微泡诱导超声空化联合血凝酶增强兔肝脏及VX2肝癌微波热消融作用的实验研究

发布时间：2018-04-23 23:23

  本文选题：微泡 + 超声空化　； 参考：《第四军医大学》2017年博士论文

【摘要】：第一部分微泡诱导超声空化联合血凝酶增强兔正常肝脏微波热消融作用的实验研究目的:探讨微泡诱导超声空化联合血凝酶对兔正常肝脏微波热消融的增强作用。方法:健康新西兰大白兔40只,随机分为4组,每组均为10只。4组均经相应的空化假辐照或空化治疗:A组,生理盐水组(空化假辐照+生理盐水);B组,血凝酶组(空化假辐照+生理盐水+血凝酶);C组,微泡空化组(超声空化+微泡);D组,微泡联合血凝酶组(超声空化+微泡+血凝酶)。空化治疗后行微波热消融治疗,消融后立即处死动物取出肝脏,以大体病理学方法测量消融区体积。分别提取消融区、过渡区及周围区组织,通过光镜(HE)观察及透射电镜显示评估4组之间微波热消融效果。结果:大体病理结果显示,微波热消融后D组的肝脏凝固坏死体积(3.13±0.78cm~3)大于其它3组(P0.05);C组凝固坏死体积(1.65±0.51cm~3)大于A组(0.53±0.10 cm~3)和B组(0.46±0.09 cm~3)(P0.05);A组和B组凝固坏死体积比较无明显统计学意义。光镜(HE)显示消融区与未消融区区域分界明显,4组消融区均未见大片凝固性坏死。透射电镜显示D组内可见大量的细胞核和细胞膜的破坏。结论:微泡诱导超声空化联合血凝酶可更显著地增加低能量微波消融兔正常肝脏体积,与对照组相比可明显增加热消融的效率。第二部分微泡诱导超声空化联合血凝酶对兔VX2肝癌微血管阻断作用及机制的实验研究目的:探讨微泡诱导超声空化联合血凝酶对兔VX2肝癌肿瘤微血管的损毁及阻断的作用机制。方法:健康新西兰大白兔接受开腹种植法建立兔VX2肝癌肿瘤模型。VX2肝肿瘤种植14 d后,存活的32只荷瘤兔随机分为4组:A组,生理盐水组(空化假辐照+生理盐水);B组,血凝酶组(空化假辐照+生理盐水+血凝酶);C组,微泡空化组(超声空化+微泡);D组,微泡联合血凝酶组(超声空化+微泡+血凝酶),每组均为8只。4组均经相应的空化假辐照或空化治疗,在实验前后分别进行超声造影检查(Contrast Enhanced Ultrasonography,CEUS),使用定量图像分析软件分析实验前后CEUS的峰值灰阶值(Peak)的变化,通过光镜(HE)观察及透射电镜超微结构显示来比较4组的治疗效果。结果:实验前后,A组和B组超声造影图像视觉分析及定量分析Peak值比较均无明显变化(P0.05)。C组和D组视觉观察有造影灌注缺损,D组缺损更为明显,定量分析D组Peak值从(72.94±10.27)降至(15.74±8.50)(P0.05),C组Peak值从(68.79±6.48)降至(29.57±4.31)(P0.05)。HE染色显示:C组内可见局灶小片状出血;D组可见大片状出血及明显的细胞水肿。A组和B组无明显病理改变。透射电镜显示:C组和D组的微血管壁破坏,基底膜不完整。结论:微泡诱导超声空化联合血凝酶能促进内皮损伤并导致更广泛的血栓形成,同时验证了血凝酶选择性针对肿瘤组织局部损伤的血管快速血栓形成及血流阻塞具有一定的靶向性,并有增强热消融的作用。第三部分微泡诱导超声空化联合血凝酶增强兔VX2肝癌微波热消融作用的超声影像学评价方法及意义目的:通过二维灰阶超声成像、彩色多普勒血流成像(Color Doppler Flow Imaging,CDFI)和超声造影检查(Contrast Enhanced Ultrasonography,CEUS)等方法,探讨微泡诱导超声空化联合血凝酶对兔VX2肝癌微波热消融的增强作用。方法:32只VX2荷瘤动物随机分为4组,每组均为8只。4组均经相应的空化假辐照或空化治疗:A组,生理盐水组(空化假辐照+生理盐水);B组,血凝酶组(空化假辐照+生理盐水+血凝酶);C组,微泡空化组(超声空化+微泡);D组,微泡联合血凝酶组(超声空化+微泡+血凝酶)。空化治疗完毕后行微波热消融治疗。并在整个治疗前后分别进行二维灰阶超声成像、CDFI和CEUS等检查。结果:实验前,4组分别用二维灰阶超声与CEUS测量肿瘤体积,2种方法所得结果比较无明显统计学差异。微波热消融治疗后,二维灰阶超声成像显示:消融区出现弥漫性分布的片状强回声光点;CDFI显示:消融区未见明显血流信号,A组、B组和C组消融区边缘可见少量血流信号;CEUS显示:消融区造影剂未充盈。二维灰阶超声和CEUS测量均显示D组消融区体积大于其它3组(P0.05),CEUS显示4组消融区体积明显大于二维灰阶超声测量结果(P0.05)。结论:二维灰阶超声和CEUS均能显示和测量肿瘤体积和热消融区范围及体积大小,尤其是CEUS能更准确地显示肿瘤和消融区体积大小,并可检测消融区是否有残留组织,对评估肿瘤消融后的疗效具有较高的准确性。第四部分微泡诱导超声空化联合血凝酶增强的微波热消融治疗兔VX2肝癌的转归及生物学效应目的:通过组织病理学、免疫组织化学及透射电镜显示等方法,探讨微泡诱导超声空化联合血凝酶增强微波热消融技术治疗兔VX2肝癌一个时间段内的连续性生物效应及转归。方法:80只VX2荷瘤动物随机分为4组,每组均为20只。4组均经相应的空化假辐照或空化治疗:A组,生理盐水组(空化假辐照+生理盐水);B组,血凝酶组(空化假辐照+生理盐水+血凝酶);C组,微泡空化组(超声空化+微泡);D组,微泡联合血凝酶组(超声空化+微泡+血凝酶)。空化治疗完毕后行微波热消融治疗,于消融后0,3,7,14 d 4个时间点每组随机选取5只动物行超声造影测量消融区体积。通过大体病理学方法测量肿瘤体积,对消融区、过渡区和周围区组织行光镜(HE)观察及透射电镜显示组织细胞超微结构,采用免疫组织化学方法观测细胞PCNA表达,采用脱氧核苷酸末端转移酶介导的生物素化脱氧三磷酸尿苷末端缺口标记法(Terminal deoxynucleotidyl transferase-mediated biotin-d UTP nick end labeling,TUNEL)观测细胞凋亡的表现,并通过视觉观察对肿瘤转移进行分级。结果:消融后0,3,7,14 d,D组消融区体积(3.059±0.476cm~3,2.734±0.407cm~3,2.796±0.482cm~3,10.159±1.444cm~3)明显大于其他3组(P0.05);C组消融区体积(1.086±0.337cm~3,0.919±0.174cm~3,1.087±0.354cm~3,3.707±0.850cm~3)明显大于A组(0.447±0.180cm~3,0.454±0.161cm~3,0.472±0.154cm~3,0.765±0.204cm~3)和B组(0.467±0.168 cm~3,0.290±0.075 cm~3,0.304±0.080cm~3,0.652±0.151cm~3)(P0.05)。消融后7 d和14 d,D组肿瘤体积(1.166±0.468cm~3,1.445±0.593cm~3)明显小于其他3组(P0.05),C组肿瘤体积(2.447±0.793 cm~3,5.477±1.703cm~3)明显小于A组(6.859±1.126cm~3,10.698±4.910cm~3)和B组(6.475±1.068 cm~3,11.821±1.885cm~3)(P0.05)。