摘要
背景:
随着超声造影应用研究的深入,微泡造影剂(以下简称微泡)在超声场中引发的相关生物学效应日益引起人们的关注,超声联合微泡在治疗领域显示出新的应用前景。微泡介导下的超声辐照可以用于提高基因的转染率、增强药物疗效、提高肿瘤治疗效果等,大多数学者认为是超声空化效应所致。本课题组前期已制备出适于瘤内注射的卡铂/乳酸羟基乙酸共聚物(PLGA)缓释微球(以下简称卡铂微球),动物实验表明卡铂微球组较卡铂原药组有较好的抑瘤效果,生存时间延长,副作用小。但病理结果肿瘤组织未达到完全原位灭活。因此寻求一种能使肿瘤达到完全原位灭活并有效防止其转移、复发的方法是需迫切解决的问题。基于此,我们设想将卡铂微球与造影剂微泡联合瘤内注射而后经超声辐照,利用空化效应毁损部分瘤细胞,增加化疗药进入细胞内的量,提高化疗药的生物利用度,从而提高局部化疗的疗效。
目的:
1.从病理学角度观察超声辐照瘤内注射微泡的生物学效应。
2.探讨超声辐照瘤内注射微泡对卡铂缓释微球局部化疗的增效作用。
方法:
1.建立大鼠Walker-256皮下移植瘤模型以及用超声评价其生长特性分别采用浓缩腹水注射法和肿瘤组织块皮下包埋法建立皮下肿瘤模型,超声观察成瘤率、肿块大小及声像图特征。用三维能量多普勒显像反映两组移植瘤血管指数(VI)的变化。
2.超声辐照瘤内注射微泡空化效应的病理学研究将已建立Walker-256皮下移植瘤模型的SD大鼠随机分为3组。A组超声监视下瘤内注射微泡,注射剂量依肿瘤体积而定,为1ml/cm3,并随即采用频率1 MHz,强度2.0 W/cm2的超声辐照10 min;B组单纯采用与A组相同强度超声辐照相同时间;C组单纯瘤内注射与A组相同剂量微泡。各组作用后1 h取肿瘤组织,HE染色观察病理改变以及用透射电镜硝酸镧示踪法观察边缘瘤组织细胞膜通透性的变化。
3.超声辐照瘤内注射微泡对局部化疗增效作用研究已建立Walker-256皮下移植瘤模型的SD大鼠分为5组,每组8只。对照组不做治疗,只观察肿瘤体积及血流变化情况;微泡+超声组(MB+US组)瘤内注射微泡(按肿瘤体积,1ml/cm3)后用超声经皮辐照;卡铂微球组(CBPMs组)只瘤内注射卡铂缓释微球(按肿瘤体积,241.1mg/cm3);卡铂微球+超声组(CBPMs+US组)瘤内注射卡铂缓释微球后超声(1MHz,2W/cm2,连续波)经皮辐照10min;卡铂微球+微泡+超声组(CBPMs+MB+US组)瘤内注射卡铂缓释微球及微泡后超声经皮辐照。二维超声动态监测肿瘤大小、声像图变化,并计算其体积。三维能量多普勒超声观察肿瘤治疗前后肿瘤血管变化情况,计算血管指数,量化评价治疗效果。治疗后第18d切取肿瘤标本,HE染色观察肿瘤坏死范围及程度。
结果:
1.浓缩腹水注射组接种成功率100%高于组织块皮下包埋组的成功率75%(P<0.05),且肿瘤生长速度快于组织块皮下包埋组。不同观察时间点浓缩腹水注射组VI均高于组织块包埋组(P<0.05),分别于第16d、第20d时达峰值。
2.超声辐照瘤内注射微泡空化效应的病理学研究中A组光镜可见肿瘤中心大片瘤细胞凝固性坏死,B、C组光镜均未见坏死。A组电镜可见镧颗粒进入肿瘤边缘瘤细胞内、以及血管内皮细胞连接。电镜B组偶可见少量镧颗粒进入细胞内,C组仅见镧颗粒分布于细胞间隙。
3.治疗后18d各组肿瘤体积和血管指数由小至大均依次为CBPMs+MB+US组、CBPMs+US组、CBPMs组、MB+US组、对照组,其中CBPMs+US组和CBPMs组治疗后18d肿瘤体积和血管指数差异均无统计学意义,其余各组间两两比较差异均有统计学意义。病理结果显示CBPMs+MB+US组为广泛的凝固性坏死,CBPMs+US组和CBPMs组可见散在瘤细胞,并可见炎细胞侵润,MB+US和对照组可见增生活跃的瘤细胞,肿瘤中心可见片状坏死。
结论:
1.浓缩腹水注射法较肿瘤组织块包埋法成瘤率高,成瘤周期短,生长特性更稳定,瘤细胞排列紧密呈多形性,生长活跃,肿瘤间质少,血供丰富,故本研究采用浓缩腹水注射法建立皮下移植瘤模型用于介入超声治疗疗效评估。
2.瘤内注射微泡后,采用频率1MHz,强度2.0W/cm2的超声距离皮肤5mm处辐照肿瘤部位10min肿瘤中心可产生局部凝固性坏死,可引起肿瘤边缘瘤细胞细胞膜通透性增高。
3.卡铂微球联合微泡瘤内注射后给予超声辐照可取得较单纯瘤内注射注射卡铂微球或注射卡铂微球后给予超声辐照更好的疗效,皮下移植瘤体积呈负增长,病理学观察可见肿瘤组织坏死广泛,肿瘤血流明显减少甚至消失,可望成为肿瘤治疗的新方法。
Background:
With the wide application of ultrasound contrast agent (UCA), increasing attention had been paid to the bioeffects of ultrasound. Ultrasound-combined microbubble has shown its new prospect in clinical therapy. It has been applied to increase gene transfection efficiency, enhance therapeutic effect of anti-tumor drugs, etc, which are generally accepted by most of researchers to be the induction of acoustic cavitation. Our preliminary study showed that, compared with simple carboplatin by intratumoral injection, it was better to employ carboplatin-loaded polylactic-co-glycolic acid microspheres (CBP-PLGA Ms, abbreviate CBPMs) to treat tumors in animal models, and there were less side effects as well as longer life span. But the pathology showed that the neoplastic cells were not completely destroyed in situ. So it is urgent to find a new to completely destroy neoplastic cells in situ as well as effectively prevent the metastasis and recurrence of tumors. Based on the presumption, we assumed that , after we inject the combination of CBPMs and contrast agent intratumoraly the acoustic cavitation would destroy partial neoplastic cells, and then increase the intracellular amount of chemo and enhance the bioavailability of chemo, consequently, augment the local therapeutic effect.
