蝎毒多肽提取物联合雷帕霉素增强自噬抑制S180肉瘤机制的研究

发布时间：2018-02-16 11:11

本文关键词： S180肉瘤蝎毒多肽提取物自噬肿瘤干细胞　出处：《济南大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:以小鼠S180肉瘤为研究对象,观察蝎毒多肽提取物(PESV)联合雷帕霉素(RAPA)对S180肉瘤生长的影响及自噬相关因子Beclin1和MAP1LC3A和肿瘤干细胞标记物CD133和CD90的蛋白表达变化,探讨蝎毒多肽提取物抑制S180肉瘤的作用机制,为临床抗肿瘤治疗提供理论依据。方法:1.建立小鼠S180肉瘤皮下荷瘤模型,随机分为荷瘤对照组、PESV高、低剂量组、雷帕霉素(RAPA组)和联合组(即PESV高+RAPA组),每组10只,连续干预21天。隔日用游标卡尺测量肿瘤体积和用天平测小鼠体重,绘制肿瘤体积增长曲线并计算抑瘤率,观察各实验用药组对S180肉瘤移植瘤生长的抑制作用。2.常规HE染色,光镜下观察S180肉瘤组织病理学改变。3.激光共聚焦显微技术检测S180肉瘤组织中Beclin1、MAP1LC3A、CD133和CD90蛋白表达。4.免疫组织化学法检测S180肉瘤组织中Beclin1、MAP1LC3A、CD133和CD90蛋白表达。5.Western blot法检测S180肉瘤组织中Beclin1、MAP1LC3A、CD133和CD90蛋白表达水平。结果:1.各个实验用药组对小鼠S180肉瘤移植瘤生长的影响药物干预后,pesv高、低剂量组、rapa组和联合组小鼠s180肉瘤移植瘤体积增长速度较荷瘤对照组均减慢,瘤重亦降低,抑瘤率分别为25.4%、15.3%、34.0%和42.5%,差异均有统计学意义(p0.05,p0.01);与rapa组比较,联合组的瘤重和肿瘤体积亦均存在显著差异(p0.05);与pesv低剂量相比,pesv高剂量组肿瘤体积减小,肿瘤生长减慢(p0.05)。2.各个实验组肿瘤组织的病理学变化he染色后光镜下观察,可见对照组坏死区较少且面积较小,少见核固缩等形态学变化。联合组、rapa组和pesv高、低剂量组可见多个大面积片状坏死区,大部分肿瘤细胞呈呈现核固缩、核碎裂等多种细胞死亡形态学改变。联合组肿瘤组织坏死区域最大。给药组亦可见肿瘤细胞胞质无定形,核碎断的表现,可能是肿瘤细胞自噬的结果。癌巢周围有单核细胞等炎细胞的浸润。3.pesv联合rapa对s180肉瘤组织beclin1、map1lc3a、cd133和cd90蛋白表达水平的影响应用激光共聚焦显微镜技术可见,beclin1、map1lc3a、cd133和cd90在胞浆与胞膜上均有表达,cd133与cd90主要表达于细胞膜上,为绿色荧光着色。与荷瘤对照组相比,其它给药组的beclin1和map1lc3a荧光强度均增高,而cd133与cd90荧光强度降低。联合组beclin1和map1lc3a荧光强度最高,cd133与cd90荧光强度最低。免疫组织化学法染色结果显示,与荷瘤对照组比较,给药组beclin1、map1lc3a表达均增多(p0.05,p0.01);cd133和cd90表达降低(p0.05,p0.01)。联合组beclin1、map1lc3a表达显著增多(p0.01),cd133和cd90表达显著降低(p0.01)。与pesv低剂量组相比,pesv高剂量组beclin1和map1lc3a表达增多,cd133和cd90表达明显降低(p0.05)。westernblot半定量法检测结果显示,联合组、rapa组,pesv高低剂量组与荷瘤对照组比较,beclin1、map1lc3a的表达条带均增高,cd133和cd90的表达条带均减弱。联合治疗组beclin1、map1lc3a的表达条带表达显著增高(p0.01),cd133和cd90的表达条带显著减弱(p0.01)。检测结果与免疫组织化学法和免疫荧光共聚焦法检测结果基本一致。结论:1.PESV联合RAPA可明显抑制小鼠S180移植瘤的生长;2.PESV与RAPA可分别上调Beclin1、MAP1LC3A蛋白表达,可能与促进S180肉瘤细胞自噬性死亡有关。3.PESV与RAPA可分别下调CD133和CD90蛋白表达,可能与抑制S180肉瘤中肿瘤干细胞生长有关。4.PESV联合RAPA可能通过协同增强细胞自噬和抑制肿瘤干细胞发挥抗肿瘤效应。
[Abstract]:Objective: to S180 sarcoma in mice as the research object, the observation of polypeptide extract from scorpion venom (PESV) combined with rapamycin (RAPA) on the expression of S180 sarcoma growth and autophagy related genes Beclin1 and MAP1LC3A and tumor stem cell markers CD133 and CD90 protein and explore the mechanism of PESV inhibits S180 sarcoma, theory on the basis of clinical treatment of tumor. Methods: 1. to establish a model of S180 sarcoma in mice subcutaneous xenograft tumor, were randomly divided into control group, high PESV, low dose group, rapamycin (RAPA group) and combination group (PESV + RAPA group), 10 rats in each group. The intervention lasted 21 days. The next day with a vernier caliper. Measurement of tumor volume and body weight of mice by measuring tumor volume, draw the growth curve and calculate the inhibitory rate, observe the treatment group on the growth of S180 sarcoma in the inhibition of.2. by routine HE staining, observed under light microscope and S180 sarcoma tissue disease Beclin1, science.3. laser scanning confocal microscopy to detect S180 sarcoma tissue MAP1LC3A, CD90 protein expression of CD133 and Beclin1, detection of S180 sarcoma.4. immunohistochemical method in MAP1LC3A, the expression of CD133 and CD90 Beclin1 protein,.5.Western blot detection of S180 sarcoma in MAP1LC3A, the levels of CD133 and CD90 protein expression. Results: each experiment group 1. drugs on the growth of mice S180 sarcoma of the intervention, high PESV, low dose group, Rapa group and combined group of S180 sarcoma in mice transplanted tumor volume growth rate higher than that in tumor control group decreased, tumor