低功率高能聚焦超声对胰腺癌移植瘤凋亡和基因表达影响的初步研究

发布时间：2018-02-10 06:44

本文关键词： 胰腺癌高强度聚焦超声低功率凋亡表达谱芯片信号通道　出处：《天津医科大学》2014年博士论文　论文类型：学位论文

【摘要】：研究目的评测低功率高强度聚焦超声(high intensity focused ultra-sound, HIFU)治疗人胰腺癌YY-1移植瘤的疗效、安全性,探讨低功率HIFU对肿瘤细胞凋亡的影响及其机制,以期为临床应用提供理论依据。研究方法 1.BALB/cnu裸小鼠30只,随机分为低功率组、高功率组、对照组,每组10只。建立人胰腺癌YY-1移植瘤裸鼠模型,并行HIFU辐照治疗,治疗期间观察肿瘤体积变化、肿瘤生长速率,记录不良反应；各组分别于第7天、第14天收集肿瘤标本,Western blot方法分析Bax、Bcl-2表达情况,TUNEL法检测各组肿瘤细胞凋亡率。 2.治疗第7天分别收集低剂量组及对照组肿瘤组织,通过Agilent人类基因表达谱芯片检测差异性基因表达。通过实时定量PCR(Quantitative Real time PCR)对筛选出的差异性基因进行验证。结果 1.治疗后,低功率组、高功率组的肿瘤体积均小于对照组(P0.05),两组间未见显著性差异(P0.05)；在第3天、第7天、第14天,低功率组、高功率组肿瘤生长速率明显低于对照组,具有统计学差异(P0.001);两组之间比较无统计学差异(P0.05)。与低功率组比较,高功率组不良反应增多,主要为皮肤烧伤和通道组织损伤(P0.05)； 2.治疗后第7天,与对照组比较,低功率组、高功率组Bax均表达上调,Bcl-2表达均下调,有统计学差异(P0.05)；治疗后第14天,与对照组比较,低功率组、高功率组Bax表达均进一步上调,Bcl-2表达均进一步下调,差异有统计学意义(P0.05)；TUNEL结果显示,治疗后第7天、第14天,低功率组和高功率组细胞凋亡率均高于对照组,差异有统计学意义(P0.05),两组间细胞凋亡率无统计学差异(P0.05)。 3.表达谱芯片结果显示,与对照组比较,低功率HIFU辐照后发生差异表达50倍以上的基因共4767个,其中上调3958个,下调809个。GO分析(Gene Ontology,GO)和pathway分析结果提示,HIFU通过上调Fas、Bid、IL1-R、Apaf-1、Caspase-8、 Caspase-9等关键基因表达,直接促进凋亡,还通过影响p53信号通道、MAPK信号通道、PI3K-Akt信号通道等多个信号通道中的关键基因间接促进肿瘤凋亡。 4.选取Caspase-8、Caspase-9、Fas、Bcl-xL行实时定量PCR验证,结果显示扩增良好,并与表达谱芯片结果一致。结论： 1.低功率、高功率HIFU治疗胰腺癌移植瘤均有效,高功率组局部不良反应较多；低功率HIFU在疗效不差于高功率HIFU的基础上,安全性更好。 2. HIFU可上调促凋亡基因Bax的表达,下调抗凋亡基因Bcl-2的表达,并具有时间和剂量依赖关系。 3.低功率、高功率HIFU均可促进肿瘤细胞凋亡,两组间肿瘤细胞凋亡率未见统计学差异。 4.低功率HIFU可通过内源性途径和外源性途径促进细胞凋亡,同时,还通过影响多种信号通道、多个环节直接或间接促进肿瘤组织凋亡,并可能存在着双向调控机制。
[Abstract]:Research purpose. To evaluate the efficacy and safety of low power high intensity focused ultrasound (HIFU) in the treatment of human pancreatic carcinoma YY-1 transplanted tumor, and to investigate the effect of low power HIFU on apoptosis of tumor cells and its mechanism, in order to provide theoretical basis for clinical application. Research method. 1. Thirty BALB / cnu nude mice were randomly divided into low power group, high power group and control group with 10 mice in each group. The nude mice model of human pancreatic carcinoma YY-1 transplanted tumor was established and treated with HIFU irradiation. The tumor volume and tumor growth rate were observed during the treatment. On the 7th and 14th day, the expression of BaxanBcl-2 was analyzed by Western blot and Tunel method was used to detect the apoptosis rate of tumor cells in each group. 2. The tumor tissues of low dose group and control group were collected on the 7th day of treatment, the differentially expressed genes were detected by Agilent human gene expression microarray, and the differentially screened genes were verified by real-time quantitative PCR(Quantitative Real time PCR. Results. 1. After treatment, the tumor volume of low power group and high power group were all smaller than that of control group (P 0.05), there was no significant difference between the two groups, and the tumor growth rate of low power group and high power group was significantly lower than that of control group on day 3, day 7 and day 14. There was no statistical difference between the two groups (P 0.05). Compared with the low power group, the adverse reactions in the high power group were more than those in the low power group, mainly in the skin burn and channel tissue injury (P0.05). 2. On the 7th day after treatment, compared with the control group, the expression of Bax in low power group and high power group were all down-regulated, with statistical difference (P 0.05), and on the 14th day after treatment, the expression of Bax in low power group was lower than that in control group. The expression of Bax was further up-regulated and the expression of Bcl-2 was further down-regulated in high power group. The results of Tunel showed that the apoptosis rate of low power group and high power group was higher than that of control group on the 7th and 14th day after treatment. The difference was statistically significant (P 0.05), and there was no significant difference in apoptosis rate between the two groups (P 0.05). 3. Compared with the control group, there were 4767 differentially expressed genes in low power HIFU irradiation, 3 958 of which were up-regulated. The down-regulation of 809 gene ontology and pathway analysis showed that HIFU could directly promote apoptosis by up-regulating the expression of caspase-8, Caspase-9 and so on. In addition, the key genes of PI3K-Akt signal pathway and other signal channels can indirectly promote tumor apoptosis by affecting p53 signal channel and MAPK signal channel. 4. Caspase-8 caspase-9 Fas-Fas-xL was selected for real-time quantitative PCR verification. The results showed that the amplification was good, and the results were consistent with the results of expression microarray. Conclusion:. 1. Low power and high power HIFU were effective in the treatment of pancreatic cancer transplanted tumor, and the local adverse reactions were more in the high power group, and the low power HIFU was more safe than the high power HIFU. 2. HIFU could up-regulate the expression of pro-apoptotic gene Bax and down-regulate the expression of anti-apoptotic gene Bcl-2 in a time-and dose-dependent manner. 3. Low power and high power HIFU could promote the apoptosis of tumor cells, but there was no statistical difference between the two groups. 4. Low power HIFU can promote apoptosis through endogenous pathway and exogenous pathway. At the same time, it can directly or indirectly promote apoptosis of tumor tissue by affecting many signal channels, and there may be bidirectional regulation mechanism.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R735.9;R445.1

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