【作者】 李卓；

【导师】 周晓东； 曹兵生；

【作者基本信息】 第四军医大学 ， 影像医学与核医学， 2013， 博士

【摘要】 第一部分兔VX2后肢肌内肿瘤模型的建立及其生物学评价目的：兔VX2肿瘤细胞株已广泛应用在兔的肝、肾、骨及脑等部位制成肿瘤模型，但是接种于肌肉内建立的肿瘤模型经常作为传种之瘤株很少用于实验研究。建立兔VX2肌肉肿瘤模型并观察其生物学特性。方法：①实验材料：2-3个月龄纯种健康新西兰兔30只，体重1.5～2.0kg,②实验过程：采用组织块包埋法，将VX2肿瘤组织移植于30只新西兰大白兔大腿股外侧肌肉内建立肌肉内肿瘤模型。③评估：一组随机取15只荷瘤兔，分别在接种肿瘤后7、10、14、21、26d行超声检查；计算肿瘤体积(V)=0.5×ab2其中a、b表示肿瘤的长径和短径以平均TV值绘制肿瘤生长曲线。计算肿瘤生长率（TGR），根据公式TGR=(a2b2-a1b1)/a1b1×100%计算。另一组15只荷瘤兔分别于10、15、20、25、30d随机处死3只荷瘤兔，剥除肿瘤，测量肿瘤体积，并与超声测量结果比较。病理检查肿瘤的组织学特性。结果：30只兔肌肉肿瘤模型均制作成功进入结果分析。①建立的兔VX2肌肉肿瘤模型成瘤率高达100%，接种7～10d，肿瘤生长迅速，生长率为56.7%，14～21d肿瘤再次迅速增长生长率为97.2%。②移植肿瘤组织后7天，二维超声示部分荷瘤兔后肢肌肉内可见等回声或偏低回声结节；至14天左右，所有荷瘤兔肌肉均内探及等回声或偏低回声结节，轮廓规整边界清，其内回声分布均匀，并可见稀疏彩色血流信号，肿物周边可见较丰富的彩色血流环绕，20天左右，一部分肿瘤内，回声分布不均，可探及小片状液性暗区及斑片样强回声钙化灶；并且随着肿时间的延长，坏死区域逐渐增多。③病理组织学观测：镜下可见肿瘤细胞生长旺盛，与片状坏死区分界清晰，坏死区域内肿瘤细胞体积明显增大，细胞密度降低，排列不齐，状态不规整，细胞核深染，分裂象较多，坏死区域内肿瘤细胞核固缩、碎裂。④剥除肿瘤，测量肿瘤体积，并与超声测量结果比较，差异无统计学意义。结论：此模型移植成功率高，建模时间短且肿瘤生长迅速，肿瘤血供丰富。是一种较为理想的用于研究肿瘤局部治疗的实验模型。第二部分低功率射频靶向释放长春瑞滨长循环热敏脂质体在兔VX2肿瘤的治疗研究目的：利用射频技术精确的定位和热效应靶向释放携载化疗药物的热敏脂质体在兔VX2肿瘤治疗中的疗效。方法：应用组织块包埋法建立新西兰大白兔股部肌肉内VX2肿瘤模型。待肿瘤生长至0.8～1.0cm时，根据肿瘤大小按区组随机的方法将36只荷瘤兔分成4组。对照组（CON）：静脉注射等量生理盐水。低功率射频组（RF）：输出功率1W，30min，靶温度42～43℃。长春瑞滨长循环热敏脂质体组（V-LCTL）：给予1.8mg/kg长春瑞滨长循环热敏脂质体。长春瑞滨长循环热敏脂质体联合低功率射频组（RF+V-LCTL）：功率1W,时间30min，低功率射频前给予1.8mg/kg长春瑞滨长循环热敏脂质体。治疗间隔时间为2天，即：在荷瘤兔分组后的第1、4、7天。治疗前及治疗后超声连续观测肿瘤的大小，治疗后采用高效液相色谱（HPLC）法测定长春瑞滨静脉注射后在体内各组织内的分布。治疗结束后8d，处死实验兔，处死动物前抽取VX2兔静脉血，行血常规检查，获得不同实验组白细胞及血小板的计数。标本分别做HE染色和Western blotting方法检测蛋白表达。结果：所有实验兔均能耐受治疗完成实验。RF+V-LCTL组抑瘤率为70.3％，显著高于RF和V-LCTL组，差异有统计学意义（P<0.05）；低功率射频组无明显消融效果。病理检查显示低功率射频作用无明显肿瘤坏死，而RF+V-LCTL组可见明显的肿瘤细胞损伤及坏死。并且实验结果表明RF+V-LCTL组在给药24h后相比V-LCTL组在肿瘤组织长春瑞滨浓度最高，而心脏、肺脏组织中的长春瑞滨浓度最低。肿瘤细胞凋亡的检测。V-LCTL组和RF+V-LCTL组均可明显升高Caspase-3活性片段，Bax的表达水平，降低Bcl-2的表达水平。结论：与低功率射频治疗组及长春瑞滨热长循环热敏脂质体治疗组相比，低功率射频靶向释放长春瑞滨热长循环热敏脂质体对兔VX2肿瘤具有明显的抑瘤效果，较好的药物靶向性、和最低的心肺毒副作用。该联合治疗可扩大射频消融的治疗区域，对减少射频消融后肿瘤复发有重要意义。第三部分高强度聚焦超声靶向释放长春瑞滨长循环热敏脂质体抗肿瘤的实验研究目的：应用HIFU作为靶向释放热源，探讨HIFU靶向释放长春瑞滨长循环热敏脂质体联合抗肿瘤的疗效。方法：应用组织块包埋法建立新西兰大白兔股部肌肉内VX2肿瘤模型。待肿瘤直径至0.8～1.0cm时，按区组随机的方法将24只荷瘤兔分成4组。对照组（CON）、单纯HIFU组、长春瑞滨热敏脂质体组（V-LCTL）、HIFU联合长春瑞滨热敏脂质体组(HIFU+V-LCTL)。治疗间隔时间为2天，即：在荷瘤兔分组后的第1、3、5天。治疗后，观察肿瘤体积和重量变化，计算抑瘤率。治疗结束后8d，处死动物，处死动物前抽取VX2兔静脉血，行血常规检查，获得不同实验组白细胞及血小板的计数。并对肿瘤进行Western blotting方法检测蛋白表达，同时送病理进行HE染色，观察肿瘤组织病理形态学变化。结果：各组荷瘤兔的肿瘤生长曲线：对照组肿瘤生长最快，HIFU组与对照组肿瘤体积增长趋势相当，V-LCTL组和HIFU+V-LCTL组肿瘤生长均受到明显抑制，且肿瘤体积较其他两组明显缩小。并且HIFU+V-LCTL组与V-LCTL组相比，HIFU+V-LCTL组肿瘤体积缩小显著。HIFU+V-LCTL组的抑瘤率为71.0％，显著高于HIFU组和V-LCTL组（0.8%和47.1%，P<0.05）。V-LCTL组和HIFU+V-LCTL组Caspase-3活性片段和Bax的表达水平明显升高，同时Bcl-2的表达水平显著降低。病理结果显示对照组和单纯HIFU组肿瘤细胞紧密排列呈实体巢状，细胞大，胞浆丰富，瘤体内微血管完整；HIFU+V-LCTL组细胞明显固缩变小，细胞核出现固缩、碎裂和溶解。毛细血管壁结构模糊，嗜伊红染色增加，血管腔内有大量红细胞集聚。结论： HIFU技术和携载化疗药物的热敏脂质体联合应用，通过HIFU靶向释放载药热敏脂质体在肿瘤局部的高度浓集，实现化疗药物增强HIFU对肿瘤消融的作用。扩大了HIFU的热消融范围及适应征。并减轻抗肿瘤化疗药物的全身毒副反应。上述结果为寻求一种有效、安全、靶向性高的肿瘤治疗手段提供了新的思路。更多还原

