【作者】 张静；

【导师】 叶彬；

【作者基本信息】 重庆医科大学 ， 病原生物学， 2012， 博士

【摘要】 背景和目的：细粒棘球绦虫,又称包生绦虫,其续绦期幼虫(棘球蚴)寄生于人和多种食草家畜引起严重的人兽共患病,称棘球蚴病或包虫病(cystic echinococcosis, CE)。目前该病的治疗方法主要以外科手术手术为主、介入性治疗、药物治疗等也同时在开展,但各治疗方法仍然存在不少问题需要解决,外科手术虽然疗效较好,但对多发性包虫与转移者难以达到理想疗效；阿苯哒唑为WH0推荐使用的有效治疗包虫病的药物,但其在体内分布广泛,无法集中于靶组织,而且由于包虫囊壁较厚角质层的阻隔作用,使药物难以进入棘球蚴囊造成局部不能达到有效的药物浓度,加之包虫囊内囊液对药物的稀释导致治疗剂量增大,从而造成转氨酶升高、黄疸、蛋白尿等药物毒性反应,甚至引起致死性的全血细胞减少。其次,我们的前期研究证明了HIFU治疗对棘球蚴有杀伤效果,发现在HIFU作用后棘球蚴囊壁生发层和角质层断裂、脱落甚至缺损,生发层细胞肿胀且有空泡形成甚至生发细胞层缺损,囊壁的通透性增加,失去其对寄生虫的屏障保护作用,而对囊内原头蚴有一定杀灭作用,但效果不尽如人意。由于囊液聚焦性差,同时液体散热又比较快,使得高强度聚焦超声波作用后囊液温度明显不如囊壁实体组织升得高且快。本研究的目的在于探索能增强高强度聚焦超声杀伤棘球蚴的有效材料和方法。通过制备出具有高强度、较快吸水速度和较好保水能力的复合高吸水性树脂吸收棘球蚴囊液水分、改变囊内液体为凝胶状态,使其成为“实体”结构,达到增强超声能量沉积效果,从而完全彻底杀死棘球蚴。材料和方法：1.采用水溶液法,以丙烯酸(AA),淀粉等为原料,通过过硫酸铵引发玉米淀粉接枝丙烯酸及加入交联剂合成复合高吸水性树脂(SAR),研究了单体组成比(淀粉/丙烯酸)以及聚合方法对树脂性能的影响。并通过光镜观察吸水前后的凝胶结构,红外光谱表征SAR颗粒结构组成。2.采用过滤法,分别测定高吸水性树脂在不同环境温度(37℃、25℃、20℃)、不同粒径对去离子水、0.9%生理盐水、无水酒精和棘球蚴囊液吸水倍率,并测定了不同粒径高吸水性树脂吸水后凝胶的保水性能。3.观察高吸水性树脂对离体囊液和体外培养原头节的影响。将不同剂量高吸水性树脂加入离体新鲜囊液中,观察囊液性状改变和主要生化指标；并将原头节悬液(液体为生理盐水)置于37℃温箱中培养,观察原头节的存活力。4.观察高吸水性树脂增强高强度聚焦超声杀伤体外原头蚴效应。将分离的原头节分为4组,对照组(超声假照组)、单纯HIFU照射组、SAR(半饱和量)+HIFU照射组、SAR(饱和量)+HIFU照射组,功率100w,作用时间分别为5s、10s、20s、30s、40s、50s、60s。光镜下观察原头节形态变化及计算死亡率,并检测了原头节内细胞琥珀酸脱氢酶和葡萄糖-6-磷酸酶的活性改变。5.观察SAR增强高强度聚焦超声杀伤肝包虫囊的效应。将选择的棘球蚴随机分为4组,分别作如下处理：,对照组(超声假照组)、单纯HIFU照射组、SAR(半饱和量)+HIFU照射组、SAR(饱和量)+HIFU照射组,功率200w,按计划治疗完一个囊即为作用时间。观察了经处理后各组原头节死亡率、原头节和生发层形态在光镜和电镜下的改变,并用Hoechst33258染色、荧光显微镜检测了原头节体细胞的凋亡情况。同时也检测了半饱和组照射后囊液的化学性质变化。用热敏温度计测量HIFU照射后包虫病肝各点温度。结果：1.结果表明,用微波法合成的高吸水性树脂(SAR)操作过程简单、后处理容易,其吸水性能与氮气保护合成的复合高吸水性树脂(SAR)具有相等的吸水性能。红外光谱证实淀粉与丙烯酸接枝成功形成高分子聚合物。2.自制的淀粉类高吸水性树脂在蒸馏水中瞬时吸水倍率在700倍以上,在棘球蚴囊液中为200～300倍,生理盐水中为25倍左右,在无水乙醇中树脂颗粒几无吸水能力。160～180目大小的颗粒其对去离子水的最大吸水倍率可达到700倍,在4～8min内即可达到吸水高峰；80～120目大小的颗粒吸水倍率亦可达700倍,但在吸水后16min时才达到吸水高峰。160～180目大小的颗粒常压室温(25℃)放置2天保水率为55%,0.5×0.5×0.5mm颗粒常压室温(25℃)放置5天保水率为58%。20℃,25℃,37℃时同样大小的树脂颗粒其吸水倍率接近。3.高吸水性树脂对在37℃、生理盐水中的原头蚴短期内(8小时)不会引起其明显的死亡；对囊液的化学成分影响不确定。4.HIFU单纯照射后前40S内内原头蚴悬液温度上升较慢,原头节死亡率为73.7%;而加入了SAR的原头蚴悬液在同样的时间内温度上升较快,40s原头蚴死亡率可达100%。死亡虫体表现为台盼兰蓝染,虫体外形尚完整无活动,或者蓝染呈皱缩,甚至虫体呈碎片状。琥珀酸脱氢酶和葡萄糖-6-磷酸酶活性明显较正常对照组和HIFU单纯照射组弱。5.各实验组棘球蚴内原头节平均死亡率依次(从Ⅰ组至Ⅳ组)为：22.94%、55.28%、63.4%、72.4%,原头节不活动、皱缩或呈碎片状。荧光显微镜观察到原头蚴内有大量折光性强的蓝白色颗粒,加入SAR后的棘球蚴内原头节经作用后折光性颗粒较单纯照射组多。透射电镜观察到加入SAR组的原头蚴体细胞内糖原颗粒减少、出现黑色凋亡小体、胞核碎裂边聚。光镜和电镜均观察到生发层细胞层变薄、生发层中断、与角皮层分离、角皮层出现空泡和同心圆条纹。囊液主要成分中蛋白质浓度依次(从Ⅰ组至Ⅲ组)为：1g/100ml、2g/100ml、2g/100ml.囊液平均温度21.3℃、31.1℃、38.9℃、44.6℃；囊壁温度(从Ⅰ组至Ⅳ组)分别为：21.3℃、38.9℃、49℃、55℃；距囊5mm处肝组织温度(从Ⅰ组至Ⅳ组)分别为：21.3℃、32.28℃、34.5℃、37.88℃。结论：1.成功制备各种粒径的高吸水性树脂颗粒,其吸水倍率可达700倍；树脂颗粒越小,其吸水速度越快,但保水效果较差；颗粒越大,吸水速度越慢,但保水效果好。温度变化对吸水速率影响不大。溶液性质,尤其是溶液含水量对树脂颗粒吸水率影响较大,因此树脂颗粒在去离子水中吸水倍率最高,其次棘球蚴囊液,生理盐水,对无水酒精几乎无吸水能力。2.高吸水性树脂在短期内(8小时)对体外生理盐水中原头节活力无明显影响。但对囊液化学成分的影响作用不确切。3.事先加入SAR再行HIFU照射,可较单纯HIFU照射明显提高囊液温度,增强HIFU对离体原头节的杀伤效果,抑制原头节SDH和G-6-P的活性。4.事先加入SAR再行HIFU照射,可较单纯HIFU照射提高棘球蚴内囊液温度,增强HIFU对离体原头节的杀伤效果,促进原头蚴体细胞凋亡。更多还原

