多功能超声造影剂显像及治疗肿瘤转移淋巴结实验研究

发布时间：2018-01-21 07:53

本文关键词： 阿霉素超顺磁性氧化铁超声造影剂多功能多功能造影剂超声染料淋巴结巨噬细胞阿霉素超顺磁性超声造影剂多功能转移淋巴结　出处：《重庆医科大学》2013年博士论文　论文类型：学位论文

【摘要】：目的分别制备两种多功能超声造影剂：载染料苏丹黑B的聚乳酸羟基乙酸超声微泡造影剂（SB-PLGA）和载阿霉素的超顺磁性聚乳酸羟基乙酸超声微泡造影剂（DOX-SPLGA），并检测其一般特性。方法采用双乳化法和真空冷冻干燥法分别制备出SB-PLGA及不同铁浓度的DOX-SPLGA,并对其进行粒径，形态，包封率等性质检测；其中对载药微泡DOX-SPLGA还进行了体外释药实验观察，采用原子吸收分光光度计检测造影剂的铁含量；用振动样品磁强计及磁共振仪检测造影剂的磁学性质；体外超声和磁共振显像评价造影剂的显像效果。结果SB-PLGA外观呈蓬松的蓝色粉末状，溶于水后为均匀的深蓝色乳液，分散度好。扫描电镜观察微泡粒径1～2μm，其形态规则，呈球形，表面光滑，大小均匀。马尔文电位检测其Zeta电位为（20.80±1.60）mV，SB的包封率为（85.67±1.44）%，载药量为（3.43±0.06）%，分散度好。DOX-SPLGA外观呈黄褐色粉末，溶于水后为均匀的乳液，分散度较好。扫描电镜观察微泡呈球形，表面有孔，大小较均匀，透射电镜显示微泡为壳核结构，SPIO纳米颗粒分布于微泡的壳膜中。原子吸收光谱法得到7种不同铁浓度制得的DOX-SPLGA中的铁含量分别为（1.47±0.07）μg/ml（,2.83±0.08）μg/ml,（5.76±0.33）μg/ml（,14.77±0.32）μg/ml,（28.06±0.62）μg/ml，（56.71±0.44）μg/ml，（185.45±1.06）μg/ml，其相对应的DOX的包封率分别为（58.10±1.61）%（,57.73±3.45）%,（60.30±2.67）%,（57.53±0.80）%,（61.13±0.95）%,（56.07±1.55）%,（59.60±1.61）%；DOX的载药量分别为（5.81±0.16）%,（5.77±0.35）%,（6.03±0.27）%,（5.75±0.08）%,（6.11±0.10）%,（5.61±0.16）%,（5.96±0.16）%。造影剂的粒径为800~900nm，具有超顺磁性，其磁敏感效应与壳膜中装载的SPIO的含量有关。体外超声显像实验显示，DOX-SPLGA能够较普通PLGA造影剂产生更强的超声回声。体外磁共振显像实验显示，T2*信号随着造影剂内铁浓度的增高而降低。结论SB-PLGA造影剂为蓝色粉末，粒径均匀，分散好，为其之后既能外科手术前进行肿瘤转移淋巴结内超声显像，又能手术活检时指示定位淋巴结提供了基础。DOX-SPLGA造影剂具有形态规则,大小较均一，表面光滑有孔，分散均匀,超顺磁性等优点，在体外能够增强超声、磁共振显像，同时能载药辅助化疗，是一种有应用前景的多功能超声造影剂。目的探讨SB-PLGA造影剂的细胞毒性及被巨噬细胞吞噬的能力；探讨SB-PLGA造影剂对兔肿瘤转移淋巴结的超声显像和手术示踪效果，及其在淋巴结组织内的分布情况。方法体外培养小鼠单核巨噬细胞RAW264.7，加入同一浓度SB-PLGA造影剂孵育3h、24h和48h后，光镜下观察巨噬细胞对造影剂的吞噬情况，用MTT法检测造影剂对巨噬细胞增殖活性的影响。采用瘤块组织悬液种植法建立兔VX2乆窝肿瘤转移淋巴结模型，建模后14天用于肿瘤淋巴结超声显像。选取肿瘤转移淋巴结模型兔8只，共16个肿瘤淋巴结（实验组8个，对照组8个），实验组给予SB-PLGA造影剂，，对照组给予等量生理盐水，分别经足垫皮下间隙注射，行超声造影检查评价乆窝肿瘤转移淋巴结及第二站腹股沟淋巴结的增强效果；并分别于30min后行乆窝和腹股沟淋巴结清扫术，切除蓝染及未染色淋巴结，进行冰冻切片HE染色。结果光镜下观察，巨噬细胞对SB-PLGA造影剂的吞噬量随着造影剂与细胞孵育时间的增加而增加。MTT结果显示，SB-PLGA造影剂被巨噬细胞吞噬后对其增殖活性无明显影响。超声造影可明显增强兔乆窝肿瘤转移淋巴结显像，而同侧腹股沟淋巴结及注射生理盐水组显像不明显。乆窝淋巴结清扫术中可见，注射SB-PLGA造影剂组淋巴结染色效果好；而腹股沟淋巴结清扫术中均未见第二站淋巴结蓝染。淋巴结冰冻切片HE染色可见，SB-PLGA存在于淋巴窦内，并有大量进入巨噬细胞。结论SB-PLGA在体外能够被巨噬细胞吞噬，且对细胞增殖活性无明显影响。SB-PLGA能够被淋巴结内巨噬细胞吞噬，增强淋巴结超声显像，同时又能通过染色来指示定位淋巴结，是一种良好的多功能超声造影剂。目的探讨DOX-SPLGA造影剂对兔肿瘤转移淋巴结的超声和磁共振显像效果；探讨DOX-SPLGA造影剂联合低频超声促进药物释放对兔肿瘤转移淋巴结的治疗效果。方法新西兰大白兔42只，采用瘤块种植法于大腿外侧植入VX2肿瘤组织块悬液，建立兔乆窝肿瘤转移淋巴结模型。建模后第15天，取12只模型兔（24个肿瘤淋巴结），随机分为三组（每组8个肿瘤淋巴结），分别为DOX-SPLGA组（经足垫皮下注射DOX-SPLGA微泡）、PLGA组（经足垫皮下注射普通PLGA微泡）及对照组（经足垫皮下注射等量生理盐水），行超声和磁共振扫描，并应用图像分析软件评价造影效果。扫描结束后，取淋巴结组织进行HE染色和普鲁士蓝染色，以及透射电镜检查。建模后第15天，取30只模型兔（60个肿瘤淋巴结），随机分为6组（每组10个肿瘤淋巴结），包括空白对照组（C）、载药微泡（DOX-SPLGA）、单纯药物组（DOX）、单纯微泡+超声组（PLGA+US）、药物+超声组（DOX+US）、载药微泡+超声组（DOX-SPLGA+US）。治疗后取肿瘤淋巴结组织，用免疫组化法检测淋巴结组织PCNA、CD34及LYVE-1的表达；TUNEL法检测淋巴结内肿瘤细胞凋亡。