【Abstract】 Part One Establishment and biological evaluation of rabbitVX2muscle tumor modelsObjectives: Rabbit VX2tumor cell lines have been widely used in tumor modelsestablished in rabbit livers, kidneys, bones and brains and other parts. While vaccination inestablishing tumor models within the muscles is often used for the transmission of tumorstrains, it is rarely applicable to present experiments. Establish rabbit VX2muscle tumormodels and observation of their biological characteristics. Methods:①Experimental materials:30purebred New Zealand white rabbits, two or three months old,1.5to2.0kg.②Experimental processes: with tissue blocks embedded, VX2tumor tissues weretransplanted in30New Zealand white rabbits’ thigh vastus lateralis muscles to establishmuscle tumor models.③Evaluation: A group of15randomly selected tumor-bearingrabbits were under Ultrasound examination after the7,10,14,21,26d tumor inoculationrespectively; calculate the tumor volume (V)=0.5×ab2, wherein a, b represents the longdiameter and short diameter of tumors respectively. Tumor growth curves were drawnwith regard to the average TV. Calculate the tumor growth rate (TGR) according to theformula TGR=(a2b2-a1b1)/a1b1×100%. As for another group of15tumor-bearingrabbits, three tumor-bearing rabbits were randomly killed at10,15,20,25,30d; theirtumors were stripped of and the tumor volumes measured and compared with ultrasoundmeasurements. Pathologically examine the tumors’ histological characteristics. Results:30rabbits were under the final analysis.①The rate of establishing rabbit VX2muscletumor models reached up to100%. After vaccination for10-14d, the tumors grew rapidlyat the rate of66.9%; for14to21d, the tumors grew at the rate of97.2%.②On7th daysafter vaccination, by ultrasound, the small isoechoic nodules were found visible in someexperimental rabbits’ muscle tissues. On the14th days low echo or isoechoic nodules werefound visible in the rabbits’ muscle tissues with clear boundary and average echoes. ColorDoppler showed, inside the tumors, blood flow signals in the form of sparse rods and, anamount of blood flow signals can be seen surrounding around. On the21st days a smallpart of the tumor echoes was uneven and, there were small pieces of echo-free zones andcalcification pots scattered in the hyperechoic zones; as the growth of tumors, tumornecrosis increased.③Pathological observation: with low magnification microscope, tumorcells were discovered in the nested distribution crossed with pieces of necrosis distribution.Tumor cells in the necrosis distribution were in low density. With high-poweredmicroscope, tumor cell volumes increased in irregular shape, with irregular arrangement.And nuclei were large and deeply stained; cytoplasm less, mitotic more and, nuclearcondensation and fragmentation were present in the necrotic region.