消融后即刻,HE染色显示4组微波消融区内均未发现明显的大面积凝固性坏死。消融后3 d,HE染色显示4组消融区内可见组织细胞坏死,过渡区可见纤维包裹带形成。消融后3,7,14 d,D组纤维包裹带厚度明显大于其它3组(P0.05)。透射电镜显示,消融后即刻,D组消融区内组织细胞结构较其它3组破坏严重;消融后7 d,D组过渡区细胞内线粒体损伤更严重。消融后0,3,7,14 d,4组过渡区均显示PCNA阳性细胞和凋亡细胞;消融后7 d,4组PCNA阳性细胞均达峰值。消融后0,3,7,14 d,C组和D组过渡区阳性细胞指数低于其他2组(P0.05)。TUNEL结果显示,消融后3 d,4组过渡区凋亡细胞达到顶峰。消融后0,3,7 d,D组过渡区凋亡细胞比值较其他3组明显增多(P0.05)。消融后7 d和14 d,相比其他3组,D组肿瘤转移程度轻,转移时间晚。结论:微泡诱导超声空化联合血凝酶可增加兔VX2肝癌微波热消融作用、扩大消融体积、增强组织细胞的破坏、抑制肿瘤的生长和转移,并促进过渡区细胞的凋亡、抑制肿瘤细胞增殖。同时,过渡区纤维包裹带形成可促进消融区周边组织的机化和修复。
[Abstract]:The first part is to study the effect of microbubble induced ultrasonic cavitation combined with hemagglutinin on microwave heat ablation of normal liver in rabbits. Objective: To explore the enhancement effect of microbubble induced ultrasonic cavitation combined with hemagglutinin on microwave heat ablation of normal liver in rabbits. Methods: 40 healthy New Zealand white rabbits were divided into 4 groups, each group was 10.4 groups. The treatment of cavitation false irradiation or cavitation: A group, saline group (cavitation false irradiation + physiological saline); B group, hemagglutination group (cavitation false irradiation + physiological saline + hemagglutination enzyme); C group, microbubble cavitation group (ultrasonic cavitation + microbubble); D group, microbubble combined hemagglutination group (ultrasonic cavitation + microbubble + hemagglutination). After cavitation treatment, microwave heat ablation treatment, ablation after ablation, erect after ablation The liver was taken out and the ablation area volume was measured by general pathological method. The ablation area, the transition zone and the surrounding tissue were extracted respectively. The effects of microwave heat ablation between the 4 groups were evaluated by light microscopy (HE) and transmission electron microscopy. Results: gross pathological results showed that the volume of liver coagulation necrosis in group D after micro wave heat ablation (3.13 + 0.78cm) ~3) was larger than the other 3 groups (P0.05); the volume of coagulation necrosis (1.65 + 0.51cm~3) in group C was greater than that in group A (0.53 + 0.10 cm~3) and B group (0.46 + 0.09 cm~3) (P0.05); there was no significant statistical significance in the volume of coagulation necrosis in A and B groups. A large number of nuclei and cell membranes were observed in the D group. Conclusion: microbubble induced ultrasound cavitation combined with hemagglutination enzyme could increase the normal liver volume of rabbits with low energy microwave ablation more significantly. Compared with the control group, the efficiency of heat ablation was significantly increased. Second microbubbles induced ultrasound cavitation combined with hemagglutinin on the microvascular obstruction of rabbit VX2 liver cancer The experimental study of the effect and mechanism: To explore the mechanism of microbubble induced ultrasound cavitation combined with hemagglutinin on the damage and blocking of the tumor microvessels of VX2 hepatocellular carcinoma in rabbits. Methods: Healthy New Zealand rabbits were treated with open implant