高强度聚焦超声波杀伤细粒棘球绦虫棘球蚴的研究
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摘要
研究背景:囊性棘球蚴病是细粒棘球绦虫后绦幼虫棘球蚴寄生所致的一种严重的人兽共患慢性寄生虫病,也称包虫病。棘球蚴寄生引起的包虫病呈世界性分布。目前包虫病的治疗手段包括手术和药物治疗,效果均不理想。无论是传统开腹手术还是腹腔镜内、经皮穿刺介入治疗等微创治疗等手术治疗方式均具侵入性。术中包囊液及棘球蚴砂溢漏会引起过敏性休克甚至死亡,术后因头节的外漏和残留导致棘球蚴病继发感染以及引起残腔感染、积液、胆漏等多种严重并发症。苯并咪唑衍生物阿苯达唑是现有抗棘球蚴病的治疗首选药物,但因其低溶解性和在体内广泛的分布性,阿苯达唑治疗包虫病存在着病人服药治疗时间长、药物的副作用较大、治疗不彻底等问题。
高强度聚焦超声波(High Intensity Focused Ultrasound,HIFU)技术是利用超声波声束良好的穿透性、汇聚性和方向性等特点,将体外低能量超声波汇聚于机体内特定靶区,通过在焦点处产生高温效应,空化效应、声化学效应等高能效应破坏组织细胞。HIFU作为一种新兴的无创性治疗技术,已在临床试用于肿瘤治疗。
本课题将这种非侵入性高强度聚焦超声波治疗技术应用于囊性棘球蚴,观测高强度聚焦超声波对细粒棘球蚴细胞,局部结构原头节以至整个寄生虫生长的影响作用及其可能机制,探索高强度聚焦超声波这一新兴治疗技术运用于棘球蚴病以至其它囊性病变的可能性和可行性。
研究方法:
1、从自然感染的绵羊棘球蚴获取内囊分离生发细胞进行体外培养,电镜及免疫荧光试验鉴定生发细胞;MTT法筛选HIFU对生发细胞增殖影响的最佳作用参数;以致10%急性生发细胞死亡率的HIFU剂量处理细胞后,AnnexinV/PI荧光染色、电镜、荧光显微镜结合流式细胞仪观测HIFU对细胞凋亡、细胞周期的影响;检测HIFU作用后细胞内Caspase-3蛋白酶活性;Western blot检测P53、BcL-2/Bax和Fas等凋亡相关蛋白表达。
2、分离原头节,观察不同剂量的HIFU照射原头节后的即刻杀灭作用,获得剂量-效应关系;以不致原头节即刻杀伤的超声剂量作用后,根据台盼蓝排斥法染色结果以及原头节形态变化确定原头节死亡率,了解HIFU对原头节生长的迟发性影响;将此种辐照原头节接种小鼠进行返种实验,根据棘球蚴在体内囊湿重及包囊形态学改变,了解HIFU对原头节的感染能力的影响;同时行酶化学染色检测原头节酸性磷酸酶、葡萄糖6-磷酸酶和琥珀酸脱氢酶的活性变化。
3、用采自自然感染的绵羊棘球蚴的原头节接种小鼠腹腔,建立小鼠继发性棘球蚴病动物模型;配对动物模型小鼠进行HIFU辐照实验,HIFU焦点紧贴包囊内壁做多层面的曲线闭环连续扫描,层面间距4mm,直至辐照完整个包囊;小鼠继续饲养2月,通过比较对照组和HIFU处理组的小鼠棘球蚴包囊减重率,观察囊壁的形态改变及TUNEL法检测囊壁细胞凋亡等,评价HIFU杀伤棘球蚴的效果;并通过ELISA法检测血清IgG抗体水平及细胞因子IFN-γ, IL-2,IL-4,IL-10的变化,流式细胞仪淋巴检测淋巴细胞亚群的变化,RT-PCR检测小鼠脾淋巴细胞细胞因子mRNA表达情况等实验手段,了解HIFU作用后小鼠免疫功能的改变。
研究结果:
1、分离的细胞电镜下表现为分化较低,较符合生发细胞功能特点,免疫荧光试验鉴定细胞表面确有细粒棘球蚴抗原成分;20W×20s,40W×10s和160W×5s为致10%急性生发细胞死亡率的HIFU剂量;经此剂量的HIFU处理后,AnnexinV/PI荧光染色、电镜观察、荧光显微镜结合流式细胞仪实验结果一致:HIFU对超声照射后存活生发细胞生长具有明显的抑制作用并可诱导生发细胞凋亡;胞内Caspase-3蛋白酶活性增加;未见P53、BcL-2/Bax和Fas等凋亡相关蛋白蛋白表达。
2、HIFU对原头节有明显的急性杀伤作用,随着超声照射剂量增大,其生物学效应越明显,声功率大于200W短时照射就极易致原头节全部即刻死亡;不致原头节即刻杀伤的最大超声剂量为25W×20s和50W×10s。经此剂量的HIFU作用后全部存活的原头节,体外培养过程中死亡率明显较对照组增高,而原头节返种实验时在小鼠体内形成棘球蚴的数量/能力亦降低;原头节葡萄糖6-磷酸酶和琥珀酸脱氢酶活性较对照组明显减弱,而酸性磷酸酶反应产物与对照组相比无明显变化。
3、成功建立小鼠继发性棘球蚴病动物模型;经HIFU以多层面贴壁曲线闭环扫描方式作用后,继续饲养至观察期满的小鼠体内的棘球蚴包囊明显减重,有缩小、实变等形态改变,囊壁结构明显不如对照组正常、完整并观测到细胞凋亡;动物模型血清IgG抗体水平无明显变化,小鼠血清及脾淋巴细胞CD4+ /CD8+比值升高,小鼠血清及脾细胞IFN-γ, IL-2等Th1型细胞因子含量升高,IL-4,IL-10等Th2型细胞因子水平降低。
研究结论:
1、HIFU辐照不但在体外对生发细胞和原头节这两种棘球蚴组成成分有明显的急性杀伤作用,而且能够抑制辐照后的生发细胞和原头节生长;
2、HIFU辐照能够抑制棘球蚴在小鼠动物模型体内的生长;
3、HIFU的杀灭机制与其高能效应直接破坏、促进生发细胞凋亡、抑制原头节酶的活性以及重新激发宿主对棘球蚴有效的免疫反应有关。
Background and purpose:
