纳秒脉冲消融细粒棘球蚴感染的体内外实验治疗学评价

发布时间：2018-07-10 08:58

本文选题：细粒棘球蚴 + 纳秒脉冲　；参考：《新疆医科大学》2017年博士论文

【摘要】：目的:通过纳秒脉冲电场(nanosecond pulsed electric filed,nsPEF)消融细粒棘球蚴(echinococcus granulosus,E.g)的体外和体内实验,开展新型非致热微创消融技术-纳秒脉冲针对E.g的治疗学评价。筛选纳秒脉冲消融技术有效治疗参数,评估其应用于肝囊型包虫病(cystic echinococcosis,CE)临床治疗的安全性和有效性。通过门静脉注射E.g原头蚴,模拟自然感染途径,建成小鼠肝E.g模型,经调控门静脉注射原头蚴数量,优化模拟临床CE致病模型,重现小鼠门静脉到肝脏的感染路径和病程特征。采用纳秒脉冲消融仪对该模型病灶进行治疗与评价,在体外实验中筛选在固定脉宽、频率下,有效消融E.g的电场强度和脉冲个数,并在小鼠模型上验证其剂量效应,获得适宜治疗E.g的电极布局。方法:(1)采集临床B超和CT诊断为单囊型肝囊型包虫病CE,通过手术获取CE患者的原头蚴,经过1%浓度胃蛋白酶消化后判断原头蚴活性,配制浓度分别为2000个/100ul、200个/100ul、100个/100ul原头蚴混悬液,经C57BL/6小鼠门静脉主干注射后观察小鼠的死亡率,计算小鼠E.g的成囊率和分布,B超随访观察E.g囊肿的生长速率;(2)B超筛查C57BL/6小鼠肝E.g囊肿,观察固定脉宽350nm,频率1Hz/s,50脉冲数下,不同场强(21、14、7、0kV/cm)作用后1周、4周、8周肝E.g囊肿的大小、形态改变、囊壁结构改变判断纳秒脉冲对小鼠肝E.g囊肿的消融抑制效应。(3)观察固定脉宽350nm,频率1Hz/s下,不同场强(29、21、14、7、0kV/cm)和脉冲数(50、100)作用后第1、3、7天原头蚴的形态、活动度、完整性,判断纳秒脉冲对其的杀伤效应,HE染色观察原头蚴结构的变化,扫描电镜观察原头蚴超微结构改变;采集临床B超和CT诊断为多子囊型CE患者,选取直径5-10mm、光亮透明,弹性好的CE囊肿,观察固定脉宽350nm,频率1Hz/s下,不同场强(29、21、14、0kV/cm)和不同脉冲数(50、100)作用后第1、3、7天E.g囊肿内层塌陷情况判断纳秒脉冲对其的杀伤效应,HE染色观察E.g囊肿壁结构的变化。结果:(1)建立门静脉接种原头蚴的小鼠模型:2000个/100ul、200个/100ul、100个/100ul原头蚴混悬液经门静脉主干注射后小鼠感染率分别为90%(9/10)、100%(10/10)、63.6%(7/11),成囊率分别为0.7%~4.1%,0.6%~1.9%,0.6%~3.9%。病理切片显示注射后1周内,大部分原头蚴体积固缩,7天时原头蚴周围形成明显的炎症带,21天时可见存活的原头蚴发育出囊泡,42天时原头蚴细胞团消失,发育成一囊型病灶;(2)获得纳秒脉冲治疗小鼠肝脏原位e.g囊肿的有效剂量:小鼠e.g囊肿在纳秒脉冲治疗仪脉冲后21kv/cm脉冲组小鼠e.g直径的增长在脉冲后1、4、8周与对照组存在差异(p0.05),呈缩小趋势,脉冲后1、4、8周的直径分别缩小1.4mm、1.2mm、1.7mm,肉眼观e.g囊肿壁增厚,透明度消失,而对照组小鼠e.g囊肿光滑透明,壁较薄、弹性较好,腔内囊液充盈清亮张力高,病理切片显示脉冲后8周e.g外纤维组织增厚,炎症反应带增宽明显;14kv/cm脉冲组小鼠e.g直径的增长在脉冲后8周与对照组存在差异(p0.05),呈缩小趋势,脉冲后1、4、8周的直径增长分别为-0.4mm、0.4mm、-0.1mm;7kv/cm脉冲组小鼠e.g直径的增长与对照组无差异(p0.05);对照组在脉冲后1、4、8周的直径增长分别为0.2mm、1.3mm、4.3mm。(3)纳秒脉冲对体外培养的原头蚴和e.g囊肿的的杀灭存在剂量效应:脉冲后第1、3天,29、21kv/cm脉冲组的原头蚴的杀伤率与对照组存在差别(p0.05),50脉冲数下29、21kv/cm脉冲组第1天杀伤率分别为16.5%、12.9%,第3天的杀伤率分别为19%、12.6%;脉冲后第7天,29、21、14kv/cm脉冲组的原头蚴的杀伤率与对照组存在差别(p0.05),50脉冲数下29、21、14kv/cm脉冲组第7天杀伤率分别为71.7%、64.3%、48.3%。100脉冲数下29、21kv/cm脉冲组第1天杀伤率分别为32.3%、15.6%,第3天的杀伤率分别为32.3%、16.7%,100脉冲数下29、21、14kv/cm脉冲组第7天杀伤率分别为79.2%、68.7%、49.2%,透射电镜见脉冲后原头蚴体表完整性被破坏;脉冲处理后e.g囊肿第1天发生明显的内层塌陷,与对照组相比存在差异(p0.05),脉冲场强越大,塌陷率越高,50脉冲数、29kv/cm场强下,1、3天的塌陷率分别为75%、100%,21kv/cm场强下,1、3、5天的塌陷率分别为50%、75%、100%,14kv/cm场强下,1、3、5、7天的塌陷率分别为25%、62.5%、75%、75%,100脉冲数、29kv/cm场强下,1、3、5、7天的塌陷率分别为62.5%、87.5%、87.5%、100%,21kv/cm场强下,1、3、5、7天的塌陷率分别为50%、75%、75%、100%,14kv/cm场强下,1、3、5、7天的塌陷率分别为75%、87.5%、87.5%、100%。结论:(1)经小鼠门静脉注射e.g原头蚴可以成功建立小鼠e.g感染模型,注射200个原头蚴可以得到较高的感染率和较合适的囊泡个数,囊泡生长个数约1-4个,可以较好的模拟临床ce;(2)21kv/cm场强可以明显消融小鼠e.g,棘球蚴囊肿壁增厚,透明度消失,14kv/cm场强可以明显抑制小鼠e.g的生长;7kv/cm场强对小鼠e.g的生长无抑制作用。纳秒脉冲对小鼠e.g囊肿具有剂量效应,需要优化治疗参数,只有在足够高的电场强度下才能获得消融及抑制生长作用。(3)固定脉宽350nm,频率1hz/s下,29、21kv/cm电场强度下对原头蚴的杀伤效应较明显;e.g单囊肿对纳秒脉冲的消融效应较敏感,固定脉宽350nm,频率1hz/s下,29、21、14kv/cm电场强度下脉冲后第1天内层塌陷明显,场强越高、脉冲越数则e.g囊肿内层塌陷越早,提示纳秒脉冲在包虫病的消融临床应用上需要分型施治,选择恰当的适应症,根据WHO分型选择合适病例,设计个体化治疗方案。
