脉冲高强度聚焦超声联合微泡非热损伤组织的实验研究

发布时间：2018-04-27 22:56

本文选题：脉冲高强度聚焦超声 + 微泡造影剂　；参考：《重庆医科大学》2007年硕士论文

【摘要】： 背景和目的高强度聚焦超声(high intensity focused ultrasound,HIFU)是一种新兴的非侵入性局部治疗技术。HIFU治疗肿瘤的机制除了热机制外,还有机械机制和空化机制。非热机制(机械机制和空化机制)在HIFU治疗中的作用一方面认为应尽量对空化现象进行抑制,另一方面认为空化有助于热损伤和监控。目前对HIFU形成损伤的机制中仍很难确切描述热机制、空化机制和机械机制在组织损伤中相对独立作用。单纯的非热损伤(机械机制和空化机制)在HIFU治疗中的研究和应用较少。实验首先进行脉冲高强度聚焦超声(pulsed high intensity focused ultrasound,PHIFU)辐照离体牛肝组织,探讨形成非热损伤的辐照参数。然后在此辐照参数下进一步研究PHIFU联合超声微泡造影剂(ultrasound contrast agent ,UCA)非热损伤活体肿瘤组织的可行性及非热损伤的生物学行为,为HIFU治疗肿瘤提供新方法和基础理论支持,进一步推进HIFU技术在肿瘤治疗中的应用。材料和方法参数设置时维持总能量恒定,按不同工作周期1%、5%、15%、25%、50%、75%和100%分成7组,在B超图像监控下将热电偶测温探针插入牛肝,并使针尖位于PHIFU焦点,然后进行定点辐照。观察各辐照参数下焦域温度、灰度和组织学改变。采用肿瘤组织块开腹包埋接种方法,建立兔肝VX2移植瘤动物模型。将36只荷瘤兔随机分为假照组、PHIFU组和PHIFU+UCA组。用第一部分实验筛选出的辐照参数(超声频率0.87 MHz、焦距150 mm、声功率150 W、脉冲重复频率100 Hz、工作周期15%)对兔肝VX2移植瘤进行PHIFU辐照。治疗后1天取材观察肿瘤组织学改变、超微结构改变,应用末端脱氧核苷酸转移酶(TdT)介导脱氧核苷酸(dUTP)缺口末端标记技术(in situ deoxynucleotityl transferase-mediated dUTP nick end labeling,TUNEL)检测细胞凋亡和用免疫组化法检测细胞增殖核抗原(proliferating cell nuclear antigen,PCNA),并用凋亡指数(apoptosis index, PI)和增殖指数(proliferating index, AI)比较细胞凋亡和增殖情况。结果PHIFU辐照离体牛肝时,工作周期25%、50%、75%和100%组平均温升分别为63.4±9.2℃、65.0±11.5℃、66.6±9.9℃、79.6±10.6℃,最高温度大于85℃且有明显的凝固性坏死。1%、5%和15%组平均温升分别29.2±1.9℃、30.0±2.8℃、31.0±2.4℃,最高温度不高于56℃且无肉眼可见的凝固性坏死形成。在25%、50%、75%、100%组,PHIFU损伤后形成的凝固性坏死区光镜下主要表现为胞浆颜色变淡,部分核仁消失、细胞核固缩、染色质边集且随工作周期延长而增加。15%组光镜下细胞间裂隙增宽,肝细胞胞浆内见大量大小不等空泡,而1%组、5%组无明显改变,核仁清晰。 PHIFU辐照兔肝VX2移植瘤后,假照组、PHIFU组和PHIFU+UCA组经2,3,5-氯化三苯基四氮唑(2,3,5-triphenyl tetrazolium chloride ,TTC)染色后,肉眼可见肿瘤组织被均匀红染,表明各组均无肉眼可见的凝固性坏死形成。光镜下PHIFU组见肿瘤细胞胞浆嗜伊红染色浅,细胞肿胀,胞浆疏松,胞浆内大量空泡,少量细胞核固缩、染色质边集;PHIFU+UCA组见胞浆内大量大小不等空泡,可见染色质边集和细胞核固缩。电镜下见PHIFU组瘤细胞胞浆内大量线粒体肿胀和内质网扩张,PHIFU+UCA组部分细胞核固缩和染色质边集,两组肿瘤组织内均见凋亡小体及细胞胞浆内大小不等空泡。TUNEL法检测发现,假照组发现少量阳性染色的肿瘤细胞,PHIFU组和PHIFU+UCA组见较多细胞核棕褐色染色的阳性细胞。PCNA检测结果表明,假照组靶区组织见大量着色部位在肿瘤细胞核的阳性细胞,而PHIFU组和PHIFU+UCA组阳性染色细胞少。PHIFU组和PHIFU+UCA组肿瘤组织的AI比假照组高,而PI阳性表达率比假照组低。PHIFU+UCA组AI比PHIFU组高,但PHIFU+UCA组PI比PHIFU组低。结论1.脉冲工作周期长在PHIFU辐照时可形成热凝固性坏死,而脉冲工作周期短可通过非热效应损伤组织。2.一定工作周期的PHIFU(超声频率0.87 MHz、焦距150 mm、声功率150 W、脉冲重复频率100 Hz、工作周期15%)可通过非热效应损伤肿瘤,表现为胞浆内大小不等空泡和促进肿瘤细胞凋亡及抑制其增殖。3.微泡造影剂可增强超声对组织的非热效应损伤。4.可通过控制PHIFU的工作周期实现非热损伤,为HIFU治疗肿瘤提供了一种新的治疗思路。
[Abstract]:Background and objective high intensity focused ultrasound (high intensity focused ultrasound (HIFU)) is a new noninvasive local treatment technique for the treatment of tumors. Besides the thermal machine, there are mechanical mechanisms and cavitation mechanisms. The role of non thermal mechanism (mechanical mechanism and cavitation mechanism) in the treatment of HIFU should be as far as possible. On the other hand, cavitation is helpful for thermal damage and monitoring. At present, it is difficult to describe the thermal mechanism in the mechanism of HIFU damage. The cavitation mechanism and mechanical mechanism are relatively independent in tissue damage. The research and application of simple non thermal damage (mechanical mechanism and cavitation mechanism) in the HIFU treatment is less.
In the experiment, the isolated bovine liver tissues were irradiated by pulsed high intensity focused ultrasound (PHIFU), and the irradiated parameters of non thermal damage were discussed. Then, the non thermal injured living body tumor tissues of PHIFU combined with the ultrasonic microbubble contrast agent (ultrasound contrast agent, UCA) were further studied under the irradiation parameters. The biological behavior of the feasibility and non thermal damage can provide a new method and basic theory support for the treatment of tumor by HIFU, and further promote the application of HIFU in the treatment of tumor.
When the material and method parameters are set, the total energy is kept constant, and the thermocouple probe is inserted into the bovine liver under the monitoring of B ultrasonic image under the monitoring of different working cycles 1%, 5%, 15%, 25%, 50%, 75% and 100%. The needle tip is located at the focus of PHIFU and then irradiated at fixed point. The focal temperature, gray scale and histological changes under the irradiated parameters are observed.