Cystic echinococcosis (also called hydatidosis) is a health-threatening zoonosis caused by the larval stage of Echinococcus granulosus. But there is currently no agreement about the ideal therapy for hydatidosis. The accepted surgical approaches for hydatidosis are all invasive and pose a risk of recurrence and postoperative complications due to rupture of cysts and/or spillage of the contents. Albendazole is the drug of choice for chemotherapy. However, its concentrations in the target organ are not optimal, and are much lower than in plasma because of thick cyst wall. Increases in dose may increase side-effects such as diarrhea, nausea, vomiting, aminotransferase elevation, leucopenia and may even result in death. High Intensity Focused Ultrasound (HIFU) is a non-invasive technique which can kill histiocytes and induce necrosis in the targeted area. The aim of this work was to observe the effect of HIFU on echinococcus and to explore whether this new and non-invasive therapy could be more effective and with fewer complications for cystic echinococcosis.
Materials and Methods:
1. Germinal cells of Echinococcus granulosus from naturally infected sheep were primarily cultured and identified and the optimal parameters relevant to the effects of HIFU on the proliferation of germinal cells selected by MTT colorometric assay; cells being treated with 10% lethal dosages of HIFU, electron microscopy, AnnexinV/PI labeling method, fluorescence microscopy combined with flow cytometry were applied to observe apoptosis in cells and analyze cell cycle as well as to identify the activity of Caspase-3; western blot used to detect the apoptosis-related protein expressions of P53、BcL-2/Bax and Fas etc.
2. HIFU dosage-effect was obtained from protoscolices isolation. Irradiated with HIFU of maximal tolerable dosage, parasite morphology and mortality was observed by fluorescence microscopy and the inhibitory effect of HIFU on protoscolices was investigated by trypan-blue exclusion assay; the infection effect was learned from the mean weight of hydatid cysts and hydatid morphological changes through infection experiments in vivo; meanwhile, the activity of ACP、G-6-P and SDH of protoscolices were detected by enzymatic histochemistry.
