超声引导下建立兔VX2肢体软组织肿瘤模型及应用VMAT-SIB技术制作外科边界的实验研究

发布时间：2018-06-25 02:52

本文选题：兔VX2肢体软组织肿瘤模型 + 容积弧形调强放疗　；参考：《新疆医科大学》2015年博士论文

【摘要】：目的:1)通过超声引导建立兔VX2肢体软组织肿瘤模型,为后期的干预研究和影像学评价奠定基础;2)在肿瘤模型上实施新辅助放疗,应用容积弧形调强放疗-同步推量(VMAT-SIB)技术在肿瘤毗邻骨骼的区域提高照射剂量,局部产生放射性损伤,制作一个坏死带定义为外科边界,评价制作外科边界的可行性、有效性;3)在肿瘤模型上,实施大分割放疗制作外科边界,评价其安全性。方法:1)选用雄性新西兰兔30只,随机分为对照组6只(在超声引导下注射生理盐水0.5ml),模型组24只(在超声引导下注射肿瘤组织混悬液0.5ml)。两组应用磁共振弥散成像(MRDWI)和三维超声观察肿瘤的形态、信号特点及体积变化。对照组在采集完影像后处死动物观察组织学变化。模型组于第14天、21天、28天随机分批处死6只动物观察组织学变化。剩下6只动物不处死,观察带瘤生存时间;2)选用30只肿瘤模型兔,随机分为对照组:10只;调强放疗(IMRT)组:10只;VMAT-SIB组:10只。三组在放疗后一周进行影像学采集:MRDWI、三维能量多普勒超声血管成像(3D-PDA)。之后,处死动物,观察组织学变化以及采用免疫组化检查肿瘤组织的增殖、凋亡、血管密变化;3)选用40只肿瘤模型兔,随机分为对照组:10只;VMAT-SIB3Gy组:10只;VMAT-SIB4Gy组:10只;VMAT-SIB5Gy组:10只。四组在放疗后,参照CTCAE分级观察皮肤的大体情况。采用HE染色观察皮肤和骨组织的形态学改变。采用透射电镜观察骨细胞超微结构改变。采用荧光原位杂交(FISH)测定皮肤组织PDGF-B、EGF的表达情况。采用实时荧光定量PCR测定皮肤组织VEGF、bFGF的基因表达量。结果:1)三维超声和MRDWI证实,24只实验动物的种植成瘤率达100%,24只肿瘤模型兔全部存活。三维超声结果,对照组在注射部位未见异常改变。模型组随着时间的增加肿瘤体积逐渐增大,与对照组比较其差异具有统计学意义(P0.05)。MRDWI显示:对照组未见明显的异常信号。模型组在左侧后肢可见肿瘤影像,肿瘤组织在DWI图呈高信号;在ADC图呈低信号。组织学结果,对照组:HE可见正常的横纹肌及支气管肺泡组织。模型组:第14天取材,HE可见生长旺盛的瘤细胞呈不规则致密排列,侵犯横纹肌。第21天取材,HE可见肿瘤区与片状坏死区边界不清,肿瘤生长区内细胞较密集。第28天取材,HE可见部分的肿瘤细胞核固缩、细胞核碎裂。第60天取材,HE可见肿瘤边缘仍有肿瘤细胞,肿瘤实质可见坏死细胞,伴有炎细胞浸润及纤维组织增生。解剖胸腔,见两肺弥漫性转移结节,部分小结节相互融合,HE可见肿瘤细胞侵犯支气管及肺泡;2)在放疗过程中,IMRT组有2只肿瘤模型兔死亡,一只动物因麻醉过量致死,一只动物由于肿瘤破溃至皮肤给予安乐死。三组之间的感兴趣区(毗邻骨骼的肿瘤组织)在DWI图和ADC图均表现出显著的不同。对照组在DWI图呈高信号;在ADC图呈明显低信号。IMRT组在DWI图呈欠均匀低信号;在ADC图呈高信号。VMAT组在DWI图呈低信号;在ADC图呈高信号。3D-PDA观察:三组之间的VI、FI及VFI均表现出显著的不同,IMRT组和VMAT组明显低于对照组。组织学结果,对照组:HE可见肿瘤细胞侵犯横纹肌,正常的细胞结构受到损害。IMRT组:HE可见肿瘤实质及肿瘤边界组织大片的坏死细胞,伴有炎细胞浸润及纤维组织增生。VMAT组在肿瘤毗邻骨骼的区域可见一坏死带形成,镜下见细胞坏死明显,纤维组织增生,周围有炎细胞浸润。放疗后,IMRT组和VMAT组的增殖活性降低、血管密度降低、凋亡增加;3)对照组、3Gy组和4Gy组的动物在放疗期间和放疗后,局部皮肤未见异常。5Gy组有8只动物在放疗期间,局部皮肤出现了轻度红斑和干燥性脱屑。对照组、3Gy组和4Gy组在镜下可见正常的皮肤组织,皮肤表皮各层结构完整。5Gy组可见表皮有大量的角化物质,上皮下有炎细胞浸润。对照组、3Gy组和4Gy组在镜下可见骨细胞形态正常。5Gy组在镜下可见骨细胞排列松散,细胞表现出明显的萎缩。透射电镜结果显示,对照组、3Gy组和4Gy组的骨细胞形态正常。5Gy组可见粗面内质网扩张,呈囊泡状样改变。FISH:EGF和PDGF-B的mRNA结果:对照组与3Gy组、4Gy组、5Gy组比较,其差异具有统计学意义(P0.05)。实时荧光定量PCR:VEGF mRNA结果:对照组与3Gy组、4Gy组比较,其差异具有统计学意义(P0.05),但与5Gy组比较未见明显差异(P0.05)。bFGF mRNA结果:对照组与3Gy组、4Gy组比较其差异具有统计学意义(P0.05),但与5Gy组比较未见明显差异(P0.05)。结论:1)通过超声引导注射肿瘤组织悬液,成瘤率高,肿瘤生长范围较局限。影像学和组织学结果提示:后期的干预研究应在种植肿瘤后的第14天左右进行,以降低肿瘤内部液化坏死对实验结果的影响;2)应用VMAT-SIB技术在肿瘤模型上实施新辅助放疗具有一定的可行性。MRDWI、3D-PDA、组织学证实,在肿瘤毗邻骨骼的区域制作的坏死带,能够有效的定义为外科边界;3)在肿瘤模型上实施大剂量分割放疗,结果提示VMAT-SIB 3Gy组和VMAT-SIB4Gy组的动物能够耐受早期的局部毒性反应,是一个相对较安全的剂量分割方案。
[Abstract]:Objective: 1) the tumor model of VX2 limb soft tissue in rabbits was established by ultrasonic guidance to lay the foundation for the later intervention study and imaging evaluation; 2) to implement new adjuvant radiotherapy on the tumor model, and to improve the radiation dose in the region adjacent to the adjacent bone with volume arc intensity modulated radiotherapy synchronous push (VMAT-SIB) technology, and to produce radioactivity damage locally. A necrotic band was defined as the surgical boundary, the feasibility and effectiveness of making the surgical boundary was evaluated. 3) in the tumor model, the surgical boundary was made by large fractionation radiotherapy, and the safety was evaluated. Method: 30 male New Zealand rabbits were selected to be randomly divided into 6 control groups (0.5ml under ultrasound guidance), and 24 in the model group. Ultrasound guided injection of the tumor tissue suspension 0.5ml. The two groups were treated with magnetic resonance diffusion imaging (MRDWI) and three-dimensional ultrasound to observe the tumor morphology, signal characteristics and volume changes. The control group died of