The animal model of rabbit liver VX2 transplanted tumor was established by the method of open tumor tissue mass, and 36 rabbits were randomly divided into sham group, PHIFU group and PHIFU+UCA group. The irradiation parameters were selected by the first part of the experiment (ultrasonic frequency 0.87 MHz, focal length 150 mm, sound power 150 W, pulse repetition rate 100 Hz, operation cycle 15%). The tumor was irradiated by PHIFU. After 1 days of treatment, the histological changes and ultrastructural changes were observed and the terminal deoxynucleotidyl transferase (TdT) mediated deoxynucleotide (dUTP) Nick nick end labeling technique (in situ deoxynucleotityl transferase-mediated dUTP nick end labeling, TUNEL) was used to detect cell apoptosis and immunohistochemical method. Proliferating cell nuclear antigen (PCNA) was detected and apoptosis and proliferation were compared with the apoptosis index (apoptosis index, PI) and proliferation index (proliferating index, AI).
Results when PHIFU was irradiated in vitro, the average temperature rise of the 25%, 50%, 75% and 100% groups was 63.4 + 9.2, 65, 11.5, 66.6 + 9.9, 79.6 + 10.6, and the highest temperature was higher than 85, and there was obvious solidification necrosis.1%. In the 25%, 50%, 75%, 100% groups, the coagulative necrosis area in the 25%, 50%, 75%, and 100% groups was mainly characterized by the pale color of the cytoplasm, the disappearance of some nucleolus, the nuclear condensation, the aggregation of chromatin and the increase of the widening of the intercellular fissure under the light microscope of the.15% group with the prolongation of the working cycle, and a large number of sizes in the cytoplasm of the liver cells. There was no difference in vacuoles, but in the 1% group, there was no significant change in the 5% groups, and the nucleolus was clear.
After PHIFU irradiated rabbit liver VX2 transplanted tumor, in group PHIFU and PHIFU+UCA group, after 2,3,5- chlorination of three phenyl tetrazoles (2,3,5-triphenyl tetrazolium chloride, TTC), the naked eye could be found to be uniform red staining, indicating that there was no visible coagulative necrosis in all groups. The PHIFU group under light microscopy showed the cytoplasm eosinophilic staining of tumor cells. Light color, cell swelling, loose cytoplasm, a large number of vacuoles in the cytoplasm, a small amount of nuclear condensation and chromatosis, PHIFU+UCA group found a large number of different vacuoles in the cytoplasm, visible chromatin edge and nuclear condensation. Under the electron microscope, a large number of mitochondria swelling and endoplasmic reticulum dilation in the cytoplasm of the tumor cells of the PHIFU group were found, and the part of the nucleus of group PHIFU+UCA was retractable and dyed. In the two groups of tumor tissues, all the two groups of tumor tissues were found to detect apoptotic bodies and cytoplasm unequal vacuoles, and a small number of positive staining cells were found in the sham group. The results of.PCNA detection in group PHIFU and PHIFU+UCA group were found in the positive cells of multicellular nuclear brown staining. The results showed that a large number of pigmentation sites were found in the target tissue of the sham group. The positive cells of the tumor nuclei were higher in group PHIFU and group PHIFU+UCA than in group.PHIFU and group PHIFU+UCA, and AI in group PHIFU+UCA was higher than that in sham group, but the positive rate of PI was higher than that of group PHIFU in the lower.PHIFU+UCA group than that in the sham group, but the PI PHIFU+UCA group was lower than that of the PHIFU group.
Conclusion the 1. pulse working cycle can form thermocoagulation necrosis with PHIFU irradiation, and the short pulse cycle can be short of PHIFU (ultrasonic frequency 0.87 MHz, focal length 150 mm, sound power 150 W, pulse repetition rate 100 Hz, operation cycle 15%) through non thermal effect injury. Unequal vacuoles in the pulp and promoting the apoptosis of tumor cells and inhibiting the proliferation of.3. microbubbles can enhance the non thermal effect of ultrasound on tissue..4. can achieve non thermal damage by controlling the working cycle of PHIFU, which provides a new therapeutic idea for the treatment of tumor in HIFU.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R312

【引证文献】