method to establish a rabbit VX2 hepatoma tumor model.VX2 liver tumor 14 d, and 4 groups of surviving tumor rabbits were randomly divided into 4 groups: A Group, normal saline group (cavitation false irradiation + physiological saline), group B, hemagglutination group (cavitation false irradiation + physiological saline + hemagglutination enzyme), group C, microbubble cavitation group (ultrasonic cavitation + microbubble), group D, microbubble combined hemagglutination group (ultrasonic cavitation + microbubble + hemagglutinase), each group of 8.4 groups were treated with corresponding cavitation false irradiation or cavitation treatment, before and after the experiment, respectively. Contrast Enhanced Ultrasonography (CEUS) was performed and the quantitative image analysis software was used to analyze the changes of the peak gray scale (Peak) of CEUS before and after the experiment. The therapeutic effects of the 4 groups were compared by the optical microscopy (HE) observation and the ultrastructural display of transmission electron microscopy. Results: the visual analysis of the images of the contrast images of the A and B groups before and after the experiment and the visual analysis of the images of the contrast images in the A and the B groups were analyzed. There was no obvious change in Peak value (P0.05) in group.C and group D, the visual observation of group.C and D was more obvious. The Peak value of group D was decreased from (72.94 + 10.27) to (15.74 +) (P0.05). The Peak value of group C decreased from (68.79 + 6.48) to (29.57 + 4.31) (P0.05). There was no obvious pathological changes in group.A and B group. Transmission electron microscopy showed that the microvascular walls of group C and D were damaged and the basement membrane was incomplete. Conclusion: microbubble induced ultrasound cavitation combined with hemagglutinin can promote endothelial damage and lead to more extensive thrombosis, and the selectivity of hemagglutinin for tumor tissue is verified. Local injury of vascular rapid thrombosis and blood flow obstruction have certain targeting and enhancement of heat ablation. Third part of microbubbles induced ultrasound cavitation combined with hemagglutination enzyme to enhance the effect of microwave heat ablation of rabbit VX2 liver cancer by ultrasonic imaging evaluation method and significance Objective: through two-dimensional gray scale ultrasound imaging, color Doppler blood flow Color Doppler Flow Imaging (CDFI) and ultrasound contrast examination (Contrast Enhanced Ultrasonography, CEUS) were used to investigate the enhancement effect of microbubble induced ultrasonic cavitation combined with hemagglutinin on microwave heat ablation of rabbit VX2 liver cancer. Methods: 32 VX2 tumor bearing animals were randomly divided into 4 groups, each of which was 8.4 groups to be irradiated by corresponding cavitation or Cavitation therapy: group A, saline group (cavitation false irradiation + physiological saline); group B, hemagglutinase group (cavitation false irradiation + physiological saline + hemagglutination enzyme); group