[Abstract]:Objective: in vitro and in vivo experiments of nanosecond pulsed electric filed (nsPEF) ablation of Echinococcus granulosus (Echinococcus granulosus, E.g) in vitro and in vivo, a new non thermal minimally invasive ablation technique, the therapeutic evaluation of nanosecond pulse for E.g, is carried out. The effective treatment parameters of nanosecond pulse ablation technique are screened and the application of the nanosecond pulse ablation technique to the liver sac is evaluated. The safety and effectiveness of the clinical treatment of cystic echinococcosis (CE). Through the portal vein injection of the original E.g cercariae, simulating the natural infection route, establishing the mouse liver E.g model, the number of the primary cercariae injected into the portal vein, optimizing the simulated clinical CE pathogenicity model, and reproducing the infection path and course characteristics of the portal vein to the liver in mice. The nanosecond pulse ablation instrument was used to treat and evaluate the lesion of the model. In the experiment, the electric field intensity and pulse number of E.g were effectively fused under the fixed pulse width and frequency. The dose effect was verified on the mouse model and the suitable electrode layout for the treatment of E.g was obtained. Method: (1) the clinical B ultrasound and CT were collected for the diagnosis of the single cystic liver bag type bag. CE, the primary cercariae of CE patients were obtained by operation. After digestion of 1% concentrations of pepsin, the activity of echinococcosis was determined. The concentration was 2000 /100ul, 200 /100ul and 100 /100ul raw cercariae suspension respectively. The death rate of mice was observed after the injection of the main portal vein of the C57BL/6 mice. The percentage and distribution of the mouse E.g were calculated and the follow up view was calculated. Detection of the growth rate of E.g cysts; (2) screening the E.g cysts in C57BL/6 mice by B-ultrasound, observed the size of the fixed pulse width 350nm, the frequency 1Hz/s, the 50 pulse number, the size and shape of the liver E.g cysts at 1 weeks, 4 weeks, and 8 weeks after the effect of different field strength (21,14,7,0kV/cm), and the changes in the structure of the cystic wall to determine the ablation inhibition effect of nanosecond pulse on the liver E.g cysts in mice. (3) observe the fixation. Pulse width 350nm, frequency 1Hz/s, different field strength (29,21,14,7,0kV/cm) and pulse number (50100) after the action of the shape, activity and integrity of the primary cercariae on day 1,3,7, judge the killing effect of nanosecond pulse, observe the changes of the structure of the original cercariae by HE staining, observe the ultrastructural changes of the original cercariae by the scanning electron microscope, and collect the clinical B ultrasonic and CT for the diagnosis of multiple children. Cystic CE patients, select the diameter 5-10mm, bright and transparent, good elastic CE cyst, observe the fixed