本文選題：微泡 + 超聲空化　； 參考：《第四軍醫(yī)大學》2017年博士論文

【摘要】：第一部分微泡誘導超聲空化聯(lián)合血凝酶增強兔正常肝臟微波熱消融作用的實驗研究目的:探討微泡誘導超聲空化聯(lián)合血凝酶對兔正常肝臟微波熱消融的增強作用。方法:健康新西蘭大白兔40只,隨機分為4組,每組均為10只。4組均經(jīng)相應的空化假輻照或空化治療:A組,生理鹽水組(空化假輻照+生理鹽水);B組,血凝酶組(空化假輻照+生理鹽水+血凝酶);C組,微泡空化組(超聲空化+微泡);D組,微泡聯(lián)合血凝酶組(超聲空化+微泡+血凝酶)。空化治療后行微波熱消融治療,消融后立即處死動物取出肝臟,以大體病理學方法測量消融區(qū)體積。分別提取消融區(qū)、過渡區(qū)及周圍區(qū)組織,通過光鏡(HE)觀察及透射電鏡顯示評估4組之間微波熱消融效果。結(jié)果:大體病理結(jié)果顯示,微波熱消融后D組的肝臟凝固壞死體積(3.13±0.78cm~3)大于其它3組(P0.05);C組凝固壞死體積(1.65±0.51cm~3)大于A組(0.53±0.10 cm~3)和B組(0.46±0.09 cm~3)(P0.05);A組和B組凝固壞死體積比較無明顯統(tǒng)計學意義。光鏡(HE)顯示消融區(qū)與未消融區(qū)區(qū)域分界明顯,4組消融區(qū)均未見大片凝固性壞死。透射電鏡顯示D組內(nèi)可見大量的細胞核和細胞膜的破壞。結(jié)論:微泡誘導超聲空化聯(lián)合血凝酶可更顯著地增加低能量微波消融兔正常肝臟體積,與對照組相比可明顯增加熱消融的效率。第二部分微泡誘導超聲空化聯(lián)合血凝酶對兔VX2肝癌微血管阻斷作用及機制的實驗研究目的:探討微泡誘導超聲空化聯(lián)合血凝酶對兔VX2肝癌腫瘤微血管的損毀及阻斷的作用機制。方法:健康新西蘭大白兔接受開腹種植法建立兔VX2肝癌腫瘤模型。VX2肝腫瘤種植14 d后,存活的32只荷瘤兔隨機分為4組:A組,生理鹽水組(空化假輻照+生理鹽水);B組,血凝酶組(空化假輻照+生理鹽水+血凝酶);C組,微泡空化組(超聲空化+微泡);D組,微泡聯(lián)合血凝酶組(超聲空化+微泡+血凝酶),每組均為8只。4組均經(jīng)相應的空化假輻照或空化治療,在實驗前后分別進行超聲造影檢查(Contrast Enhanced Ultrasonography,CEUS),使用定量圖像分析軟件分析實驗前后CEUS的峰值灰階值(Peak)的變化,通過光鏡(HE)觀察及透射電鏡超微結(jié)構(gòu)顯示來比較4組的治療效果。結(jié)果:實驗前后,A組和B組超聲造影圖像視覺分析及定量分析Peak值比較均無明顯變化(P0.05)。C組和D組視覺觀察有造影灌注缺損,D組缺損更為明顯,定量分析D組Peak值從(72.94±10.27)降至(15.74±8.50)(P0.05),C組Peak值從(68.79±6.48)降至(29.57±4.31)(P0.05)。HE染色顯示:C組內(nèi)可見局灶小片狀出血;D組可見大片狀出血及明顯的細胞水腫。A組和B組無明顯病理改變。透射電鏡顯示:C組和D組的微血管壁破壞,基底膜不完整。結(jié)論:微泡誘導超聲空化聯(lián)合血凝酶能促進內(nèi)皮損傷并導致更廣泛的血栓形成,同時驗證了血凝酶選擇性針對腫瘤組織局部損傷的血管快速血栓形成及血流阻塞具有一定的靶向性,并有增強熱消融的作用。第三部分微泡誘導超聲空化聯(lián)合血凝酶增強兔VX2肝癌微波熱消融作用的超聲影像學評價方法及意義目的:通過二維灰階超聲成像、彩色多普勒血流成像(Color Doppler Flow Imaging,CDFI)和超聲造影檢查(Contrast Enhanced Ultrasonography,CEUS)等方法,探討微泡誘導超聲空化聯(lián)合血凝酶對兔VX2肝癌微波熱消融的增強作用。方法:32只VX2荷瘤動物隨機分為4組,每組均為8只。4組均經(jīng)相應的空化假輻照或空化治療:A組,生理鹽水組(空化假輻照+生理鹽水);B組,血凝酶組(空化假輻照+生理鹽水+血凝酶);C組,微泡空化組(超聲空化+微泡);D組,微泡聯(lián)合血凝酶組(超聲空化+微泡+血凝酶)�？栈委熗戤吅笮形⒉嵯谥委煛２⒃谡麄�治療前后分別進行二維灰階超聲成像、CDFI和CEUS等檢查。結(jié)果:實驗前,4組分別用二維灰階超聲與CEUS測量腫瘤體積,2種方法所得結(jié)果比較無明顯統(tǒng)計學差異。微波熱消融治療后,二維灰階超聲成像顯示:消融區(qū)出現(xiàn)彌漫性分布的片狀強回聲光點;CDFI顯示:消融區(qū)未見明顯血流信號,A組、B組和C組消融區(qū)邊緣可見少量血流信號;CEUS顯示:消融區(qū)造影劑未充盈。