C, microbubble cavitation group (ultrasonic cavitation + microbubble), group D, microbubble combined hemagglutinase group (ultrasonic cavitation + microbubble + hemagglutination). After cavitation treatment, microwave heat ablation treatment. Do not carry out two-dimensional gray scale ultrasound imaging, CDFI and CEUS examination. Results: before the experiment, the 4 groups were measured by two dimensional gray scale ultrasound and CEUS tumor volume respectively. The results of the 2 methods were no significant difference. After microwave heat ablation, two-dimensional gray scale ultrasound imaging showed that the diffuse distribution of strong echo light spots in the melting zone; CDFI There was no obvious blood flow signal in the ablation area, and a small amount of blood flow signals were seen on the edge of ablation area in group A, B and C. CEUS showed that the contrast agent was not filled in the ablation area. The volume of the ablation area in the D group was larger than that of the other 3 groups (P0.05), and CEUS showed that the volume of 4 groups of ablation area was obviously larger than that of the two dimensional gray scale ultrasound measurement (P0.05). Conclusion: two dimensional gray scale ultrasound and CEUS can both display and measure the size and size of tumor volume and heat ablation area, especially CEUS can more accurately show the volume of tumor and ablation area, and detect whether there is residual tissue in the ablation area, and it has high accuracy for evaluating the curative effect after the ablation. Fourth part of microbubble induced ultrasound The outcome and biological effects of cavitation combined with hemagglutinin enhanced microwave heat ablation in the treatment of rabbit VX2 liver cancer: through histopathology, immunohistochemistry and transmission electron microscopy, the continuous biological effects of microbubble induced ultrasound cavitation combined with hemagglutinin enhanced microwave heat ablation in the treatment of rabbit VX2 liver cancer in one time period were discussed. Methods: 80 VX2 tumor bearing animals were randomly divided into 4 groups, each group of 20.4 groups were treated with corresponding cavitation irradiation or cavitation treatment: group A, saline group (cavitation false irradiation + physiological saline); B group, blood coagulase group (cavitation false irradiation + physiological saline + hemagglutinase); C group, microbubble cavitation group (ultrasonic cavitation + microbubble); D group, microbubble combined hemagglutination The enzyme group (ultrasonic cavitation + microbubble + hemagglutinase). After the cavitation treatment, microwave heat ablation was performed. At the 4 time points of 0,3,7,14 d after ablation, 5 animals in each group were randomly selected to measure the volume of the ablation area. The volume of the tumor was measured by the general pathological method, and the optical microscopy (HE) of the ablation area, the transition zone and the surrounding area was observed and transmitted. The ultrastructure of tissue cells was observed by electron microscopy, and