pulse width 350nm, the frequency 1Hz/s, the different field strength (29,21,14,0kV/cm) and the different pulse number (50100) the E.g cyst inner layer collapse condition after the action of 1,3,7 day, judge the nanosecond pulse to its killing effect, HE staining observation E.g cyst wall structure change. Results: ( 1) to establish a mouse model of portal vein inoculation of the original cercariae: 2000 /100ul, 200 /100ul, 100 /100ul primary cercariae suspension after injection of the portal vein to 90% (9/10), 100% (10/10), 63.6% (7/11), respectively, 0.7%~4.1%, 0.6%~1.9%, and 0.6%~3.9%. pathological sections, respectively, within 1 weeks after injection, most of the volume of the original cercariae In 7 days, there were obvious inflammatory zones in the cercariae of the cercariae. At 21 days, the surviving cercariae developed vesicles, and the primary cercariae disappeared and developed into a cystic lesion at 42 days. (2) the effective dose of nanosecond pulse therapy in the liver in situ e.g cysts in mice was obtained: the mouse e.g cyst was small in 21kv/cm pulse group after nanosecond pulse therapy instrument pulse. The growth of rat e.g diameter was different from that in the control group (P0.05) after 1,4,8 weeks (P0.05), and the diameter of the 1,4,8 weeks after the pulse was reduced to 1.4mm, 1.2mm, 1.7mm, the naked eye e.g cyst wall thickened and the transparency disappeared, while the control group of mice was smooth and transparent, the wall was thinner, the elasticity was better, the cavity fluid filled clear tension and pathological section. 8 weeks after e.g, the fibrous tissue was thickened and the inflammatory reaction zone was widened. The growth of e.g in the 14kv/cm pulse group was significantly different from the control group (P0.05) after the pulse (P0.05), and the diameter of 1,4,8 weeks after the pulse was -0.4mm, 0.4mm, -0.1mm, and the e.g diameter of the mice in the 7kv/cm pulse group was no different than that in the control group (P) 0.05) in the control group, the growth of the diameter of 1,4,8 weeks after the pulse was 0.2mm, 1.3mm, 4.3mm. (3) nanosecond pulse had a dose effect on the killing of the original cercariae and the e.g cysts in vitro: after the pulse on the day 1,3, the killing rate of the original cercariae in the 29,21kv/cm pulse group was different from the control group (P0.05), and the 29,21kv/cm pulse group was killed at first days under the 50 pulse number. The injury rates were 16.5%, 12.9%, and third days, respectively, 19%, 12.6%. Seventh days after the pulse, the killing rate of the 29,21,14kv/cm pulse group was different from that of the control group (P0.05). The seventh day killing rate of the 29,21,14kv/cm pulse group under the 50 pulse number was 71.7%, 64.3%, and the 29,21kv/cm pulse group first day killing rate was 3 respectively under the 48.3%.100 pulse number of 3, respectively 