【Abstract】 Background and objectiveEchinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal hosts, is distributed worldwide. Echino-coccosis is an increasing public health and socioeconomic concern.. 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 ofthe 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. HIFU, in which the ultrasound is focused to a focal zone, can deposit ultrasound mechanical energy and produce clinically relevant bio-effects in the targeted tissue, and has been widely applied to treat some tumors as a non-invasive therapeutic option. In HIFU therapy, absorption of ultrasonic energy at the focus leads to local heating that can result in apoptosis and thermal necrosis when the local temperature rises above60℃. However, success of this therapeutic modality depends critically on the ability to focus HIFU energy accurately at the diseased site. Our group has previously found that HIFU could damage protoscolices in vitro, but the death rate was limited by cyst fluid with characters of less focus HIFU energy and fast loss of heat. In this study, we prospectively suggest that a kind of special particle with superabsorbent ability was added into the cyst fluid of Echinococcus granulosus and would strongly focus HIFU energy. Starch based superabsorbent polymer (SAR) of poly (sodium acry late) was prepared by reverse phase suspension polymerization. They are solid, granular or powder cross linked polymers that rapidly absorb and retain large volumes of aqueous solutions. The absorptive properties of SAR are ideally suited for the absorption and solidification of various types of liquids like water, sludge, blood etc. which has caused an increased use of this substance in the production of hygiene products and agriculture.In this study, we prospectively suggest that a kind of special particle with superabsorbent ability was added into the cyst fluid of Echinococcus granulosus and would strongly focus HIFU energy. By the present work evaluated whether or not a superabsorbent polymer (SAR) could enhance the damage efficacy of high intensity focused ultrasound (HIFU) on the viability of E. granulosus protoscolices in vitro.Materials and Methods1. A super absorbent resin was synthesized with grafting acrylic acid and corn starch as elementary materials by solution polymerization in the presence of N, N-Methylene bisacrylamide as cross-linker and potassium per-sulfate as initiator. The products synthesizing were observed through infrared spectrum and light microscope.2. The instantaneous water absorption ratios of this resin were measured when it, of which are different particle sizes, mixed with the different liquids such as demineralized water, physiological saline, anhydrous alcohol and hydatid cyst fluid at different temperatures of20℃,25℃, and37℃respectively. The water preserving capabilities of the resin of several different particle sizes were measured.3.The protoscolices were cultivated in the physiological saline with superabsorbent resin at37℃. and the major chemical constituent in the hydatid fluid with superabsorbent resin were measured.4. HIFU of100W acoustic power, and some dose of superabsorbent resin, were used to treat5000protoscolices