结果在超声显像的造影模式和传统灰阶模式下，与对照组相比，经DOX-SPLGA与普通PLGA微泡造影后，乆窝淋巴结均可见回声增强；且DOX-SPLGA组比普通PLGA组回声更强。在磁共振显像中，DOX-SPLGA组表现出明显负性增强效果，普通PLGA组负性增强效果较弱，而对照组未见明显增强。普鲁士蓝染色和透射电镜结果均证实了DOX-SPLGA存在于淋巴结组织内。免疫组化结果显示，DOX-SPLGA+US组的凋亡指数（AI）显著高于其它各组（P0.05）；DOX-SPLGA+US组的肿瘤增殖受到明显抑制，其增殖指数明显低于其它各组（P0.05）。结论DOX-SPLGA造影剂能显著增强显像超声和磁共振两种模式下的兔肿瘤转移淋巴结，同时联合低频超声促进造影剂内的药物释放，达到治疗转移淋巴结的效果，为寻求一种有效、安全、集显像和治疗
[Abstract]:Objective To prepare two kinds of multi - functional ultrasound contrast agents : the ultrasonic microbubble contrast agent ( SB - PLGA ) of poly ( lactic acid glycolic acid ) and the super - paramagnetic polylactic acid hydroxyacetic acid ultrasonic microbubble contrast agent ( GSPLGA ) loaded with adriamycin . Methods SB - PLGA and different iron concentrations were prepared by double emulsion method and vacuum freeze - drying method . The particle size , morphology , encapsulation efficiency and so on were tested . The magnetic properties of contrast agent were determined by using atomic absorption spectrophotometer . The magnetic properties of contrast agent were detected by vibrating sample magnetometer and magnetic resonance instrument , and the imaging effect of contrast agent was evaluated in vitro and magnetic resonance imaging . Results SB - PLGA appeared in a fluffy blue powder form , which was dissolved in water to form a homogeneous deep blue emulsion with good dispersion . The microvesicles were observed by scanning electron microscope . The entrapment efficiency of the microvesicles was ( 20.80 卤 1.60 ) mV , ( 5.76 卤 0 . 33 ) 渭g / ml , ( 56.71 卤 0 . 44 ) 渭g / ml , ( 57.53 卤 0 . 80 ) % , ( 61.13 卤 0.95 ) % , ( 56.07 卤 1.55 ) % , ( 59.60 卤 1 . 61 ) % , respectively . The particle size of the contrast medium was 800 - 900nm , ( 5.61 卤 0.16 ) % , ( 5.96 卤 0.16 ) % , ( 5.61 卤 0.16 ) % , ( 5.96 卤 0.16 ) % . Conclusion The SB - PLGA contrast agent is a blue powder with uniform particle size and good dispersion . It has the advantages of regular morphology , uniform size , smooth surface , uniform distribution , super paramagnetism and so on . Objective To investigate the cytotoxicity of SB - PLGA contrast agent and the ability of macrophages to phagocytize the tumor , and to investigate the ultrasound imaging and surgical tracing effect of SB - PLGA contrast agent on the metastatic lymph node of rabbit tumor and its distribution in lymph node tissue . Methods Mouse mononuclear phagocytes RAW264.7 were cultured in vitro and incubated for 3 h , 24 