animal observation after the collection of images. The model group was executed at fourteenth days, 21 days, 28 days and 6 animals were killed at random to observe histological changes. The remaining 6 animals were not killed, and the survival time of the tumor was observed; 2) 30 rabbit models were selected and randomly divided into the control group: 10; the intensity modulated radiotherapy (IMRT) group: 10; the VMAT-SIB group: 10. The three groups were collected for one week after radiotherapy: MRDWI, and three-dimensional energy Doppler ultrasound angiography (3D-PDA). Then, the animals were executed and histological changes were observed. And using immunohistochemistry to examine the proliferation, apoptosis, vascular density change of tumor tissue; 3) selected 40 tumor model rabbits, randomly divided into control group: 10 rats, group VMAT-SIB3Gy, 10; group VMAT-SIB4Gy, 10; group VMAT-SIB5Gy: 10. After radiotherapy, the general condition of skin was observed by CTCAE classification. HE staining was used to observe the skin and bone tissue. Morphological changes. The ultrastructural changes of the bone cells were observed by transmission electron microscopy. The expression of PDGF-B and EGF in skin tissue was measured by fluorescence in situ hybridization (FISH). The expression of VEGF and bFGF in skin tissue was measured by real-time fluorescence quantitative PCR. Results: 1) three dimensional ultrasound and MRDWI confirmed that the growth rate of 24 experimental animals was 100%, 24 The tumor model rabbits were all alive. The results of three-dimensional ultrasound showed no abnormal change at the injection site. The tumor volume in the model group increased gradually with the increase of time. Compared with the control group, the difference was statistically significant (P0.05).MRDWI showed that there was no obvious abnormal signal in the control group. The model group showed tumor image and tumor on the left hind limb. Tissue showed high signal on DWI map, low signal on ADC map. Histological results, control group: normal rhabdomyoma and bronchioloalveolar tissue were seen in HE. Model group: fourteenth days of extraction, HE visible growth of tumor cells were irregular and dense arrangement, invasion of rhabdomyoma. Twenty-first days, HE visible tumor area and flaky necrotic area boundary was unclear, tumor birth The cells in the long area were denser. After twenty-eighth days, the nuclei of the tumor were recovered and the nuclei were broken. The tumor cells were found on the edge of the tumor on the sixtieth day, and the tumor cells were still found on the edge of the tumor. The tumor was visible with necrotic cells, with inflammatory cells infiltration and fibrous tissue proliferation. The thoracic cavity was dissected, two pulmonary diffuse nodules were seen, and some small nodules fused, HE It can be seen that tumor cells invade bronchi and alveoli; 2) during the course of radiotherapy, 2 tumor model rabbits in group IMRT died, one animal died of overdose of anesthesia, and one animal broke into the skin for euthanasia due to the collapse of the tumor to the skin. The region of interest between the three groups (adjacent to the bone tumor group) showed significant differences between the DWI map and the ADC map. The group in the DWI map showed high signal, the ADC graph showed the low signal.IMRT group in the DWI diagram and the low signal in the DWI diagram; the high signal group in the ADC diagram showed the low signal in the