④The tumors werestripped of, tumor volumes measured and compared with ultrasound measurements; the differences were not statistically significant.Conclusion:This model can be transplanted at a high rate, with short modeling time, rapid tumorgrowth and sufficient blood supply. It is an ideal experimental model used to study tumorlocal treatment.Part two Studies on the treatment of rabbit VX2tumors bylow-power RF technique mediating targeted release ofvinorelbine long-circulating thermosensitive liposomesObjectives: Use in rabbit VX2tumor therapy targeted release of thermosensitiveliposomes with portable chemotherapy drugs through precise positioning and thermaleffects of RF technology. Methods: Establish VX2tumor models in the thigh muscles ofNew Zealand white rabbits by embedding tissue blocks. When the tumors grew to0.8to1.0cm,24tumor-bearing rabbits were randomly divided into4groups according to tumorsizes. Control group (CON): normal saline was intravenously injected. Low-frequency RFgroup (RF): Output power,1W,30min, target temperature of42~43℃. Vinorelbinetartrate long cycle thermosensitive liposomes (LCTSL):1.8mg/kg vinorelbine tartratelong-circulating thermosensitive liposomes were given. Vinorelbine tartrate long cyclethermosensitive liposomes combined with low-frequency RF (LCTSL+RF): power,1W,time30min. Before the low-frequency RF, the1.8mg/kg vinorelbine long cyclethermosensitive liposomes were given. Treatment was carried out on the1st,4th, and7thdays respectively, in the total of3times. Observe the tumor sizes without a stop underUltrasonic before and after treatment; Distribution within the internal tissues afterintravenous vinorelbine injection was determined by HPLC. After8d treatment, westarted extraction of VX2venous blood followed by blood routine examination before therabbits were killed, and then we obtained different data on white blood cells and plateletsof the different experimental groups. The samples were obtained for HE staining and detecting protein expression through Western blotting. Results: All rabbits werewell-tolerated in treatment, which completed the experiment. The inhibition rate ofLCTSL+RF group was70.3%, significantly higher than the RF and LCTSL group; thedifference was statistically significant (P <0.05). There was no significant ablation effectin Low-frequency RF group. With histopathological examination, less obvious tumornecrosis was revealed under a low frequency RF. But in TSL+RF group tumor cell injuryand necrosis were significantly found visible. And compared with the LCTSL group,LCTSL+RF group presented itself in the highest concentration of tumor tissue vinorelbine,24h later after administration. And the concentrations of vinorelbine in hearts and lungswere the lowest. The detection of tumor cell apoptosis: The LCTSL group and LCTSL+RF group could be seen to significantly promote tumor cells apoptosis and inhibit tumorcells proliferation. Compared with the LCTSL group, the rate of tumor cells apoptosis wasmore significant than that of the LCTSL+RF group.Conclusion:Low-power RF targeted release of the vinorelbine hot long circulating thermosensitiveliposomes was discovered to have an obvious effect on inhibiting rabbit VX2tumors, withvery good drug targeting and the lowest cardiopulmonary side effects. Thusradiofrequency ablation treatment can be applied, which is of great significance forreducing tumor recurrence after radiofrequency