Objective:
1. To explore the bioeffects of intratumoraly injected microbubbles induced by ultrasound from the point of pathology
2. To investigate the synergistic effect of intratumoraly injected microbubble and CBPMs in local chemotherapy.
Methods:
1. Twenty-four SD rats were randomly divided into two groups, twelve in each group. The subcutaneous transplantation tumor models were prepared by directly injection of enriched cancerous ascites or by the implantation of tumor tissue. The positive rate of inoculation, tumor size, growth rate were observed by ultrasound volume probe. The vascularization index (VI) of two groups was subjected to three-dimensional power Doppler angiography (3D-PDA) examinations.
2. Twelve rats were randomly divided into three groups after twenty-four Walker-256 carcinomas were subcutaneousely implant into the right and left hind legs. In group A, after intratumoral injection of microbubbles, ultrasound with the 1MHz frequency, and the intensity of 2.0W/cm2 was applied on the tumors of the rats; In group B, the intensity and time of ultrasound irridiation were the same as in group A; In group C, the same amount of microbubbles as in group A were injected into the tumor. One hour after the treatment, histopathology damage was observed under light microscope and the edge tumor was harvested for electron microscope examination by means of Lanthanum (La) tracer.
3. The Walker-256 tumor-bearing rats were divided into control group, microbubbles+ ultrasound group (MB+US), carboplatin microspheres group (CBPMs), carboplatin microspheres+ultrasound group (CBPMs+US), carboplatin microspheres+ microbubbles +ultrasound group (CBPMs+MB+US), eight in each group. Two-dimensional ultrasonography was applied to survey the tumor size. Three-dimensional power Doppler angiography was performed pre-and post-treatmently to evaluate the change of vascular perfusion in tumors and the numbers of vessels in tumors so as to evaluate the therapeutic effect of the treatment. The tumor was excised and the pathologic characterization was observed to evaluate the intensity and extent of the necrosis of tumor after eighteen days.
Results:
1. The positive rate of inoculation in the injection group was higher than that in the implantation group, which were 100% vs. 75% respectively (P<0.05). And the growth rate was faster in the injection group than in the implantation group. The time of peak amplitude of VI of the injection group and the implantation group were 12 days and 16 days respectively.
2. In group A, many coagulated necrotic areas were spotted in the tumors under light microscope, and La particles were seen in the cells and vascular endothelial cellular space as well as intercellular space under electronic microscope;there were no necrotic cancer cells seen under light microscope in group B and C, and under electronic microscope little La particles were seen in the cells in group B, and La particle appeared only in the intercellular space in group C.
3. The sequences of tumor volume and VI from small to big were CBPMs+MB+US, CBPMs+US, CBPMs, MB+US, and control group. There was no significant difference in the tumor volume between group CBPMs+US and CBPMs, and there was no significant difference in the VI between group CBPMs+US and CBPM s either. The histopathology showed that there were severe coagulation necrosis in group CBPMs+MB+US, a few scatter neoplastic cells and inflammatory cell were found in CBPMs+US and CBPM group, while there were a lot of active neoplastic cells in MB+US and control group
Conclusion:
1. Both the injection and the implantation technique can prepare subcutaneous transplantation tumor model in rats, the positive rate of inoculation of the injection group was higher than that of the implantation group. Three-dimensional sonography was useful in the evaluation of subcutaneous tumors.
2. Ultrasound-mediated intratumoral injection of microbubbles and destruction could enhance the permeability of tumor cell membrane and could cause coagulate necrosis areas in the tumors.
3. Tumor growth inhibition was most significant in CBPMs+MB+US Group. The method significantly inhibited the growth of subcutaneous tumors with the intensive and complete necrosis of neoplastic tissues, and disappearing of neoplastic vessels. The CBPMs+MB+US way gain a bright future in the treatment of cancer.
引文
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