摘要
高强度聚焦超声(high intensity focused ultrasound, HIFU)利用超声波在组织内良好的穿透性、方向性和可聚焦性,将体外低能量的超声波聚焦于生物体内,形成高能量的生物学焦域(biological focal region, BFR),在焦域处达到65-100℃的瞬态高温,导致组织瞬间凝固性坏死,而周围组织并无显著损伤。HIFU作为一种非侵入性局部治疗实体肿瘤的新技术,现已广泛应用于多种肿瘤的临床治疗。但由于超声波本身的特性,随着传播距离的增加,超声能量衰减越大,到达靶区的能量越低；若靶组织较均匀,其声阻抗(acoustic impedance,AI)差异小,超声能量更不易沉积,因此,HIFU治疗多采用大剂量、长时间的方法以达到消融目的。这虽然有效,但由于长时间使操作人员易疲劳,大剂量则易造成皮肤、声通道及周围组织损伤,不安全因素大大增加。所谓组织声环境(acoustic environment in tissue, AET),是指HIFU治疗时,治疗靶区组织的声学特性状况,或者组织特有的密度、结构、功能状态及血液供应等所表现出来的超声性质,即影响超声能量沉积的媒质(组织)因素。本研究通过改变组织声学环境(changing acoustic environment in tissue, CAET),改变组织的固有声学特性,从而增加组织超声能量沉积,达到提高HIFU治疗效率的目的。
目的
①探讨采用瘤块种植建立兔肝VX2肿瘤模型的可行性,并研究该模型的特点及生物学特性,为该模型的应用提供实验依据；②研究兔肝VX2移植瘤经低剂量聚焦超声辐照后的病理学转归,探讨低剂量聚焦超声辐照改变兔肝VX2移植瘤内在声环境的作用及机制；③探讨高强度聚焦超声二次辐照对兔肝VX2移植瘤的疗效及影响,为肝癌的HIFU治疗提供一种新思路或新方法。
材料与方法
纯种新西兰大白兔70只,雌雄不限,体重1.5-2.0kg,兔龄2-3个月。采用瘤块种植建立兔肝VX2肿瘤模型。
随机选取30只荷瘤兔,分别于建模后第1、2、3、4、5周各处死5只荷瘤兔,观察肿瘤生长情况及转移情况,同时取部分肿瘤组织行光镜(light microscope, LM)与电镜(electron microscope, EM)检查。剩余5只荷瘤兔观察自然生存时间。
另随机选取10只荷瘤兔作为A组(一次辐照组),于肿瘤建模后第16天进行高强度聚焦超声(HIFU)辐照消融,功率250W。
余下30荷瘤兔,于建模后第15天进行一遍低剂量聚焦超声辐照,功率100W。分别于辐照后第1(即刻)、2、3、5、7天各处死2只荷瘤兔,取辐照组织进行光镜与电镜观察。剩余20只荷瘤兔随机平均分为两组：B组(连续两日二次辐照组),于低剂量辐照后第2天再次辐照消融；C组(间日二次辐照组),于低剂量辐照后第3天再次辐照消融。消融处理同A组。
HIFU辐照按照“线-面-体”的方式,由深至浅逐层进行扫描,每层间距3mm,两线间距8mm,治疗头移动速度3 mm/s。比较A、B、C三组的治疗时间、皮肤改变、能效因子(energy efficiency factor, EEF)、肿瘤复发转移以及生存时间等情况。
计量资料以均数±标准差表示,计数资料以构成比表示；计量资料采用t检验进行统计学分析,计数资料采用x2检验进行统计学分析。检验水准a=0.05。统计过程由SAS8.1统计软件包处理。
结果
兔肝VX2肿瘤接种后第2周均可见到肿瘤生长,肿瘤移植成功率为100%。肿瘤接种后第4周肝内开始出现转移灶,第5周所有荷瘤兔均出现肺部转移。肉眼观,肿瘤呈灰白色,扪之质地较硬,无明显包膜,切面呈鱼肉状,直径超过2cm的肿瘤中心可出现灶性坏死；光镜下,肿瘤细胞体积较大,胞浆丰富,核肥大,核浆比例大,细胞排列紊乱；电镜下,肿瘤细胞呈不规则形,胞浆内细胞器少,核大畸形,核内常染色质丰富。荷瘤兔的自然生存时间为39-57d,死亡原因多为肿瘤生长过大,以及广泛转移所导致的多器官功能衰竭。
兔肝VX2移植瘤经低剂量聚集超声辐照后,光镜下组织学变化：辐照后即刻,可见肿瘤细胞水肿；第2、3天水肿加重,并可见淤血；随后肿瘤细胞逐渐恢复至辐照前表现。电镜下超微结构变化：辐照后即刻,可见肝血窦内皮细胞脱落,肿瘤细胞胞浆内细胞器肿胀；第2、3天可见肝血窦内红细胞聚集,肿瘤细胞胞浆内出现空泡；随后细胞器肿胀逐渐减轻,直至恢复至辐照前表现。
二次辐照组的首次低剂量辐照功率仅为消融功率的2/5,并且二次辐照组分两次进行辐照,故每次辐照的时间缩短,剂量降低,而总的治疗时间(首次低剂量辐照与再次辐照消融的时间之和)并不比一次辐照组延长。消融后,三组的肿瘤组织均发生了典型的凝固性坏死,并有效控制了复发与转移,但二次辐照组的皮肤红斑发生率明显比一次辐照组降低(P<0.05),且EEF大大减小(P<0.05)。其中,连续两日二次辐照的总治疗时间、皮肤红斑发生率、消融时间、EEF最小,最为安全有效。
结论
兔肝VX2移植瘤的病理改变、生长过程、转移情况以及病理转归等都与临床人体肝癌相似,并且生长周期短,成瘤率高,是一种较理想的肝肿瘤动物模型。
低剂量聚焦超声辐照可使兔肝VX2移植瘤发生一系列病理生理改变,从而改变组织声环境,提高再次HIFU消融的损伤效率,并且其表现出来的声学特性与组织发生病理生理变化的程度有关。
二次辐照在达到一次性消融相同的疗效时,每次辐照的时间与剂量减少,并发症减少,且再次辐照消融的损伤效率提高,但总的治疗时间并不延长。其中,以连续两日二次辐照的方式最为安全有效,有可能成为一种新的HIFU治疗方法。
