放射治疗棘球蚴病的体内外实验研究

发布时间：2018-05-09 13:34

本文选题：放射治疗 + 棘球蚴　；参考：《新疆医科大学》2016年博士论文

【摘要】：研究目的:棘球蚴病俗称包虫病,流调表明我国有38万包虫病患者,现有手术资源很难完成所有治疗,30余年的药物治疗效果亦不尽人意,需另辟治疗途径。本研究旨在探明X线杀伤包虫的有效性和安全性,做为新技术的开发为临床应用提供证据。1)建立E.g体外成囊培养系统,为放射治疗棘球蚴的体外实验研究提供实验材料。2)明确X线照射体外培养的原头节和包囊的杀伤作用。3)探讨放射治疗对大鼠继发泡球蚴(E.m)的疗效和安全性。4)探讨放射治疗对自然感染羊细粒棘球蚴(E.g)的疗效和安全性。研究方法:1)从屠宰场自然感染E.g的绵羊肝脏采集新鲜原头蚴(PSC),经1%的胃蛋白酶消化后检测虫体活性并计数,于37℃、5%C02条件下进行体外培养,不同时间点(第1天、第7天、第15天、第60天)进行形态学观察,超微结构观察,计算包囊的成囊率。2)将原头蚴分装于7组(每组3瓶),每瓶约2000个原头蚴,对7组原头蚴分别进行0Gy、20Gy、40Gy、60Gy、80Gy、100Gy、120Gy剂量照射,机架角180度,射野大小10×10cm,SSD=100cm,剂量率300cGy/min,将包囊分装于4组(每组3瓶),每瓶约200个包囊,1组为对照组,对其他3组包囊分别进行30Gy/3f、45Gy/3f、60Gy/3f剂量照射,照射后电镜及光镜下观察包囊的病理变化判断疗效,观察半数致死量和时间,qRT-PCR检测受X线照射前后关键基因表达的差异。3)建立雌性S-D大鼠继发性泡球蚴病动物模型,随机分为4组,每组15只,分别为低、中、高剂量放射治疗组和对照组,对照组不予任何处理,治疗组给予6-MeV线照射,总剂量分别为30Gy、45Gy和60Gy,照射3次,每次间隔2天,放疗结束1个月后检测各组大鼠泡球蚴囊湿重、抑囊率,并对泡球蚴组织进行病理组织学和超微结构观察。4)从新疆牧区用B超筛选53只自然感染肝细粒棘球蚴病的羊,经CT验证后将感染羊随机抽取20只分配到四个实验组中,高剂量组(60Gy)5只、中剂量组(45Gy)5只、低剂量组(30Gy)5只、对照组(0Gy)5只。每只羊麻醉好后采用高分子低温水解塑料热压成型体位固定技术固定,GE大孔径螺旋CT扫描,放疗医师勾画预照射包囊部位的靶区,物理师做照射计划,核准位置后实施图像引导下的精确放疗。照射组共照射3次,隔两日一次,一周内完成照射,达到预照射总量。放疗后三月复查CT,对比放疗前后目标病灶的变化。处死各组绵羊,取出放疗区肝内细粒棘球蚴囊,用于光镜和电镜下观察,qRT-PCR检测受X线照射前后关键基因表达的差异,比较宿主和包囊的免疫反应。结果:1)E.g原头蚴在体外培养15d时微囊逐渐形成,60d时包囊外围形成透明角质层结构,超微结构显示E.g囊泡外部有较厚的无细胞薄片层,并可见生发囊及囊内PSC产生。2)不同剂量X线照射后14天后原头节的成囊率P0.05,比较有统计学意义。6MV-X线照射原头蚴后14天后,对照组成囊发育中,照射组发生了钩突崩解,正常结构消失。6MV-X线照射已成囊的包囊7天后,对照组包囊囊壁结构完整,致密,照射组囊壁萎缩塌陷,正常结构紊乱消失,内质网扩张,部分为异常浓缩的染色团块。照射后7天,EgTPX、EgHSP70、EgEPC1、Csapase-3、Gadd45的随着照射剂量的升高表达增强。3)低、中、高剂量放射治疗组泡球蚴囊湿重分别为(1.82±0.74)g、(1.04±0.38)g和(0.76±0.33)g,抑囊率分别为50%、72%和82%,对照组泡球蚴囊湿重为(3.65±0.79)g,4组大鼠泡球蚴的平均湿重有显著性差异(P0.05),光镜结果显示对照组泡球蚴结构基本正常,角质层、生发层清晰,育囊内有多少不等的原头节,治疗组泡球蚴呈不同程度的改变和破坏,结构失常,角质层、生发层普遍变性肿胀或分离脱落,原头节少见,囊壁病理改变程度与对照组比较差异均有显著性(P0.05),电镜结果显示治疗组泡球蚴囊壁角质层和生发层均有不同程度的损伤,其中60Gy放射治疗组损害最重。4)CT结果显示高剂量组中有4只羊的受照射包囊囊壁钙化,HE染色结果显示受照射包囊的角质层及生发层结构遭到不同程度破坏,普遍变性肿胀或变薄,部分分离、断裂、脱落,生发层细胞核溶解或消失,很少见育囊及原头蚴。提取受照射包囊的RNA行荧光定量PCR检测结果显示:EgTPX表达随着剂量的升高表达降低,P0.05(P=0.04);EgEPC1表达低剂量组明显高于中高剂量组,P0.05(P=0.03);EgHSP70表达随着剂量的升高表达有降低的趋势,但无统计学意义,P0.05(P=0.22);说明随着放疗照射剂量的增加,包囊的活性被抑制。辐射相关的凋亡基因caspase-3和Gadd45基因的表达随着剂量的升高表达降低。放疗前后感染羊的一般生活状况、体重、血常规、肝功、电解质无明显变化(P0.05)。结论:1)建立了稳定的E.g体外成囊培养平台,为后续放射线抗棘球蚴的体外实验研究提供了充足的实验材料。2)X线对体外培养的原头节和包囊确有杀伤作用,3)放射线有破坏大鼠泡球蚴囊壁结构和抑制原头节增殖的作用,且作用强度与剂量有关。4)放射治疗后患羊的一般生活状况、血常规和肝功未受到影响,证明放射治疗羊肝包虫病是安全的。从CT影像学、病理学、分子生物学不同角度证实放射治疗对肝包虫病有一定疗效。60Gy/20Gy/3f是对肝包虫病有效且安全的剂量分割方式。
[Abstract]:Research objective: echinococcosis is commonly known as echinococcosis, and the flow modulation indicates that there are 380 thousand echinococcosis patients in our country. The existing surgical resources are difficult to complete all the treatment. The effect of drug treatment for more than 30 years is also unsatisfactory. The purpose of this study is to explore the effectiveness and safety of X-ray killing echinococcosis, and to develop new techniques for clinical application. For evidence.1) establish an in vitro culture system of E.g in vitro, provide experimental material.2 for the experimental study of Echinococcus radiosaciae in vitro) the killing effect of X ray irradiation on the original sac and cysts in vitro; the effect of radiation therapy on the secondary alveolar echinococcus (E.m) in rats and the safety of.4) to explore the effect of radiation therapy on the natural infection of fine spinous balls of sheep The efficacy and safety of E.g. Study methods: 1) collect fresh proto cercariae (PSC) from the sheep liver of the slaughterhouse which naturally infected E.g. After 1% pepsin digestion, the insect body activity was detected and counted, under the