h and 48 h with the same concentration of SB - PLGA contrast medium . The effect of contrast agent on the proliferation of macrophages was observed by MTT assay . Results The results showed that SB - PLGA contrast medium had no significant effect on the proliferation of SB - PLGA contrast medium . The results showed that SB - PLGA contrast media had no obvious effect on the proliferation activity of lymph node and injected physiological saline group . Conclusion SB - PLGA can be phagocytosed by macrophages in vitro and has no significant effect on cell proliferation activity . SB - PLGA can be phagocytosed by macrophages in lymph nodes , enhanced lymph node ultrasound imaging , and can be used to indicate the localization of lymph nodes by staining , which is a good multi - functional ultrasound contrast agent . Objective To investigate the ultrasound and magnetic resonance imaging of tumor metastasis lymph nodes in rabbits by using the contrast medium - SPLGA contrast agent , and to investigate the effect of the combination of the combined low - frequency ultrasound in the treatment of tumor metastasis lymph nodes in rabbits . Methods 42 model rabbits ( 24 tumor nodes ) were randomly divided into three groups ( 10 tumor nodes in each group ) , including blank control group ( C ) , drug - loaded microvesicle ( PLGA + US ) , drug + ultrasound group , drug + ultrasound group and drug - carrying microbubble + ultrasound group . The expression of PCNA , CD34 and LYVE - 1 in lymph nodes was detected by immunohistochemistry . Results Compared with the control group , the echogenic enhancement was observed in the lymph nodes of the common PLGA group compared with the control group . In the magnetic resonance imaging , there was a significant negative enhancement effect on the lymph nodes of the common PLGA group . The results of both blue staining and transmission electron microscopy showed that the apoptosis index ( AI ) of the common PLGA group was significantly higher than that in the other groups ( P0.05 ) . Conclusion The contrast agent can significantly enhance the metastatic lymph nodes of rabbit tumors in both the imaging and magnetic resonance modes . Meanwhile , the combination of low frequency ultrasound can promote the drug release in contrast agent , so as to achieve the effect of treating metastatic lymph node , and to seek an effective , safe , set imaging and treatment .

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R730.55

【参考文献】