【作者】 张红梅；

【导师】 姜宏宇；

【作者基本信息】 吉林大学 ， 临床医学硕士（专业学位）， 2017， 硕士

【摘要】 研究背景及目的:多柔比星(doxorubicin,DOX)作为临床常用的蒽环类抗肿瘤药物,在治疗肿瘤的同时,对正常组织也有损伤,尤以其心脏毒性更为显著。如何在不减弱DOX抗肿瘤作用的同时,降低DOX引起的心脏毒性,是临床面临的棘手问题。有研究证明低剂量辐射(low dose radiation,LDR)可诱导机体正常组织的兴奋效应和适应性反应,同时对肿瘤组织亦有一定的抑瘤效应。也有学者证实m TOR(mammalian target of rapamycin,哺乳动物雷帕霉素靶蛋白)信号通路不仅参与肿瘤细胞的增殖和凋亡,而且在心肌细胞的生长、代谢过程中发挥重要作用。另有研究发现LDR可通过调节m TOR上游信号通路促进机体正常组织的生长发育,而LDR是否通过调节m TOR信号通路对肿瘤组织及心肌组织产生影响目前国内外尚未见报道。本课题组前期研究发现,LDR可通过对抗DOX诱导的氧化应激与线粒体损伤降低DOX的心脏毒性,另外发现LDR与DOX对肿瘤组织的生长具有一定的协同抑瘤效应。本研究选用女性发病率最高的肿瘤——乳腺癌作为研究对象,构建乳腺癌BALB/c小鼠皮下移植瘤模型,通过观察荷瘤鼠体重及瘤重变化,研究移植瘤组织细胞增殖、血管生成及细胞凋亡情况,检测分析移植瘤组织及心肌组织m TOR信号通路蛋白及凋亡相关蛋白的表达水平,探讨LDR对DOX抑瘤效应及心肌损伤的影响及其可能的机制。实验材料及试验方法:选取24只8周龄的BALB/c健康雌性小鼠,每只小鼠右腋下注射无血清的4T1乳腺癌细胞悬液0.1 ml(2×107/毫升),构建乳腺癌BALB/c小鼠皮下移植瘤模型,当移植瘤长径为3-5毫米时进行实验。荷瘤鼠随机分为4个实验组:Control组、LDR组(75m Gy)、DOX组(DOX腹腔注射)、LDR+DOX组(LDR72小时后腹腔注射DOX)。称量荷瘤鼠体重及瘤重,观察LDR与DOX对荷瘤鼠的体重及瘤重的影响;免疫组化法检测各实验组Ki-67与CD34的表达水平,研究肿瘤组织的细胞增殖及血管生成情况;HE染色、TUNEL法检测各实验组肿瘤组织细胞凋亡情况;Western Blot法分析各实验组肿瘤组织及心肌组织凋亡相关蛋白及m TOR信号通路蛋白的表达水平。实验结果:荷瘤鼠体重结果:DOX组与LDR+DOX组体重均下降,DOX组下降显著。瘤重结果:DOX组与LDR+DOX组均下降,LDR+DOX组下降显著。HE染色结果:DOX组肿瘤组织巢状坏死;LDR+DOX组肿瘤组织片状坏死。TUNEL检测结果:DOX组与LDR+DOX组肿瘤组织绿色荧光面积均增多,以LDR+DOX组绿色荧光面积增多明显。免疫组化结果:DOX组与LDR+DOX组肿瘤组织Ki67、CD34阳性染色比例均减少,以LDR+DOX组减少明显。Western Blot结果:肿瘤组织:DOX组与LDR+DOX组Bcl-2表达均下调,以LDR+DOX组下调显著;DOX组与LDR+DOX组Bax和Caspase3表达均上调,其中Caspase3以LDR+DOX组上调显著;与DOX组比,LDR+DOX组m TOR、EIF4E和4EBP1表达均下调。心肌组织:DOX组Bcl-2表达下调,LDR+DOX组表达上调;DOX组Bax和Caspase3表达上调,LDR+DOX组表达下调;DOX组m TOR、EIF4E和4EBP1表达下调,LDR+DOX组m TOR和EIF4E表达上调,4EBP1表达下调。结论:LDR可增强DOX的抑瘤效应,减轻DOX所致的心脏毒性。其机制可能为:LDR可通过调节移植瘤组织m TOR信号通路,阻碍移植瘤组织细胞增殖与血管生成,促进肿瘤细胞凋亡,增强DOX抑瘤效应;LDR可通过调节心肌组织m TOR信号通路,影响凋亡相关基因蛋白的表达水平,减少心肌细胞凋亡,对抗DOX所致心脏毒性。LDR有望成为临床治疗肿瘤的辅助手段。更多还原