ablation.Part three Anti-tumor experimental study on HIFU targetedrelease of vinorelbine long-circulating thermosensitiveliposomesObjectives: Study the anti-tumor efficiency of HIFU targeted release of vinorelbinelong-circulating thermosensitive liposomes, with HIFU as targeted release of heat.Methods: Establish VX2tumor models in New Zealand white rabbits’ thigh muscles with tissue blocks embedded. Until the tumors grew to0.8～1.0cm,24tumor-bearing rabbitswere divided randomly into4groups according to tumor sizes: control group (CON),HIFU group, V-LCTL group, HIFU+V-LCTL group. Treatment was carried out every twodays or on the1st,3rd, and5th days respectively. Western blotting were be used fordetection of protein expression in the tumors, which would then obtained for pathologicalHE staining. And tumor pathomorphological changes would be observed. After treatment,observe tumor volumes and weight changes and calculate inhibitory rates. After8dtreatment, we started extraction of VX2venous blood followed by blood routineexamination before the rabbits were killed, and then we obtained different data on whiteblood cells and platelets of the different experimental groups. Results: Tumor growthcurves for tumor-bearing rabbits in each group: Control group presented itself with thefastest tumor growth; the tumor volumes of HIFU group and control group wereequivalent when growing. The tumor growth was significantly inhibited in HIFU+VBLgroup and HIFU+LCTSL group. And the tumor volumes were significantly reduced ascompared to the other groups. Compared with HIFU+VBL group, the tumor volumes inHIFU+LCTSL group were also more significantly reduced. The rate of tumor inhibitionwas71.0%in LCTSL+HIFU group and significantly higher than HIFU group and HIFU+VBL group (0.8%and48.9%, P <0.05). In TV-LCTL group and HIFU+the V-LCTLgroup caspase-3active fragments and bax expression levels were significantly increasedwhile bcl-2expression levels were significantly decreased. The pathological resultsshowed that, in control group and HIFU group, tumor cells, big, with abundant cytoplasmand complete intratumoral microvasculars, were closely arranged in nested shape. Thecells of HIFU+LCTSL group were obviously condensed smaller, accompanied withnuclear condensation, fragmentation and dissolution. The capillary wall structures becamefuzzy with eosinophilic staining increasing and a large number of red blood cells werefocused in endovascular.Conclusion: Chemotherapeutic drugs-carrying thermosensitive liposomes were combinedwith HIFU technique in our study. By HIFU targeted release, with highly concentration ofchemotherapeutic drugs-carrying thermosensitive liposomes in local tumors, chemotherapeutic drugs can be used to enhance the tumor ablation through HIFU. Theranges of HIFU thermal ablation were enlarged as well as its adaptation and the systemictoxicity of anti-tumor chemotherapy drugs was relieved. This study provides a new way ofseeking an effective, safe and highly targeting tumor treatment.更多还原