3. The killing rates of 2.3%, 15.6%, and third days were 32.3%, 16.7%, and 100 pulses in the 29,21,14kv/cm pulse group were 79.2%, 68.7%, 49.2%, respectively. The transmission electron microscope showed that the body surface integrity of the cercariae was destroyed after the pulse treatment. After pulse treatment, the e.g cyst had an obvious inner layer collapse, which was different from the control group (P0.05), and the pulse field intensity was different (P0.05). The larger the collapse rate is, the higher the collapse rate, the 50 pulse number and the 29kv/cm field strength, the collapse rates of 1,3 days are respectively 75%, 100%, and 21kv/cm field strength, the collapse rates of 1,3,5 days are respectively 50%, 75%, 100%, and 14kv/cm field strength, the collapse rates of 1,3,5,7 days are 25%, 62.5%, 75%, 75%, 100 pulses, respectively, under the 29kv/cm field, the collapse rates of 1,3,5,7 days are 62.5%, 87.5%, 87.5%, 62.5%, respectively, under the 29kv/cm field strength. Under the kv/cm field, the collapse rates of 1,3,5,7 days were 50%, 75%, 75%, 100% and 14kv/cm, and the collapse rates of 1,3,5,7 days were 75%, 87.5%, 87.5%, 100%. conclusion: (1) the e.g infection model of mice could be successfully established by injection of e.g original cercariae in the portal vein of mice, and the high infection rate and the appropriate number of vesicles could be obtained by injection of 200 original cercariae. There are about 1-4 vesicles, which can simulate clinical CE better. (2) 21kv/cm field strength can obviously melt mouse e.g, the hydatid cyst wall thickening, transparency disappears, and 14kv/cm field strength can obviously inhibit the growth of e.g in mice; 7kv/cm field strength has no inhibitory effect on the growth of e.g in mice. Nanosecond pulse has a dose effect on mouse e.g cysts, which needs a dose effect. In order to optimize the treatment parameters, only the ablation and growth inhibition can be obtained only under high electric field strength. (3) the effect of the fixed pulse width 350nm, the frequency 1hz/s, the 29,21kv/cm electric field strength to the original cercariae is more obvious; the e.g single cyst ablation effect of nanosecond pulse is more sensitive, the fixed pulse width 350nm, the 29,21,14kv/cm electric field under the frequency 1hz/s, the 29,21,14kv/cm electric field After first days of pulse intensity, the inner layer collapse is obvious. The higher the field strength is, the higher the field strength, the earlier the collapse of the e.g cyst. It is suggested that the nanosecond pulse in the clinical application of echinococcosis need to be treated by typing, choosing appropriate indications, selecting appropriate cases according to the WHO classification, and designing a physical therapy scheme.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R532.32

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