二維灰階超聲和CEUS測量均顯示D組消融區(qū)體積大于其它3組(P0.05),CEUS顯示4組消融區(qū)體積明顯大于二維灰階超聲測量結(jié)果(P0.05)。結(jié)論:二維灰階超聲和CEUS均能顯示和測量腫瘤體積和熱消融區(qū)范圍及體積大小,尤其是CEUS能更準確地顯示腫瘤和消融區(qū)體積大小,并可檢測消融區(qū)是否有殘留組織,對評估腫瘤消融后的療效具有較高的準確性。第四部分微泡誘導超聲空化聯(lián)合血凝酶增強的微波熱消融治療兔VX2肝癌的轉(zhuǎn)歸及生物學效應目的:通過組織病理學、免疫組織化學及透射電鏡顯示等方法,探討微泡誘導超聲空化聯(lián)合血凝酶增強微波熱消融技術治療兔VX2肝癌一個時間段內(nèi)的連續(xù)性生物效應及轉(zhuǎn)歸。方法:80只VX2荷瘤動物隨機分為4組,每組均為20只。4組均經(jīng)相應的空化假輻照或空化治療:A組,生理鹽水組(空化假輻照+生理鹽水);B組,血凝酶組(空化假輻照+生理鹽水+血凝酶);C組,微泡空化組(超聲空化+微泡);D組,微泡聯(lián)合血凝酶組(超聲空化+微泡+血凝酶)�？栈委熗戤吅笮形⒉嵯谥委�,于消融后0,3,7,14 d 4個時間點每組隨機選取5只動物行超聲造影測量消融區(qū)體積。通過大體病理學方法測量腫瘤體積,對消融區(qū)、過渡區(qū)和周圍區(qū)組織行光鏡(HE)觀察及透射電鏡顯示組織細胞超微結(jié)構(gòu),采用免疫組織化學方法觀測細胞PCNA表達,采用脫氧核苷酸末端轉(zhuǎn)移酶介導的生物素化脫氧三磷酸尿苷末端缺口標記法(Terminal deoxynucleotidyl transferase-mediated biotin-d UTP nick end labeling,TUNEL)觀測細胞凋亡的表現(xiàn),并通過視覺觀察對腫瘤轉(zhuǎn)移進行分級。結(jié)果:消融后0,3,7,14 d,D組消融區(qū)體積(3.059±0.476cm~3,2.734±0.407cm~3,2.796±0.482cm~3,10.159±1.444cm~3)明顯大于其他3組(P0.05);C組消融區(qū)體積(1.086±0.337cm~3,0.919±0.174cm~3,1.087±0.354cm~3,3.707±0.850cm~3)明顯大于A組(0.447±0.180cm~3,0.454±0.161cm~3,0.472±0.154cm~3,0.765±0.204cm~3)和B組(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組腫瘤體積(1.166±0.468cm~3,1.445±0.593cm~3)明顯小于其他3組(P0.05),C組腫瘤體積(2.447±0.793 cm~3,5.477±1.703cm~3)明顯小于A組(6.859±1.126cm~3,10.698±4.910cm~3)和B組(6.475±1.068 cm~3,11.821±1.885cm~3)(P0.05)。消融后即刻,HE染色顯示4組微波消融區(qū)內(nèi)均未發(fā)現(xiàn)明顯的大面積凝固性壞死。消融后3 d,HE染色顯示4組消融區(qū)內(nèi)可見組織細胞壞死,過渡區(qū)可見纖維包裹帶形成。消融后3,7,14 d,D組纖維包裹帶厚度明顯大于其它3組(P0.05)。透射電鏡顯示,消融后即刻,D組消融區(qū)內(nèi)組織細胞結(jié)構(gòu)較其它3組破壞嚴重;消融后7 d,D組過渡區(qū)細胞內(nèi)線粒體損傷更嚴重。消融后0,3,7,14 d,4組過渡區(qū)均顯示PCNA陽性細胞和凋亡細胞;消融后7 d,4組PCNA陽性細胞均達峰值。消融后0,3,7,14 d,C組和D組過渡區(qū)陽性細胞指數(shù)低于其他2組(P0.05)。TUNEL結(jié)果顯示,消融后3 d,4組過渡區(qū)凋亡細胞達到頂峰。消融后0,3,7 d,D組過渡區(qū)凋亡細胞比值較其他3組明顯增多(P0.05)。消融后7 d和14 d,相比其他3組,D組腫瘤轉(zhuǎn)移程度輕,轉(zhuǎn)移時間晚。結(jié)論:微泡誘導超聲空化聯(lián)合血凝酶可增加兔VX2肝癌微波熱消融作用、擴大消融體積、增強組織細胞的破壞、抑制腫瘤的生長和轉(zhuǎn)移,并促進過渡區(qū)細胞的凋亡、抑制腫瘤細胞增殖。同時,過渡區(qū)纖維包裹帶形成可促進消融區(qū)周邊組織的機化和修復。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.7
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本文編號：1794112