本文選題：細粒棘球蚴 + 納秒脈沖��； 參考：《新疆醫(yī)科大學》2017年博士論文

【摘要】：目的:通過納秒脈沖電場(nanosecond pulsed electric filed,nsPEF)消融細粒棘球蚴(echinococcus granulosus,E.g)的體外和體內實驗,開展新型非致熱微創(chuàng)消融技術-納秒脈沖針對E.g的治療學評價。篩選納秒脈沖消融技術有效治療參數,評估其應用于肝囊型包蟲病(cystic echinococcosis,CE)臨床治療的安全性和有效性。通過門靜脈注射E.g原頭蚴,模擬自然感染途徑,建成小鼠肝E.g模型,經調控門靜脈注射原頭蚴數量,優(yōu)化模擬臨床CE致病模型,重現小鼠門靜脈到肝臟的感染路徑和病程特征。采用納秒脈沖消融儀對該模型病灶進行治療與評價,在體外實驗中篩選在固定脈寬、頻率下,有效消融E.g的電場強度和脈沖個數,并在小鼠模型上驗證其劑量效應,獲得適宜治療E.g的電極布局。方法:(1)采集臨床B超和CT診斷為單囊型肝囊型包蟲病CE,通過手術獲取CE患者的原頭蚴,經過1%濃度胃蛋白酶消化后判斷原頭蚴活性,配制濃度分別為2000個/100ul、200個/100ul、100個/100ul原頭蚴混懸液,經C57BL/6小鼠門靜脈主干注射后觀察小鼠的死亡率,計算小鼠E.g的成囊率和分布,B超隨訪觀察E.g囊腫的生長速率;(2)B超篩查C57BL/6小鼠肝E.g囊腫,觀察固定脈寬350nm,頻率1Hz/s,50脈沖數下,不同場強(21、14、7、0kV/cm)作用后1周、4周、8周肝E.g囊腫的大小、形態(tài)改變、囊壁結構改變判斷納秒脈沖對小鼠肝E.g囊腫的消融抑制效應。(3)觀察固定脈寬350nm,頻率1Hz/s下,不同場強(29、21、14、7、0kV/cm)和脈沖數(50、100)作用后第1、3、7天原頭蚴的形態(tài)、活動度、完整性,判斷納秒脈沖對其的殺傷效應,HE染色觀察原頭蚴結構的變化,掃描電鏡觀察原頭蚴超微結構改變;采集臨床B超和CT診斷為多子囊型CE患者,選取直徑5-10mm、光亮透明,彈性好的CE囊腫,觀察固定脈寬350nm,頻率1Hz/s下,不同場強(29、21、14、0kV/cm)和不同脈沖數(50、100)作用后第1、3、7天E.g囊腫內層塌陷情況判斷納秒脈沖對其的殺傷效應,HE染色觀察E.g囊腫壁結構的變化。結果:(1)建立門靜脈接種原頭蚴的小鼠模型:2000個/100ul、200個/100ul、100個/100ul原頭蚴混懸液經門靜脈主干注射后小鼠感染率分別為90%(9/10)、100%(10/10)、63.6%(7/11),成囊率分別為0.7%~4.1%,0.6%~1.9%,0.6%~3.9%。病理切片顯示注射后1周內,大部分原頭蚴體積固縮,7天時原頭蚴周圍形成明顯的炎癥帶,21天時可見存活的原頭蚴發(fā)育出囊泡,42天時原頭蚴細胞團消失,發(fā)育成一囊型病灶;(2)獲得納秒脈沖治療小鼠肝臟原位e.g囊腫的有效劑量:小鼠e.g囊腫在納秒脈沖治療儀脈沖后21kv/cm脈沖組小鼠e.g直徑的增長在脈沖后1、4、8周與對照組存在差異(p0.05),呈縮小趨勢,脈沖后1、4、8周的直徑分別縮小1.4mm、1.2mm、1.7mm,肉眼觀e.g囊腫壁增厚,透明度消失,而對照組小鼠e.g囊腫光滑透明,壁較薄、彈性較好,腔內囊液充盈清亮張力高,病理切片顯示脈沖后8周e.g外纖維組織增厚,炎癥反應帶增寬明顯;14kv/cm脈沖組小鼠e.g直徑的增長在脈沖后8周與對照組存在差異(p0.05),呈縮小趨勢,脈沖后1、4、8周的直徑增長分別為-0.4mm、0.4mm、-0.1mm;7kv/cm脈沖組小鼠e.g直徑的增長與對照組無差異(p0.05);對照組在脈沖后1、4、8周的直徑增長分別為0.2mm、1.3mm、4.3mm。