condition of 37 and 5%C02, the morphological observation was carried out at different time points (first days, seventh days, fifteenth days, sixtieth days), and the ultrastructure was observed. To observe and calculate the cyst formation rate of the cyst.2), the original cercariae were divided into 7 groups (3 bottles in each group), about 2000 cercariae in each bottle. The 7 groups of original cercariae were treated with 0Gy, 20Gy, 40Gy, 60Gy, 80Gy, 100Gy, 120Gy, 180 degrees, 10 x 10cm, SSD=100cm, and 300cGy/min. The capsule was divided into 4 groups (3 bottles of each group), about 200 capsules each bottle, 1 The group was the control group. The other 3 groups were irradiated with 30Gy/3f, 45Gy/3f, 60Gy/3f, and the pathological changes of the capsule were observed under the electron microscope and the light microscope, and the median lethal dose and time were observed. The difference of the key gene expression before and after the X-ray was detected by qRT-PCR.3). The animal model of secondary alveolar echinococcosis in female S-D rats was established. 4 groups were randomly divided into 4 groups, 15 in each group, which were low, medium and high dose radiation therapy group and control group. The control group was not treated with any treatment. The treatment group was given 6-MeV line irradiation, the total dose was 30Gy, 45Gy and 60Gy, irradiation 3 times, each interval was 2 days. After 1 months of radiotherapy, the wet weight and inhibition rate of alveolar echinococcus were detected and the tissues of alveolar echinococcus were detected. 53 sheep with natural infection with echinococcosis of liver were screened from Xinjiang pastoral area by B-ultrasound. After CT verification, 20 sheep were randomly selected from four experimental groups, with high dose group (60Gy) 5, medium dose group (45Gy) 5, low dose group (30Gy) 5, and control group (0Gy) 5. GE large aperture spiral CT scanning with high polymer low temperature hydrolysis plastic, spiral CT scan of large aperture, radiation therapist to draw the target area of pre irradiation capsule, the physicist to do the radiation plan, and the precise radiotherapy under the image guidance after the approval position. The irradiated group is irradiated 3 times, every time, the radiation is completed within one week to reach the total pre irradiation. After radiotherapy, CT was reviewed in March to compare the changes of the target lesion before and after radiotherapy. The sheep were killed and the echinococcosis cyst in the liver of the radiotherapy area was taken out for observation under light microscope and electron microscope. QRT-PCR was used to detect the difference of key gene expression before and after X-ray irradiation, and compared the immune response of the host and cyst. Results: 1) E.g prophylla was cultured for 15d in vitro. The microcapsules were formed gradually, and the outer layer of the capsule formed a transparent cuticle structure at 60d. The ultrastructure showed that there was a thicker cell layer outside the E.g vesicle, and the PSC produced.2 in the germinal sac and the capsule. 