【Abstract】 Background and purpose: Doxorubicin(DOX)as a commonly used anthracycline anti-tumor drugs,in the anti-tumor at the same time,the normal tissues were also damaged and its cardiotoxicity was particularly evident.How to reduce DOX-induced cardiotoxicity without compromising its anti-tumor effect is a difficult problem facing clinical practice.Studies have shown that low dose radiation(LDR)can stimulate the normal tissue of the excitatory effect and adaptive response,LDR also has a certain anti-tumor effect.Some scholars have confirmed that m TOR(mammalian target of rapamycin)signaling pathways is not only involved in the proliferation and apoptosis of tumor cells,but also plays an important role in the growth and metabolism of cardiomyocytes.Another study found that LDR can promote the growth and development of normal tissue by regulating the upstream of the m TOR signaling pathways,and how LDR impacts the tumor tissue and myocardial tissue through m TOR signaling pathways,at home and abroad has not been reported yet.In our previous study,we found that pre-LDR could rescue the cardiotoxicity by alleviating oxidative stress and reducing mitochondrial damage DOX-induced,and found that LDR combined with DOX had some synergistic anti-tumor effect on tumor tissue growth.In view of the highest incidence of female tumor,the breast cancer was chosen as the research object in this study.We establish the subcutaneous transplanted tumor model of breast cancer BALB/c mice,by observing the weight of tumor-bearing mice and transplanted tumor,studying the proliferation and angiogenesis and apoptosis of transplanted tumor cells,detecting and analyzing theexpression of m TOR signaling pathways protein and apoptosis-related protein in both transplanted tumor tissue and myocardium,to explore the effect of LDR on anti-tumor effect and myocardial injury induced by DOX and its possible mechanism.Materials and methods: Twenty-four BALB/c healthy female mice(8 week-old)were selected.Each mouse was injected right-armpit with serum-free 4T1 breast cancer cell suspension 0.1 ml(2x107/ml)to construct breast cancer BALB/c mice subcutaneous transplanted tumor model.When tumor diameter for 3-5 mm,the tumor-burdened rats were randomly divided into four experimental groups: Control group,LDR group(75 m Gy),DOX group(intraperitoneal injection of DOX),LDR+DOX group(LDR 72 hours later intraperitoneal injection of DOX).We observe how LDR and DOX affect the rat weight and tumor weight by weighing the tumor-bearing rat weight and tumor weight.The expression levels of Ki-67 and CD34 were detected by immunohistochemical method,so as to measure the cell proliferation and angiogenesis of tumor;HE staining and TUNEL method to detect the tumor cell apoptosis;Western Blot method to analysis the apoptosis-related proteins and m TOR signaling pathways protein expression in the experimental tumor tissue and the myocardial tissue.Results: Tumor-burdened rat weight results: DOX and LDR+DOX group weight decreased,DOX group decreased significantly.Tumor weight results: DOX group and LDR+DOX group decreased,LDR+DOX group decreased significantly.HE staining results: tumor tissue nests necrosis in DOX group;LDR+DOX group flaky necrosis.TUNEL test results: green fluorescent area increased both in DOX group and LDR+DOX group of tumor tissue and was significantly in LDR+DOX group.Immunohistochemical results: both Ki67 and CD34 positive staining rate in DOX group and LDR+DOX group of tumor tissue decreased and was significantly in LDR+DOX group.Western Blot results: Tumor tissue: The expression of Bcl-2 in LDR+DOX group was significantly lower than that in DOX group;The expression of both Bax and Caspase3 in DOX group and LDR+DOX group increased,Caspase3 increased significantly in LDR+DOX group;Compared to the DOX group,the expression of m TOR,EIF4 E and 4EBP1 in LDR+DOX group decreased.Myocardial tissue: The expression of Bcl-2 in DOX group was down-regulated,was up-regulated in LDR+DOX group;both Bax and Caspase3 was up-regulated in DOX group,was down-regulated in LDR+DOX group.The expression of m TOR,EIF4 E and 4EBP1 in DOX group was down-regulated,the expression of m TOR and EIF4 E in LDR+DOX group was up-regulated,while 4EBP1 was down-regulated.Conclusion: LDR could enhance the anti-tumor effect and mitigate cardiotoxicity induced by DOX.The mechanism may be that LDR could regulate the m TOR signaling pathways of transplanted tumor tissue,blocking tumor cell proliferation and angiogenesis,accelerating tumor cell apoptosis and therefore enhance DOX-induced tumor-inhibition effect.LDR could affect the expression of apoptosis-related gene protein expression by regulating m TOR signaling pathways in myocardial tissue so as to reduce myocardial cell apoptosis and cardiotoxicity induced by DOX.LDR is expected to become an auxiliary means in the clinical treatment of tumor.更多还原