in2milliliter protoscolices suspension. The temperature of protoscolices suspension was investigated at different HIFU exposure time (5,10,20,30,40,50,60seconds) respectively, the structures of protoscolices were observed by light microscopy. To better understand the biological mechanisms responsible for the regulation of protoscolices death, we examined the activity of succinate dehydrogenase (SDH) and Glucose-6-phosphatase of the protoscolices treated with HIFU.5. HIFU of200W acoustic power, and some dose of superabsorbent resin, were used to treat hydatid of E. granulosus. All the experimental hydatid of E. granulosus were randomly assigned into four groups by intervention study as follows:A)with no exposure to HIFU was considered as the blank group; B)with exposure to HIFU was considered as the test control group (200power); C)with exposure to HIFU combined with lg SAR in the hydatid liquid and D) with exposure to HIFU combined with2g SAR werconsidered as the test group (the parameter same as the forward). The changes of structure of procolices and cyst ball of hydatid were by light microscope, scanning and transmission electric microscope, and detected the apoptosis of protolices by Hochest assay. The temperature of monitoring points in the hydatid liver were measured by temperature sensitive thermometer.Results1. A transparent graticule ultrastructures was observed in the water-absorbing resin under light microscope and through infrared spectrum.2. The absorption ratio of the resin to deionized water, and hydatid cyst fluid, physiological saline were the maximum, being of700g/g and200-300g/g respectively. It’s ratio to physiological saline, however, was only25times of its mass, and less absorptive to anhydrous alcohol. Of which160-180mesh size particles, to deionized water could reach to700times, and a peak of water absorption was observed within4to8minutes. Although a same maximum absorption ratio was observed in the resin with a particle size of80-120mesh, but it took16minutes for the water absorption peak. At room temperature of25℃, the water-preserving rate of the resin with a particle size of160-180mesh was55%in2days, but it was58%in5days for the resin of which size was0.5×0.5×0.5mm. The water-absorbing rate was almost same for a resin with a particular size mixed the above mentioned liquids at different temperature of20℃,25℃, and37℃respectively.3. The lively ability of protoscolices in the physiological saline at37℃had not been reflected by Super Absorbent Resin (SAR) within the8minutes. The major chemical constituent had not preonunced changes by Super Absorbent Resin (SAR).4. The rising effect of temperature of protoscolices suspension treated with HIFU was slow, and the death rate of protoscolices was73.7%in the group of HIFU treatment time of40seconds; In the group of HIFU treatment time of40seconds, the rising effect was quick, and the death rate of protoscolices was100%in the group of HIFU combined with SAR. The best protoscolicidal