(3)納秒脈沖對體外培養(yǎng)的原頭蚴和e.g囊腫的的殺滅存在劑量效應:脈沖后第1、3天,29、21kv/cm脈沖組的原頭蚴的殺傷率與對照組存在差別(p0.05),50脈沖數下29、21kv/cm脈沖組第1天殺傷率分別為16.5%、12.9%,第3天的殺傷率分別為19%、12.6%;脈沖后第7天,29、21、14kv/cm脈沖組的原頭蚴的殺傷率與對照組存在差別(p0.05),50脈沖數下29、21、14kv/cm脈沖組第7天殺傷率分別為71.7%、64.3%、48.3%。100脈沖數下29、21kv/cm脈沖組第1天殺傷率分別為32.3%、15.6%,第3天的殺傷率分別為32.3%、16.7%,100脈沖數下29、21、14kv/cm脈沖組第7天殺傷率分別為79.2%、68.7%、49.2%,透射電鏡見脈沖后原頭蚴體表完整性被破壞;脈沖處理后e.g囊腫第1天發(fā)生明顯的內層塌陷,與對照組相比存在差異(p0.05),脈沖場強越大,塌陷率越高,50脈沖數、29kv/cm場強下,1、3天的塌陷率分別為75%、100%,21kv/cm場強下,1、3、5天的塌陷率分別為50%、75%、100%,14kv/cm場強下,1、3、5、7天的塌陷率分別為25%、62.5%、75%、75%,100脈沖數、29kv/cm場強下,1、3、5、7天的塌陷率分別為62.5%、87.5%、87.5%、100%,21kv/cm場強下,1、3、5、7天的塌陷率分別為50%、75%、75%、100%,14kv/cm場強下,1、3、5、7天的塌陷率分別為75%、87.5%、87.5%、100%。結論:(1)經小鼠門靜脈注射e.g原頭蚴可以成功建立小鼠e.g感染模型,注射200個原頭蚴可以得到較高的感染率和較合適的囊泡個數,囊泡生長個數約1-4個,可以較好的模擬臨床ce;(2)21kv/cm場強可以明顯消融小鼠e.g,棘球蚴囊腫壁增厚,透明度消失,14kv/cm場強可以明顯抑制小鼠e.g的生長;7kv/cm場強對小鼠e.g的生長無抑制作用。納秒脈沖對小鼠e.g囊腫具有劑量效應,需要優(yōu)化治療參數,只有在足夠高的電場強度下才能獲得消融及抑制生長作用。(3)固定脈寬350nm,頻率1hz/s下,29、21kv/cm電場強度下對原頭蚴的殺傷效應較明顯;e.g單囊腫對納秒脈沖的消融效應較敏感,固定脈寬350nm,頻率1hz/s下,29、21、14kv/cm電場強度下脈沖后第1天內層塌陷明顯,場強越高、脈沖越數則e.g囊腫內層塌陷越早,提示納秒脈沖在包蟲病的消融臨床應用上需要分型施治,選擇恰當的適應癥,根據WHO分型選擇合適病例,設計個體化治療方案。
[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.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R532.32

【相似文獻】

相關期刊論文 前2條

1 姚陳果;郭飛;李成祥;肖德友;唐麗靈;;納秒脈沖誘導腫瘤細胞縫隙連接通訊恢復的實驗研究[J];中國生物醫(yī)學工程學報;2011年06期

2 ;[J];;年期

相關會議論文 前9條

1 邵濤;嚴萍;張適昌;章程;;重頻納秒脈沖絕緣與放電研究中的電信號測試[A];2010電工測試技術學術交流會論文集[C];2010年

2 王守國;;納秒脈沖常壓輝光放電等離子體及薄膜表面改性研究[A];TFC’09全國薄膜技術學術研討會論文摘要集[C];2009年

3 邵濤;章程;龍凱華;王玨;張東東;嚴萍;周遠翔;;重復頻率納秒脈沖介質阻擋放電PET表面改性研究[A];中國核科學技術進展報告——中國核學會2009年學術年會論文集（第一卷·第7冊）[C];2009年

4 聶晶;;納秒脈沖產生器觸發(fā)控制器[A];第9屆全國核電子學與核探測技術學術年會論文集[C];1998年

5 龍sハ，

本文編號：2112866