weight decreased, the inhibition rates were 25.4%, 15.3%, 34% and 42.5%, the difference there was statistically significant (P0.05, P0.01); compared with Rapa group, the tumor volume and weight in the combined group there were significant difference (P0.05); compared with the low dose of PESV, PESV in high dose group decreased tumor volume, tumor growth slowed down ( P0.05).2. pathology in each experimental group to observe the pathological change of tumor tissue with HE staining, observed in control group the necrosis area is less and smaller, rare nuclear pyknosis morphological changes. The United Group, Rapa group and PESV high and low dose group showed many large areas of necrosis, a large part of the tumor cells are present nuclear pyknosis, nuclear fragmentation and death of a variety of morphological changes of cells. The combination group of tumor necrosis area. The drug group also showed the tumor cell cytoplasm of amorphous, nuclear fragmentation performance may be tumor cell autophagy results. Mononuclear cells infiltration of inflammatory cells in.3.pesv combined with Rapa on S180 sarcoma Beclin1, map1lc3a around the cancer nests, affect the expression level of CD133 and the application of laser scanning confocal microscopy of CD90 protein Beclin1, map1lc3a, visible, CD133 and CD90 in the cytoplasm and cell membrane expressed CD133 and CD90 mainly expressed in the cell membrane On the green fluorescence staining. Compared with the tumor bearing group, other treatment group of Beclin1 and the fluorescence intensity of map1lc3a was increased, and the fluorescence intensity of CD133 and CD90 decreased. Beclin1 and map1lc3a combined group the highest fluorescence intensity, fluorescence intensity of CD133 and CD90 is the lowest. Immunohistochemical staining showed that compared with the control group charge tumor, drug group Beclin1, the expression of map1lc3a were increased (P0.05, P0.01); CD133 and the decreased expression of CD90 (P0.05, P0.01). Group Beclin1, the expression of map1lc3a was significantly increased (P0.01), the expression of CD133 and CD90 significantly decreased (P0.01). Compared with the PESV low dose group, high dose group increased the expression of PESV Beclin1 and map1lc3a, the expression of CD133 and CD90 were significantly lower (P0.05) test results of semi quantitative.Westernblot method showed that the combined group, Rapa group, PESV low dose group and the tumor control group, Beclin1, map1lc3a expression bands were increased, the expression of CD133 and CD90 bands The combined treatment group was decreased. The expression of Beclin1, map1lc3a protein bands increased significantly (P0.01), the expression of CD133 and CD90 bands decreased significantly (P0.01). The detection results of immunohistochemistry and immunofluorescence and confocal method are consistent with the test results. Conclusion: 1.PESV combined with RAPA could obviously inhibit S180 tumor the growth of 2.PESV and RAPA respectively; the upregulation of Beclin1 protein expression, MAP1LC3A, and S180 may promote autophagy sarcoma cell death related to.3.PESV and RAPA were down regulated by CD133 and CD90 protein expression might be related to the inhibition of tumor stem cells in S180 sarcoma growth on.4.PESV combined with RAPA may be through the synergistic enhancement of autophagy and inhibit tumor stem cells play anti the effect of tumor.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R738.1

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