the expression of PCNA was observed by immunohistochemistry. The table of apoptosis was observed by Terminal deoxynucleotidyl transferase-mediated biotin-d UTP nick end labeling, TUNEL, using deoxynucleotidyl terminal transferase. The tumor metastasis was classified by visual observation. Results: the ablation zone volume of 0,3,7,14 d after ablation (3.059 + 0.476cm~3,2.734 + 0.407cm~3,2.796 + 0.482cm~3,10.159 + 1.444cm~3) was significantly greater than that of the other 3 groups (P0.05), and the volume of the C group ablation zone (1.086 + 0.337cm~ 3,0.919 + 0.174cm~3,1.087 + + +) was significantly greater than that of the C group. Group A (0.447 + 0.180cm~3,0.454 + 0.161cm~3,0.472 + 0.154cm~3,0.765 + 0.204cm~3) and B group (0.467 + 0.168 cm~3,0.290 + 0.075 cm~3,0.304 + 0.080cm~3,0.652 + 0.151cm~3) (P0.05). 7 d and 14 respectively after ablation. The volume of tumor (1.166 + +) was significantly smaller than that of the other 3 groups (2.447 + 0.793). 7 + 1.703cm~3) was significantly less than group A (6.859 + 1.126cm~3,10.698 + 4.910cm~3) and B group (6.475 + 1.068 cm~3,11.821 + 1.885cm~3) (P0.05). Immediately after the ablation, HE staining showed that no obvious large area coagulation necrosis was found in the 4 groups of microwave ablation areas. 3 d after ablation, HE staining showed necrosis of tissue cells in the 4 groups of ablation areas, and the transition area was visible. After ablation, the thickness of 3,7,14 D and D group was significantly greater than that of other 3 groups (P0.05). Transmission electron microscopy showed that the tissue cell structure in the ablation area of D group was more serious than that of the other 3 groups immediately after ablation, and 7 d after ablation, and the mitochondria injury in the transitional region of D group was more serious. 0,3,7,14 d after ablation, 4 groups of transition regions all showed PCNA Yang. After ablation 7 d, 4 groups of PCNA positive cells reached peak value. After ablation, 0,3,7,14 D, C and D group positive cells index were lower than the other 2 groups (P0.05).TUNEL results, 3 d after ablation, 4 groups of apoptotic cells reached the peak. 0,3,7 d after ablation, the ratio of apoptotic cells in the transitional zone of D group was significantly higher than that of the other 3 groups. 05). 7 d and 14 d after ablation, compared with other 3 groups, the metastasis degree of tumor in group D is light and the time of metastasis is late. Conclusion: microbubble induced ultrasound cavitation combined with hemagglutination enzyme can increase the effect of microwave heat ablation in rabbit VX2 liver cancer, enlarge the ablation volume, enhance the destruction of tissue cells, inhibit the growth and metastasis of tumor, and promote the apoptosis of the transitional cells and inhibit the fine tumor. Cell proliferation, meanwhile, the formation of fibrous wrapping zone in the transitional zone can promote the healing and repair of the surrounding tissues of the ablation area.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
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本文编号：1794112