High intensity focused ultrasound (HIFU) focused low-energy ultrasound on biological focal region (BFR) in vivo utilized it's penetrability, directionality and focalization, and created transient temperature about 60 to 100 degrees Celsius to cause tissue coagulation necrosis without surrounding tissues injury. As a new non-invasive local therapy technology of solid tumors, HIFU has been widely used in clinical treatment of various tumors. As the ultrasound characteristics, the greater the propagation distance, the greater the ultrasonic energy attenuation, and the lower the energy reached target; if the target tissue was uniform, the acoustic impedance (AI) difference was small, ultrasonic energy would be more difficult to deposit. Therefore, HIFU therapy used high dose and long time to achieve ablation purpose. That was effective, but long time leading to operator fatigue, and high dose leading to injury of skin and surrounding tissue, which increased the insecurity. Acoustic environment in tissue (AET) indicated the acoustic characteristics of target tissue during HIFU therapy, or the ultrasound features showed by the density, structure, function and blood supply of tissue, that were the medium (tissue) factors impacting the deposition of ultrasonic energy. In this study, ultrasonic energy deposition were increased and efficacy of HIFU therapy were enhanced by changing acoustic environment in tissue (CAET) to change the acoustic characteristics.
①To explore the feasibility of the establish of rabbit VX2 hepatic tumor model by tumor lump implantation and to study the feature and the biology characteristics of the model, to offer the experimental evidence for the application of the model.②To observe the pathological regression of rabbit VX2 hepatic tumors after low-dose irradiation with focused ultrasound, and to explore the role and mechanism of the irradiation in changing acoustic environment of rabbit VX2 hepatic tumors.③To investigate the efficacy and impact of the twice irradiation on rabbit VX2 hepatic tumors using high intensity focused ultrasound (HIFU), and to offer a new way of HIFU treatment for liver cancer.
70 purebred New Zealand rabbits, male or female, were all 1.5 to 2.0 kg by weight,2 to 3 months by age. To establish rabbit VX2 hepatic tumor model by tumor lump implantation.
5 of 30 tumor-bearing rabbits selected Randomly were sacrificed to observe the tumor growth and metastasis respectively in 1,2,3,4 and 5 weeks after modeling, then the tumor tissue were observed under light microscope. The spontaneous survival time of the remaining 5 tumor-bearing rabbits were observed.
