿瘤恶病质的效果,是一个非常有潜力的恶病质治疗药物。
[Abstract]:Background: tumor cachexia (cancer cachexia) is a functional lesion of progressive skeletal muscle consumption (with or without fat consumption), and can not be completely reversed through nutritional support. Its mechanism is complex and current therapeutic effects are limited. There is no unified standard and systemic in vivo and in vivo biology for the study of antitumor drugs. The screening and evaluation model, expensive and time-consuming screening delayed the discovery and study of antitumor cachexia drugs, and the establishment and optimization of a representative biological evaluation model of cachexia in vivo and in vitro, will bring great convenience to the.NF- kappa B signaling pathway and tumor cachexia for muscle atrophy and fat consumption of tumor cachexia. Cut correlation, pyrrolidine two thiocarbamate (PDTC) can alleviate the weight decline of cachexia mice by inhibiting the expression of NF- kappa B in tumor tissue, but its direct effect on adipocytes and skeletal muscle cells and the molecular mechanism of cancerous cachexia are still unclear. In vivo and in vivo tumor cachexia model, used for the research and development of new antitumor cachexia drugs. Use the established model in vivo and in vitro to evaluate the remission of PDTC on cachexia, explore its direct effect on fat and skeletal muscle and possible molecular mechanism. Methods: (1) MTT, glycerol detection kit, immunofluorescence The effects of TNF- alpha on mature adipocytes and myotubes were detected by light method and Western Blot, and the model of Myoatrophy and lipolysis induced by TNF- alpha was constructed. (2) C26 cell culture directly acted on mature adipocytes, or C26 cell culture and 2% HS differentiation liquid 1:1 diluted to myotubes, using oil red O staining and glycerol three fat GPO-POD enzyme assay reagent, respectively. The effects of C26 cell culture on mature adipocyte and myotube were detected by immunofluorescence and Western Blot, and the model of Myoatrophy and adipization induced by C26 cell culture was constructed. (3) C26 cell suspension was used to inoculate SPF grade BALB/c male rat axillary, to monitor mouse weight, tumor volume and intake, and to construct the mouse model of C26 cachexia; (4) MTT method or The effect of PDTC on the proliferation of mature adipocytes or mature myoductus cells was detected by CCK8. (5) oil red O staining, glycerol content detection kit and glycerol three fat GPO-POD enzyme detection kit were used to determine the alleviating effect of PDTC on TNF- alpha and C26 cells induced by culture, Western Blot detection of NF- kappa B activation, Perilipin changes and correlation Protein expression; (6) after immunofluorescence staining,]mage J software statistical differentiation myotube diameter detection: PDTC to TNF- alpha and C26 cells induced myotube atrophy, Western Blot detection of NF- kappa B activation status, atrophy associated protein MuRF-1 and Atrogin-1, and myogenerative protein MyoD expression; (7) monitoring The weight of the tumor bearing mice, the weight of the gastrocnemius and epididymal adipose tissue, biochemical indicator and HE staining to observe the microstructural changes of the gastrocnemius and epididymal adipose tissue, and evaluate the remission of PDTC on the cachexia of C26 tumor bearing mice. Results: (1) glycerol test and MTT method showed the maximum glycerol release of TNF- alpha 50 ng/mL There was no effect on the activity of adipocyte, and the expression of p-p65 increased, and the expression of Perilipin protein decreased. The Image J and immunofluorescence test found that the diameter of the myotube was obviously smaller than the control group when TNF- alpha 100 ng/mL was less than the control group, and the expression of p-p65 was up and the expression of MHC was down regulated. (2) the test of glycerol three fat and immunoblot test showed that the fat was fine under the stimulation of C26 cell culture. The content of glycerol three in the cell decreased significantly, the expression of p-p65 increased, and the expression of Perilipin was down. The Image J measurements found that the myotube diameter decreased and the p-p65 expression increased, and the MHC expression decreased. (3) the C26 bearing tumor model group had the cachexic phenotype around twelfth days of experiment, the tumor volume continued to increase, and the weight and the gastrocnemius weight decreased: (4) PDTC (0) was measured by MTT method (0 The effect of -100 pM on mature adipocytes was 24 h and 48 h, which had no effect on cell activity. CCK8 method showed that when PDTC concentration was more than 50 state, the effect of 48 h on mature myotubes and the activity of myotube cells had a certain effect. (5) the release of glycerol induced by TNF- a could be reduced when PDTC 5 micron was in the lipid solution, and the lipid release was greatly relieved when 100 um M. With the increase of PDTC concentration, the content of glycerol three fat in the adipocytes was significantly higher than that of the C26 cell culture group. With the increase of PDTC concentration, the content of glycerol three fat increased gradually, and the expression of p-p65 and p-HSL decreased with the increase of PDTC concentration, and the expression of lipid peri protein (Perilipin) was followed by PDTC concentration. Increase gradually; (6) in the Myoatrophy model, PDTC can significantly improve the myotube atrophy caused by TNF- alpha and C26 cell culture, p-p65, E3 ubiquitin linked enzyme MuRF-1 and Atrogin-1 expression gradually decreased with the increase of PDTC concentration, MyoD and MI-IC expression increased in a dose-dependent manner; (7) the dose of the tumor cachexia model in mice. Reducing the weight of cachexia mice, the decrease of the weight of the gastrocnemius and epididymis, and the improvement of TG, GLU, ALB and glycerol release in the blood. Conclusion: a successful establishment of an in vitro adipose model, a model of muscle atrophy and a cachexia model in the body of C26 mice induced by TNF- alpha and C26 cells, and a model of cachexia in the body of C26 mice, are provided for the research of cachexia drugs. A convenient system platform; PDTC can reduce the activation of NF- kappa B and inhibit the activation of esterase and ubiquitin proteasome system, improve the myogenesis pathway, thus alleviating the lilipsis of mature adipocytes induced by TNF- alpha and C26 culture and the atrophy of mature myotubes, and also better retarding the weight of tumor cachexia mice, gastrocnemius The decrease of body weight and epididymal fat weight has shown a significant effect on relieving cancer cachexia, and is a potential therapeutic agent for cachexia.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

【相似文献】