泮托拉唑缓解肿瘤恶病质骨骼肌消耗及其机制

发布时间：2018-02-28 09:11

本文关键词： 泮托拉唑肿瘤恶病质炎症泛素蛋白酶体途径　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的本实验通过建立肿瘤恶病质小鼠模型,研究泮托拉唑对恶病质状态下的骨骼肌消耗的保护作用及其可能的相关机制,为恶病质的进一步研究提供新的思路,并为临床应用提供理论依据。方法48只雄性BALB/c小鼠,按照随机数字表分为6个组,每组8只,分别为:正常对照组(NC)、恶病质组(CC)、低浓度治疗组(CL)、中浓度治疗组(CM)、高浓度治疗组(CH)、生理盐水组(CS)。荷瘤小鼠于右前肢腋下接种小鼠结肠腺癌细胞c26,以建立小鼠肿瘤恶病质模型;正常对照组于相同部位注射等量的PBS缓冲液。每日监测小鼠的体重、肿瘤的大小以及各组小鼠的自发活动。待接种肿瘤细胞的小鼠进入到恶病质状态后,治疗组采用不同浓度的泮托拉唑灌胃,生理盐水组采用等量生理盐水灌胃,NC组和CC组不作任何干预。待药物治疗2周后,收集标本检测相关指标。ELISA法检测血清IL-6及TNF-α水平。Real-time PCR检测腓肠肌JAK2及STAT3 m RNA表达情况;Western blot检测腓肠肌p-JAK2,p-STAT3,Fbx32和Mu RF1蛋白表达情况。免疫组化法检测腓肠肌组织中Fbx32和Mu RF1的表达。HE染色观察腓肠肌形态变化。采用SPSS17.0软件分析数据。结果1肿瘤恶病质模型成功建立荷瘤小鼠在接种肿瘤细胞后第5天,皮下可触及肿瘤结节生长;接种后第12天,荷瘤小鼠肿瘤明显增大,体重较正常对照组明显下降(p0.05),并且出现了自发性活动减少(p0.05)、毛发暗淡无光、精神虚弱等症状,提示荷瘤小鼠进入恶病质状态。2小鼠体质量及腓肠肌变化接种肿瘤细胞后第5天,荷瘤小鼠皮下可触及肿瘤结节,但各组小鼠体重无差异(p0.05);接种c26细胞后第12天,荷瘤小鼠较正常组体重明显减轻(p0.05);在泮托拉唑干预2周后,治疗组小鼠较生理盐水组体重有不同程度增加,且高浓度组小鼠体重增加最为明显(p0.05)。实验结束时,治疗组小鼠较生理盐水组腓肠肌重量及横切面积均有不同程度增加,且高浓度组小鼠体重增加最为明显(p0.05);高浓度组小鼠的肿瘤重量较生理盐水组也有明显减轻(p0.05)。3小鼠血清中炎性因子水平变化与正常对照组相比,血清炎性因子IL-6和TNF-α水平在恶病质小鼠中显著升高(p0.05)。在使用泮托拉唑干预后,各浓度组较生理盐水组血清炎性因子都有不同程度降低,尤其高浓度组降低得最为显著(p0.05)。4小鼠炎性通路JAK2/STAT3表达变化与正常对照组相比,炎性通路JAK2和STAT3的m RNA表达水平在恶病质小鼠中显著升高(p0.05),并且p-JAK2和p-STAT3的蛋白表达水平在恶病质小鼠中也明显增高(p0.05)。在使用泮托拉唑干预后,各浓度治疗组同生理盐水组比较,JAK2和STAT3的m RNA表达水平以及p-JAK2和p-STAT3的蛋白表达水平都有不同程度降低,尤其以高浓度组降低最为显著(p0.05)。5小鼠骨骼肌泛素蛋白酶体系统变化与正常对照组比较,恶病质组小鼠腓肠肌中的Mu RF1和Fbx32的蛋白表达水平明显升高(p0.05),而泮托拉唑干预后,不同浓度干预组小鼠的Mu RF1和Fbx32的蛋白表达水平较生理盐水组均降低,且高浓度组降低最为明显(p0.05)。结论小鼠C26细胞接种于BALB/c小鼠成功建立肿瘤恶病质模型。泮托拉唑能明显改善肿瘤恶病质一般情况,增加体质量,缓解骨骼肌萎缩,抑制肿瘤生长,并且高浓度泮托拉唑具有更好的保护作用。泮托拉唑缓解肿瘤恶病质骨骼肌消耗可能是通过降低炎性因子水平,抑制炎性通路JAK2/STAT3的激活,抑制泛素蛋白酶体途径。
[Abstract]:Objective to establish a mouse model of cancer cachexia by this experiment, the protective effect of pantoprazole on the consumption state of the skeletal muscle cachexia and related mechanism, to provide new ideas for the further research of cachexia, and provide theoretical basis for clinical application. Methods: 48 male BALB/c mice were randomly divided into 6 group, 8 rats in each group, respectively: normal control group (NC), cachexia group (CC), low dose treatment group (CL), the concentration of the treatment group (CM), high dose treatment group (CH), normal saline group (CS). The tumor bearing mice inoculated with mouse colon to the right forelimb adenocarcinoma cell C26, in order to establish the model of cancer cachexia mice; PBS buffer in normal control group was injected with the same amount of daily monitoring. The weight of mice, the tumor size and the mice spontaneous activity. After inoculation of tumor cells into mice to cachexia state, The treatment group was treated with different concentration of pantoprazole orally, saline group with normal saline, NC group and CC group without any intervention. 2 weeks after drug treatment, samples were collected to detect the serum IL-6 and TNF- level.Real-time.ELISA index method to detect PCR JAK2 and STAT3 m of gastrocnemius muscle RNA expression Western; blot detection of gastrocnemius p-JAK2 p-STAT3, Fbx32 Mu and RF1 protein expression. To observe the morphological changes of.HE Fbx32 expression of gastrocnemius muscle and Mu RF1 were detected by immunohistochemistry staining in gastrocnemius tissues. Data analysis by SPSS17.0 software. Results of the 1 tumor cachexia model was successfully established in mice after inoculation of tumor cells the fifth day, subcutaneous tumor nodules were