effect (100%) of HIFU of100W acoustic power combined with SAR was obtained after40,50, and60seconds respectively. The live protoscolices in the blank control group showed distinct inner structure. The membrane of dead protoscolices were stained to blue exposed by the100W acoustic power of HIFU, showed shrunken and black calcareous corpuscles, disorder and decreasing hooks though of intact membrane; some protoscolices lost hooks and teared open on membrane were observed. In the group of HIFU combined with SAR, it was found that the superabsorbent polymer around the spoiled protoscolices and the destruction of much stronger than in the group of HIFU. The dead protoscolices exhibited the reduction or absence of SDH and G-6-P staining intensity in the parenchymal cell and calcareous corpuscles undergoing HIFU irradiation, where a large number of apoptotic, necrotic cell were evident; but was markedly preserved in the blank group.5. The average death rates of procolices in the Ⅰ、Ⅱ、Ⅲ、Ⅳgroup were22.94%、55.28%、63.4%、72.4%。The dead pritoscolices such as lost-alively and crimple and teard into piece were observed by light microscope, the blue and reflected light particles of the proscolices observed by fluorescence microscope exposured by the HIFU combined with SAR were more than the simple HIFU group. The decreasing glycogenosome and appeared to black body and karyorrhexis of the proscolices were observed by transmission electron microscope, it showed germinal layer growed thinner and interruption, separated from the cuticulae layer, wherein appeared to vacuoles and concentric circles. The protein concentration of major component in the hydatid fluid ofⅠ、Ⅱ、 Ⅲgroup were lg/100ml、2g/100ml、2g/100ml。The average temperatures of hydatid fluid were21.3℃、31.1℃、38.9℃、44.6℃;were21.3℃、38.9℃、49℃、55℃in the cystwall;21.3℃、32.28℃、34.5℃、37.8℃in the which was5mm between liver and hydatid cyst。Conclusions1. Super Absorbent Resin (SAR) is very unique water-absorbing and water-holding materials. They can absorb up to700times of their own weight of pure water and even under pressure resist release of the absorbed water. The temperature change has little effect on the rate of water-absorbing. Solution properties, especially the water content of solution greatly impact the water-absorbing. Therefore, the water absorbency of the particles is the highest in the deionized water, followed by hydatid cyst fluid, normal saline, anhydrous alcohol for almost no water absorption.2. The lively ability of protoscolices in the physiological saline had not been reflected by Super Absorbent Resin (SAR) within the8minutes.3. These results suggest that HIFU induced damage and loss of viability of protoscolices and hydatid, and Super Absorbent Resin (SAR) enhanced the HIFU energy and caused more severe destruction efficacy of HIFU irradiation on protoscolices and hydatid, and that mitochondrial energetic function is involved in the regulation of cell death pathways in the pathogenesis of protoscolices.更多还原