Other 10 tumor-bearing rabbits selected randomly were ablated by HIFU (250 W) 16 days after the tumor implantation, as group A (one-time irradiation group).
The remaining 30 tumor-bearing rabbits were irradiated by low-dose focused ultrasound (100 W) 15 days after the tumor implantation.2 tumor-bearing rabbits were sacrificed and the tumor tissue were observed under light and electron microscope 1 (immediately),2,3,5,7 days after the low-dose irradiation with focused ultrasound. The remaining 20 tumor-bearing rabbits were randomly divided into two groups:group B (two-day twice irradiation group), of which rabbits were ablated by HIFU 2 days after the low-dose irradiation; group C (inter-day twice irradiation group), of which rabbits were ablated by HIFU 3 days after the low-dose irradiation. The ablation process were the same with group A.
During low-dose irradiation and ablation, the procedure "from lines-to planes; from planes to volume" was followed. The spacing was 3 mm and 8 mm respectively between two nearest planes and between two closest lines, the scan speed of ultrasound beam was set to be 3 mm/s. The treatment time, skin change, energy efficiency factor, pathologic analysis, recurrence and metastasis, and survival time of the 3 groups were compared.
Measurement data were presented as mean±standard deviation, while count data as rate; measurement data were statistically analyzed by t test, while count data by x2 test. Test level a= 0.05. Statistic were processed by the Statistical Package SAS 8.1.
The tumors could grow up in 2 weeks after implanting. The successful rate of implantation was 100%. Metastases in Liver were appeared 4 weeks after implanting, and lung metastases were appeared in all tumor-bearing rabbits 5 weeks after implanting. Macroscopic observation showed that the tumors were gray white in color and felt harder, necrotic foci was present in the center of the tumor over 2.0 cm in diameter. Light microscope observation showed that the tumor cells were relatively large with abundant cytoplasm and large nucleus, the tumor cells'nucleoplasm proportion was great, and tumor cells arranged irregularly. Electron microscope observation showed that the tumor cells were irregular within less organelles in the cytoplasm, and nucleus were large and abnomal with rich euchromatin in it. Tumor-bearing rabbits' spontaneous survival time was 39 to 57 days. The cause of their death was multiple system organ failure for the large tumor and wide metastasis.
Rabbit VX2 hepatic tumor tissue were changed after low-dose focused ultrasound irradiation. Light microscope changes:edema existed in tumor cells after irradiation instantly, congestion and aggravated edema were found at 2 and 3 days later, then the injured cells recovered gradually. Electron microscope changes:detached endothelial cells of hepatic sinusoid and swelling organelles in tumor cells cytoplasm were observed instantly after irradiation, while 2 or 3 days later, erythrocyte aggregation was found in hepatic sinusoid and cavitations were found in cytoplasm. Thereafter, organelles swelling reduced till resumed to normal.
The first low-dose irradiation power of twice irradiation group was 2/5 of the ablation power, and twice irradiation group were irradiated twice, so each irradiation time and the dose reduced, but the total treatment time (sum of the first low-dose irradiation time and re-irradiation ablation time) was not extend. After ablation, the tumor tissue of three groups was all typical coagulation necrosis, recurrence and metastasis were effective controlled, but the skin erythema occurring rate and EEF of twice irradiation group was significantly lower than one-time irradiation group (p<0.05). Among them, the total treatment time, skin erythema occurring rate, ablation time and EEF of two-day twice irradiation were the least, which was the most safe and effective.
In pathological morphology, growth process, visceral metastasis and pathological regression, rabbit VX2 hepatic tumors are similar to human hepatocarcinoma. It has such characteristics as short growth period, high success rate and so on. The model is an ideal animal hepatoma model.
Low-dose focused ultrasound irradiation can make a series of pathophysiological changes in rabbit VX2 hepatic tumors to change the acoustic environment, and to enhance the efficacy of HIFU re-irradiation to ablate. The acoustic characteristics shown were related to the degree of pathophysiological changes.
With the same effect, twice irradiation group have less irradiation time and dose, of which the complications are reduced, and the damage efficiency are enhanced. Among twice irradiation, the way of continuous two-day is the most safe and effective, which would be a new method of HIFU treatment.
引文
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