palpable; twelfth days after inoculation, the tumor bearing mice significantly increased, weight decreased significantly compared with the control group (P0.05), and the emergence of spontaneous activity reduction (P0.05), hair Dim and dark, mental weakness and other symptoms, suggesting that cachexia.2 mice body weight and gastrocnemius muscle changes fifth days after inoculation of tumor cells into nude mice bearing subcutaneous tumor bearing mice, palpable tumor nodules, but no difference in body weight of mice in each group (P0.05); twelfth days after inoculation of C26 cells, the tumor bearing mice compared with the normal group significant weight loss (P0.05); after 2 weeks of intervention in pantoprazole, treatment group mice compared with saline group weight increased to different degrees, and the weight of the high concentration group mice increased significantly (P0.05). At the end of the experiment, mice in treatment group compared with the saline group and the gastrocnemius muscle weight cutting area increased in varying degrees, and weight the high concentration group mice increased significantly (P0.05); high concentration group mice tumor weight compared with the saline group was significantly reduced (P0.05) changes in the levels of inflammatory factors in serum of.3 mice compared with the normal control group, serum inflammatory Factor IL-6 and TNF- levels increased significantly in cachexia in mice (P0.05). In the use of pantoprazole intervention groups compared with saline group serum inflammatory factors were decreased in different degree, especially in the high concentration group decreased most significantly (P0.05).4 mice JAK2/ STAT3 expression in the inflammatory pathway and normal compared with the control group, the expression level of M RNA and STAT3 JAK2 inflammatory pathway increased significantly in cachexia in mice (P0.05), and the expression of p-JAK2 and p-STAT3 protein levels were significantly higher in cachexia in mice (P0.05). In the use of pantoprazole after the intervention, the treatment group was with normal saline group, m RNA JAK2 and STAT3 and the expression of p-JAK2 and p-STAT3 protein expression levels were decreased in different degree, especially in the high concentration group was reduced significantly (P0.05) in skeletal muscle of.5 mice ubiquitin proteasome system change compared with normal control group, The expression level of cachexia mice gastrocnemius in Mu RF1 and Fbx32 protein increased significantly (P0.05), and pantoprazole intervention, different concentrations of Mu and RF1 in the intervention group Fbx32 protein expression levels compared with the saline group decreased, and decreased in high concentration group was most significant (P0.05). Conclusion C26 in mice the cells were inoculated into BALB/c mice successfully established model of cancer cachexia. Pantoprazole can significantly improve tumor cachexia in general, increased body weight, alleviate skeletal muscle atrophy, inhibit tumor growth, protective effect and high concentration of pantoprazole has better. Pantoprazole reduced the consumption of cancer cachexia skeletal muscle is likely to decrease levels of inflammatory factors the activation, inhibition of inflammatory pathways of JAK2/STAT3, inhibition of the ubiquitin proteasome pathway.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5

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