14 days after X-ray irradiation, the percentage of the sac in the sac was P0.05, and the comparison group was compared with the control group after 14 days after the X-ray irradiation of the original cercariae. In the development of cysts, the uncinate process was disintegrated in the irradiated group. The normal structure disappeared after 7 days of.6MV- X - ray irradiation, and the capsule wall structure of the control group was complete and compact, the capsule wall of the irradiated group collapsed, the normal structure disappearance disappeared, the endoplasmic reticulum dilated, and the abnormal concentration of the dyed mass. EgTPX, EgHSP70, EgEPC1, Csapase-3, G after irradiation. The wet weight of the alveolar cysts in the high dose radiation therapy group was (1.82 + 0.74) g, (1.04 + 0.38) g and (0.76 + 0.33) g, respectively (50%, 72% and 82%, respectively), and the control group was (3.65 + 0.79) g (3.65 + 0.79) g in the control group, and the average wet weight of the alveolar echinococcus in the 4 group of rats was significantly different (P0.05). The results showed that the structure of the hydatid cercariae in the control group was basically normal, the stratum corneum, the germinal layer were clear, and there were different levels of the original joint in the sac. The treatment group had different degrees of change and destruction in the treatment group. The structure disorder, the stratum corneum, the germinal layer were generally denatured swelling or separation and shedding, the primary joint was rare, and the pathological changes of the cyst wall were different from those of the control group. The results of P0.05 and electron microscopy showed that the cuticle and the germinal layer of the cyst wall of the treatment group were damaged in varying degrees, of which the 60Gy radiation therapy group damaged the heaviest.4. The CT results showed that 4 sheep were exposed to the cystic wall calcification of the capsule, and the HE staining results showed that the cuticle and the structure of the germinal layer were different in the irradiated capsule. Degenerative swelling or thinning, partial separation, breakage, shedding, dissolving or disappearance of the nucleus of the germinal layer, and rare bursa and cercariae. The results of RNA fluorescence quantitative PCR detection of the irradiated capsule showed that the expression of EgTPX decreased with the increase of dose, P0.05 (P=0.04); the low dose group of EgEPC1 expression was obviously higher than the middle and high dose. Group, P0.05 (P=0.03); the expression of EgHSP70 expression decreased with the increase of dose, but there was no statistical significance, P0.05 (P=0.22). It showed that the activity of cyst was inhibited with the increase of radiation dose. The expression of Caspase-3 and Gadd45 gene of radiation related apoptotic genes decreased with the increase of dose. There was no obvious change in the living conditions, weight, blood routine, liver work and electrolyte (P0.05). Conclusion: 1) a stable E.g in vitro culture platform was established to provide a sufficient experimental material.2 for the in vitro experimental study of echinococcosis in vitro.) x ray did have a killing effect on the cultured primary sac and capsule in vitro, and 3) radiation destroyed the rat bubble. The structure of the cyst wall of the cercariae and the inhibition of the proliferation of the primary node, and the action intensity related to the dose related to the dose of.4) the general living condition of the sheep after radiotherapy, the blood routine and the liver function were not affected. It was proved that the radiation therapy was safe. From the CT imaging, pathology and molecular biology, the radiation therapy had a positive effect on the liver hydatid disease. The curative effect of.60Gy/20Gy/3f is effective and safe dose fractionation for hepatic echinococcosis.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R532.32

【相似文献】