DWI diagram; the VI, FI and VFI were significantly different between the three groups, and the group and the group were obviously lower than the control group. Histology results, control group: control group: It can be seen that the tumor cells infringe the rhabdomyosus muscle, and the normal cell structure is damaged in the.IMRT group: HE can be seen in the tumor parenchyma and the necrotic cells of the tumor boundary tissue, with the infiltration of inflammatory cells and the proliferation of fibrous tissue in the.VMAT group, a necrotic zone is visible in the area adjacent to the adjacent bone, and the necrosis of the cells, the proliferation of fibrous tissue, and the surrounding area are observed under the microscope. After radiotherapy, the proliferation activity of IMRT group and VMAT group decreased, blood vessel density decreased, apoptosis increased; 3) control group, 8 animals in group 3Gy and 4Gy group had no abnormal.5Gy group during radiotherapy and radiotherapy, and the local skin appeared mild erythema and dryness desquamation during radiotherapy. The control group, 3Gy group and 4Gy were in the control group. The normal skin tissue was seen under the microscope, and the epidermis of all layers of the skin had a large number of keratinized substances and infiltration of inflammatory cells under the epithelium. In the control group, group 3Gy and group 4Gy showed that the bone cell morphology in the normal.5Gy group was visible under the microscope, and the cells were loosely arranged and the cells showed obvious atrophy. The transmission electron microscope showed that the transmission electron microscope showed that the cells were obviously atrophied under the microscope. In the control group, the 3Gy group and the 4Gy group, the normal.5Gy group showed the dilation of the rough endoplasmic reticulum and the mRNA results of the vesicular like change of.FISH:EGF and PDGF-B. The control group was statistically significant compared with the 3Gy group, 4Gy group and 5Gy group (P0.05). The real time fluorescence quantitative PCR:VEGF mRNA results were compared with those in the control group. Statistical significance (P0.05), but compared with the 5Gy group, there was no significant difference (P0.05).BFGF mRNA results: in the control group and the 3Gy group, the difference in the 4Gy group was statistically significant (P0.05), but there was no significant difference between the group and the 5Gy group (P0.05). Conclusion: 1) the tumor growth rate was high and the growth range of the tumor was limited by ultrasound guided injection of tumor tissue suspension. The results of image and histology suggest that the later intervention study should be carried out about fourteenth days after the tumor is planted to reduce the effect of internal liquefaction and necrosis of the tumor to the experimental results. 2) the application of VMAT-SIB technology to the neoadjuvant radiotherapy on the tumor model has a certain feasibility,.MRDWI, 3D-PDA, histologically confirmed, in the region adjacent to the bone. The necrotic zone made by the domain can be effectively defined as the surgical boundary; 3) the large dose fractionation radiotherapy on the tumor model indicates that the animals in the VMAT-SIB 3Gy group and the VMAT-SIB4Gy group can tolerate the early local toxicity, which is a relatively safe dose division scheme.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R738.6;R-332

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