HB增强低强度超声对鼻咽癌细胞杀伤作用的研究

发布时间：2018-03-11 00:02

本文选题：声动力疗法　切入点：低强度超声　出处：《重庆医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景:鼻咽癌(Nasopharyngeal carcinoma)是一种好发于中国南方的恶性肿瘤。目前的治疗方法存在一定的局限性和严重的副作用。寻找选择性好、副作用小的有效方法一直是人们追求的目标。光动力治疗(photodynamic therapy, PDT)是一种选择性好、副作用相对小的肿瘤治疗方法。它主要是利用选择性潴留于增殖活跃组织的光敏剂在一定波长的光照下产生细胞毒作用的活性氧物质摧毁肿瘤组织细胞。由于光在生物组织中的穿透能力有限,严重影响到光动力治疗的实际应用。令人欣慰的是,有研究发现一些光敏剂也能被超声活化。这一发现激起了人们对声动力治疗(Sonodynamic therapy, SDT)研究的兴趣。相继发现卟啉类光敏剂极易被超声激活产生活性氧物质,并得到广泛深入的研究。竹红菌素B(hypocrellin B, HB)是新近开发成功的一种中草药光敏剂,其成分单一,化学结构明确,性质稳定,是一种很有发展前景的新型光敏剂。开展竹红菌素B成为一种新型声敏剂引起我们的广泛兴趣,因此,本研究采用超声频率为1.7 MHz.声强为0.65 W/cm2为实验参数,探讨了低强度超声联合竹红菌素B对鼻咽癌CNE2细胞株的影响,旨在为声动力治疗提供新的声敏剂,并为鼻咽癌的临床治疗提供新的技术手段。材料与方法:采用倒置显微镜观察和CCK-8法检测低强度超声活化HB声动力作用后CNE2细胞的生长状况;应用流式细胞检测术和Hoechst33258细胞核荧光染色法观察超声活化HB介导的声动力作用对CNE2细胞凋亡的影响;通过Rhodamine123染色法了解细胞线粒体膜电位变化情况;最后通过透射电镜观察声动力作用后细胞超微结构的变化情况。结果:HB能显著增强低强度超声对人鼻咽癌CNE2细胞的杀伤效率,CCK-8法检测发现随着时间的增加细胞抑制率较对照组显著增加,倒置显微镜下观察到HB介导的声动力组细胞数量较其他对照组明显减少,细胞固缩,单纯HB对细胞没有细胞毒性作用;Hoechst33258细胞核荧光染色示US(0.65 W/cm2)活化HB(2.5uM)介导的声动力组出现核凝聚、凋亡小体;同时流式细胞术检测细胞凋亡率发现HB介导的声动力组,在声动力作用18小时候细胞早期凋亡率达18.61%; Rhodamine123染色对CNE2细胞线粒体膜电位检测显示HB介导的声动力组,在声动力作用4小时后细胞线粒体膜电位明显降低,最后透射电镜观察到HB介导的声动力组,在声动力作用18小时细胞出现线粒体肿胀溶解,大部分细胞坏死,碎裂。结论:低强度超声活化HB介导的声动力作用能够有效的杀伤人鼻咽癌CNE2细胞,提示HB可能是一种新型声敏剂,具有潜在的推广和应用价值;SDT有望发展成为一种很有潜力的治疗肿瘤的新模式。
[Abstract]:Background: nasopharyngeal carcinoma (NPC) is a malignant tumor in southern China. There are some limitations and serious side effects in the treatment of nasopharyngeal carcinoma. Photodynamic therapy (PDT) is a good selectivity. Cancer treatment with relatively small side effects. It mainly uses reactive oxygen species (Ros), which selectively trap active proliferating tissues to produce cytotoxic effects under certain wavelengths of light. Because of light, tumor cells are destroyed. With limited penetration in biological tissues, Has had a serious impact on the practical use of photodynamic therapy. Some studies have found that some Guang Min agents can also be activated by ultrasound. This discovery has aroused people's interest in the study of sonodynamic therapy (SDTs). It is a newly developed Chinese herbal medicine Guang Min. Its composition is single, its chemical structure is clear, and its properties are stable. It is a new type of Guang Min agent with great development prospects. It is of great interest to develop a new type of acoustical sensitizer. Therefore, the ultrasonic frequency is 1.7 MHz and the sound intensity is 0.65 W / cm ~ 2 as the experimental parameter in this study, and the experimental parameters are as follows: (1) the ultrasonic frequency is 1.7 MHz and the sound intensity is 0.65 W / cm ~ (2). The effects of low intensity ultrasound combined with erythromycin B on nasopharyngeal carcinoma (NPC) CNE2 cell line were studied in order to provide a new sound sensitizer for acoustic dynamic therapy and a new technique for clinical treatment of nasopharyngeal carcinoma. Materials and methods: the growth of CNE2 cells was observed by inverted microscope and CCK-8 method. Flow cytometry and Hoechst33258 nuclear fluorescence staining were used to observe the effect of ultrasonic activated HB-mediated acoustics on apoptosis of CNE2 cells, and the changes of mitochondrial membrane potential were observed by Rhodamine123 staining. At last, the ultrastructure of the cells was observed by transmission electron microscope (TEM). Results the effect of low intensity ultrasound on human nasopharyngeal carcinoma (NPC) CNE2 cells was significantly enhanced by 1: HB. The results of CCK-8 method showed that the inhibition rate of human nasopharyngeal carcinoma (NPC) CNE2 cells was significantly higher than that of the control group with the increase of time. Under inverted microscope, the number of cells in HB-mediated acoustic power group was significantly lower than that in other control groups, and cell pyknosis was observed in HB-mediated acoustical power group. There was no cytotoxic effect of HB alone on cells. Fluorescence staining of Hoechst33258 cells showed that US(0.65 W / cm 2) activated HB2. 5 uM) -mediated nuclear condensation and apoptotic corpuscles were found in the acoustic power group, and the apoptosis rate was detected by flow cytometry in the HB-mediated acoustic dynamic group, and the cell apoptosis rate was found in the HB-mediated acoustics group by flow cytometry. The rate of early apoptosis reached 18.61% at 18 hours after acoustics, and the mitochondrial membrane potential of CNE2 cells was significantly decreased by Rhodamine123 staining after 4 hours of acoustical stimulation, and the mitochondrial membrane potential of CNE2 cells was detected by Rhodamine123 staining. Finally, transmission electron microscopy showed that in HB-mediated acoustic power group, mitochondria swelling and dissolution, necrosis and fragmentation of most cells were observed at 18 hours after acoustical stimulation. Conclusion: low intensity ultrasound activated HB-mediated acoustic action can effectively kill human nasopharyngeal carcinoma (NPC) CNE2 cells, suggesting that HB may be a new type of sound sensitizer. SDT is expected to become a potential new model for tumor treatment.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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