本文選題：放射治療 + 棘球蚴��； 參考：《新疆醫(yī)科大學》2016年博士論文

【摘要】：研究目的:棘球蚴病俗稱包蟲病,流調表明我國有38萬包蟲病患者,現(xiàn)有手術資源很難完成所有治療,30余年的藥物治療效果亦不盡人意,需另辟治療途徑。本研究旨在探明X線殺傷包蟲的有效性和安全性,做為新技術的開發(fā)為臨床應用提供證據(jù)。1)建立E.g體外成囊培養(yǎng)系統(tǒng),為放射治療棘球蚴的體外實驗研究提供實驗材料。2)明確X線照射體外培養(yǎng)的原頭節(jié)和包囊的殺傷作用。3)探討放射治療對大鼠繼發(fā)泡球蚴(E.m)的療效和安全性。4)探討放射治療對自然感染羊細粒棘球蚴(E.g)的療效和安全性。研究方法:1)從屠宰場自然感染E.g的綿羊肝臟采集新鮮原頭蚴(PSC),經(jīng)1%的胃蛋白酶消化后檢測蟲體活性并計數(shù),于37℃、5%C02條件下進行體外培養(yǎng),不同時間點(第1天、第7天、第15天、第60天)進行形態(tài)學觀察,超微結構觀察,計算包囊的成囊率。2)將原頭蚴分裝于7組(每組3瓶),每瓶約2000個原頭蚴,對7組原頭蚴分別進行0Gy、20Gy、40Gy、60Gy、80Gy、100Gy、120Gy劑量照射,機架角180度,射野大小10×10cm,SSD=100cm,劑量率300cGy/min,將包囊分裝于4組(每組3瓶),每瓶約200個包囊,1組為對照組,對其他3組包囊分別進行30Gy/3f、45Gy/3f、60Gy/3f劑量照射,照射后電鏡及光鏡下觀察包囊的病理變化判斷療效,觀察半數(shù)致死量和時間,qRT-PCR檢測受X線照射前后關鍵基因表達的差異。3)建立雌性S-D大鼠繼發(fā)性泡球蚴病動物模型,隨機分為4組,每組15只,分別為低、中、高劑量放射治療組和對照組,對照組不予任何處理,治療組給予6-MeV線照射,總劑量分別為30Gy、45Gy和60Gy,照射3次,每次間隔2天,放療結束1個月后檢測各組大鼠泡球蚴囊濕重、抑囊率,并對泡球蚴組織進行病理組織學和超微結構觀察。4)從新疆牧區(qū)用B超篩選53只自然感染肝細粒棘球蚴病的羊,經(jīng)CT驗證后將感染羊隨機抽取20只分配到四個實驗組中,高劑量組(60Gy)5只、中劑量組(45Gy)5只、低劑量組(30Gy)5只、對照組(0Gy)5只。每只羊麻醉好后采用高分子低溫水解塑料熱壓成型體位固定技術固定,GE大孔徑螺旋CT掃描,放療醫(yī)師勾畫預照射包囊部位的靶區(qū),物理師做照射計劃,核準位置后實施圖像引導下的精確放療。照射組共照射3次,隔兩日一次,一周內完成照射,達到預照射總量。放療后三月復查CT,對比放療前后目標病灶的變化。處死各組綿羊,取出放療區(qū)肝內細粒棘球蚴囊,用于光鏡和電鏡下觀察,qRT-PCR檢測受X線照射前后關鍵基因表達的差異,比較宿主和包囊的免疫反應。結果:1)E.g原頭蚴在體外培養(yǎng)15d時微囊逐漸形成,60d時包囊外圍形成透明角質層結構,超微結構顯示E.g囊泡外部有較厚的無細胞薄片層,并可見生發(fā)囊及囊內PSC產(chǎn)生。2)不同劑量X線照射后14天后原頭節(jié)的成囊率P0.05,比較有統(tǒng)計學意義。6MV-X線照射原頭蚴后14天后,對照組成囊發(fā)育中,照射組發(fā)生了鉤突崩解,正常結構消失。6MV-X線照射已成囊的包囊7天后,對照組包囊囊壁結構完整,致密,照射組囊壁萎縮塌陷,正常結構紊亂消失,內質網(wǎng)擴張,部分為異常濃縮的染色團塊。照射后7天,EgTPX、EgHSP70、EgEPC1、Csapase-3、Gadd45的隨著照射劑量的升高表達增強。3)低、中、高劑量放射治療組泡球蚴囊濕重分別為(1.82±0.74)g、(1.04±0.38)g和(0.76±0.33)g,抑囊率分別為50%、72%和82%,對照組泡球蚴囊濕重為(3.65±0.79)g,4組大鼠泡球蚴的平均濕重有顯著性差異(P0.05),光鏡結果顯示對照組泡球蚴結構基本正常,角質層、生發(fā)層清晰,育囊內有多少不等的原頭節(jié),治療組泡球蚴呈不同程度的改變和破壞,結構失常,角質層、生發(fā)層普遍變性腫脹或分離脫落,原頭節(jié)少見,囊壁病理改變程度與對照組比較差異均有顯著性(P0.05),電鏡結果顯示治療組泡球蚴囊壁角質層和生發(fā)層均有不同程度的損傷,其中60Gy放射治療組損害最重。4)CT結果顯示高劑量組中有4只羊的受照射包囊囊壁鈣化,HE染色結果顯示受照射包囊的角質層及生發(fā)層結構遭到不同程度破壞,普遍變性腫脹或變薄,部分分離、斷裂、脫落,生發(fā)層細胞核溶解或消失,很少見育囊及原頭蚴。提取受照射包囊的RNA行熒光定量PCR檢測結果顯示:EgTPX表達隨著劑量的升高表達降低,P0.05(P=0.04);EgEPC1表達低劑量組明顯高于中高劑量組,P0.05(P=0.03);EgHSP70表達隨著劑量的升高表達有降低的趨勢,但無統(tǒng)計學意義,P0.05(P=0.22);說明隨著放療照射劑量的增加,包囊的活性被抑制。輻射相關的凋亡基因caspase-3和Gadd45基因的表達隨著劑量的升高表達降低。放療前后感染羊的一般生活狀況、體重、血常規(guī)、肝功、電解質無明顯變化(P0.05)。結論:1)建立了穩(wěn)定的E.g體外成囊培養(yǎng)平臺,為后續(xù)放射線抗棘球蚴的體外實驗研究提供了充足的實驗材料。2)X線對體外培養(yǎng)的原頭節(jié)和包囊確有殺傷作用,3)放射線有破壞大鼠泡球蚴囊壁結構和抑制原頭節(jié)增殖的作用,且作用強度與劑量有關。4)放射治療后患羊的一般生活狀況、血常規(guī)和肝功未受到影響,證明放射治療羊肝包蟲病是安全的。從CT影像學、病理學、分子生物學不同角度證實放射治療對肝包蟲病有一定療效。60Gy/20Gy/3f是對肝包蟲病有效且安全的劑量分割方式。
[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.

【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】：2016
【分類號】：R532.32

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