3. The animal model of cystic echinococcosis was established using protoscolices from naturally infected sheep. Irritated with HIFU of curve scanning mode around cysts, the morphology of hydatid walls was observed in comparison with the wet weights of hydatid and the apoptotic induction of the wall cells detected by TUNEL assay to evaluate the effects of HIFU on the cysts; the level of IgG, IFN-γ, IL-2, IL-4 and IL-10 in serum was detected by EL1SA, the counts and subsets of lymphocyte in peripheral blood detected by flow cytometry, mRNA expressions of lymphocytokines detected by RT-PCR to learn the immunoreactive change of the mice treated with HIFU.
Results:
1. The results of MTT demonstrate that HIFU does have the effects of growth inhibition on germinal cells in vitro. Cell apoptosis was similar under fluorescence microscopy EM and FCM, with 10% lethal dosages of HIFU (20W×20s,40W×10s and160W×5s), while the cell apoptosis and the activity of Caspase-3 increased, but the expression of P53、B cL-2/Bax and Fas were not be detected.
2. HIFU damage on protoscolices and the protoscolicidal effect was dose-dependent. HIFU at 200Ws (or above) could easily cause immediate death of protoscolices even with instant irradiation; HIFU at 25 W×20 s and 50 W×10 s could be used as maximal tolerable dosages. Under the effect of HIFU of maximal tolerable dosages, the growth of protoscolices that survived the exposure to HIFU was obviously suppressed in vitro and the mean weight of hydatid cysts resulting from such protoscolices was less than that in control group. The positive reactive product of G-6-P and SDH decreased markedly in HIFU treated protoscolices, and there was no difference between HIFU treated protoscolices and control group in ACP which were detected by enzymatic histochemistry.
3. The mean weight of hydatid cysts decreased obviously, and the structure of the germinal membrane of irradiated cysts was not as normal or intact as those non-irradiated ones, and morphological changes related to degeneration were observed after being irritated with HIFU of curve scanning mode around cysts. The level of IgG almot remained no difference as previous in serum by ELISA. The content of CD4+ increased and as well as the value of CD4+/CD8+ in lymphocyte. The results of RT-PCR demonstrate that the mRNA expression of IFN-γwas up-regulated while IL-4 down-regulated, in accordance with the results that the contents of IFN-γand IL-2 increasing while IL-4 and IL-10 decreasing.
Conclusions:
1. HIFU demonstrated an immediate damaging effect on both germinal cells and protoscolices of Echinococcus granulosu, and inhibited the proliferations as well in vitro.
2. HIFU was capable of inhibiting the growth of cysts in animal mode in vivo, and could be a possible therapeutic option for cystic echinococcosis.
3. The mechanism maybe related to directly damaging, promiting apoptosis, suppressing the enzymatic activities and re-motivating the efficient immune responses of the host on the parasite as well.
高强度聚焦超声波(High Intensity Focused Ultrasound,HIFU)技术是利用超声波声束良好的穿透性、汇聚性和方向性等特点,将体外低能量超声波汇聚于机体内特定靶区,通过在焦点处产生高温效应,空化效应、声化学效应等高能效应破坏组织细胞。HIFU作为一种新兴的无创性治疗技术,已在临床试用于肿瘤治疗。
本课题将这种非侵入性高强度聚焦超声波治疗技术应用于囊性棘球蚴,观测高强度聚焦超声波对细粒棘球蚴细胞,局部结构原头节以至整个寄生虫生长的影响作用及其可能机制,探索高强度聚焦超声波这一新兴治疗技术运用于棘球蚴病以至其它囊性病变的可能性和可行性。
研究方法:
1、从自然感染的绵羊棘球蚴获取内囊分离生发细胞进行体外培养,电镜及免疫荧光试验鉴定生发细胞;MTT法筛选HIFU对生发细胞增殖影响的最佳作用参数;以致10%急性生发细胞死亡率的HIFU剂量处理细胞后,AnnexinV/PI荧光染色、电镜、荧光显微镜结合流式细胞仪观测HIFU对细胞凋亡、细胞周期的影响;检测HIFU作用后细胞内Caspase-3蛋白酶活性;Western blot检测P53、BcL-2/Bax和Fas等凋亡相关蛋白表达。
2、分离原头节,观察不同剂量的HIFU照射原头节后的即刻杀灭作用,获得剂量-效应关系;以不致原头节即刻杀伤的超声剂量作用后,根据台盼蓝排斥法染色结果以及原头节形态变化确定原头节死亡率,了解HIFU对原头节生长的迟发性影响;将此种辐照原头节接种小鼠进行返种实验,根据棘球蚴在体内囊湿重及包囊形态学改变,了解HIFU对原头节的感染能力的影响;同时行酶化学染色检测原头节酸性磷酸酶、葡萄糖6-磷酸酶和琥珀酸脱氢酶的活性变化。
3、用采自自然感染的绵羊棘球蚴的原头节接种小鼠腹腔,建立小鼠继发性棘球蚴病动物模型;配对动物模型小鼠进行HIFU辐照实验,HIFU焦点紧贴包囊内壁做多层面的曲线闭环连续扫描,层面间距4mm,直至辐照完整个包囊;小鼠继续饲养2月,通过比较对照组和HIFU处理组的小鼠棘球蚴包囊减重率,观察囊壁的形态改变及TUNEL法检测囊壁细胞凋亡等,评价HIFU杀伤棘球蚴的效果;并通过ELISA法检测血清IgG抗体水平及细胞因子IFN-γ, IL-2,IL-4,IL-10的变化,流式细胞仪淋巴检测淋巴细胞亚群的变化,RT-PCR检测小鼠脾淋巴细胞细胞因子mRNA表达情况等实验手段,了解HIFU作用后小鼠免疫功能的改变。
研究结果:
1、分离的细胞电镜下表现为分化较低,较符合生发细胞功能特点,免疫荧光试验鉴定细胞表面确有细粒棘球蚴抗原成分;20W×20s,40W×10s和160W×5s为致10%急性生发细胞死亡率的HIFU剂量;经此剂量的HIFU处理后,AnnexinV/PI荧光染色、电镜观察、荧光显微镜结合流式细胞仪实验结果一致:HIFU对超声照射后存活生发细胞生长具有明显的抑制作用并可诱导生发细胞凋亡;胞内Caspase-3蛋白酶活性增加;未见P53、BcL-2/Bax和Fas等凋亡相关蛋白蛋白表达。
2、HIFU对原头节有明显的急性杀伤作用,随着超声照射剂量增大,其生物学效应越明显,声功率大于200W短时照射就极易致原头节全部即刻死亡;不致原头节即刻杀伤的最大超声剂量为25W×20s和50W×10s。经此剂量的HIFU作用后全部存活的原头节,体外培养过程中死亡率明显较对照组增高,而原头节返种实验时在小鼠体内形成棘球蚴的数量/能力亦降低;原头节葡萄糖6-磷酸酶和琥珀酸脱氢酶活性较对照组明显减弱,而酸性磷酸酶反应产物与对照组相比无明显变化。
3、成功建立小鼠继发性棘球蚴病动物模型;经HIFU以多层面贴壁曲线闭环扫描方式作用后,继续饲养至观察期满的小鼠体内的棘球蚴包囊明显减重,有缩小、实变等形态改变,囊壁结构明显不如对照组正常、完整并观测到细胞凋亡;动物模型血清IgG抗体水平无明显变化,小鼠血清及脾淋巴细胞CD4+ /CD8+比值升高,小鼠血清及脾细胞IFN-γ, IL-2等Th1型细胞因子含量升高,IL-4,IL-10等Th2型细胞因子水平降低。
研究结论:
1、HIFU辐照不但在体外对生发细胞和原头节这两种棘球蚴组成成分有明显的急性杀伤作用,而且能够抑制辐照后的生发细胞和原头节生长;
2、HIFU辐照能够抑制棘球蚴在小鼠动物模型体内的生长;
3、HIFU的杀灭机制与其高能效应直接破坏、促进生发细胞凋亡、抑制原头节酶的活性以及重新激发宿主对棘球蚴有效的免疫反应有关。
Background and purpose:
Cystic echinococcosis (also called hydatidosis) is a health-threatening zoonosis caused by the larval stage of Echinococcus granulosus. But there is currently no agreement about the ideal therapy for hydatidosis. The accepted surgical approaches for hydatidosis are all invasive and pose a risk of recurrence and postoperative complications due to rupture of cysts and/or spillage of the contents. Albendazole is the drug of choice for chemotherapy. However, its concentrations in the target organ are not optimal, and are much lower than in plasma because of thick cyst wall. Increases in dose may increase side-effects such as diarrhea, nausea, vomiting, aminotransferase elevation, leucopenia and may even result in death. High Intensity Focused Ultrasound (HIFU) is a non-invasive technique which can kill histiocytes and induce necrosis in the targeted area. The aim of this work was to observe the effect of HIFU on echinococcus and to explore whether this new and non-invasive therapy could be more effective and with fewer complications for cystic echinococcosis.
Materials and Methods:
1. Germinal cells of Echinococcus granulosus from naturally infected sheep were primarily cultured and identified and the optimal parameters relevant to the effects of HIFU on the proliferation of germinal cells selected by MTT colorometric assay; cells being treated with 10% lethal dosages of HIFU, electron microscopy, AnnexinV/PI labeling method, fluorescence microscopy combined with flow cytometry were applied to observe apoptosis in cells and analyze cell cycle as well as to identify the activity of Caspase-3; western blot used to detect the apoptosis-related protein expressions of P53、BcL-2/Bax and Fas etc.
2. HIFU dosage-effect was obtained from protoscolices isolation. Irradiated with HIFU of maximal tolerable dosage, parasite morphology and mortality was observed by fluorescence microscopy and the inhibitory effect of HIFU on protoscolices was investigated by trypan-blue exclusion assay; the infection effect was learned from the mean weight of hydatid cysts and hydatid morphological changes through infection experiments in vivo; meanwhile, the activity of ACP、G-6-P and SDH of protoscolices were detected by enzymatic histochemistry.
3. The animal model of cystic echinococcosis was established using protoscolices from naturally infected sheep. Irritated with HIFU of curve scanning mode around cysts, the morphology of hydatid walls was observed in comparison with the wet weights of hydatid and the apoptotic induction of the wall cells detected by TUNEL assay to evaluate the effects of HIFU on the cysts; the level of IgG, IFN-γ, IL-2, IL-4 and IL-10 in serum was detected by EL1SA, the counts and subsets of lymphocyte in peripheral blood detected by flow cytometry, mRNA expressions of lymphocytokines detected by RT-PCR to learn the immunoreactive change of the mice treated with HIFU.
Results:
1. The results of MTT demonstrate that HIFU does have the effects of growth inhibition on germinal cells in vitro. Cell apoptosis was similar under fluorescence microscopy EM and FCM, with 10% lethal dosages of HIFU (20W×20s,40W×10s and160W×5s), while the cell apoptosis and the activity of Caspase-3 increased, but the expression of P53、B cL-2/Bax and Fas were not be detected.
2. HIFU damage on protoscolices and the protoscolicidal effect was dose-dependent. HIFU at 200Ws (or above) could easily cause immediate death of protoscolices even with instant irradiation; HIFU at 25 W×20 s and 50 W×10 s could be used as maximal tolerable dosages. Under the effect of HIFU of maximal tolerable dosages, the growth of protoscolices that survived the exposure to HIFU was obviously suppressed in vitro and the mean weight of hydatid cysts resulting from such protoscolices was less than that in control group. The positive reactive product of G-6-P and SDH decreased markedly in HIFU treated protoscolices, and there was no difference between HIFU treated protoscolices and control group in ACP which were detected by enzymatic histochemistry.
3. The mean weight of hydatid cysts decreased obviously, and the structure of the germinal membrane of irradiated cysts was not as normal or intact as those non-irradiated ones, and morphological changes related to degeneration were observed after being irritated with HIFU of curve scanning mode around cysts. The level of IgG almot remained no difference as previous in serum by ELISA. The content of CD4+ increased and as well as the value of CD4+/CD8+ in lymphocyte. The results of RT-PCR demonstrate that the mRNA expression of IFN-γwas up-regulated while IL-4 down-regulated, in accordance with the results that the contents of IFN-γand IL-2 increasing while IL-4 and IL-10 decreasing.
Conclusions:
1. HIFU demonstrated an immediate damaging effect on both germinal cells and protoscolices of Echinococcus granulosu, and inhibited the proliferations as well in vitro.
2. HIFU was capable of inhibiting the growth of cysts in animal mode in vivo, and could be a possible therapeutic option for cystic echinococcosis.
3. The mechanism maybe related to directly damaging, promiting apoptosis, suppressing the enzymatic activities and re-motivating the efficient immune responses of the host on the parasite as well.
引文
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