【作者】 逄鑫；

【导师】 邹利光；

【作者基本信息】 第三军医大学 ， 影像医学与核医学， 2010， 硕士

【摘要】 目的(1)观察大鼠乳腺癌生长分化病理特点及淋巴管生成;(2)合成靶向podoplanin的MRI金磁微粒对比剂,体外观察该纳米粒对比剂理化特性;(3)Podoplanin分子靶向MRI活体显像,评价乳腺癌淋巴管生成。方法(1)利用金磁微粒(GoldMag-Coreshell, GoldMag)兼有磁性粒子的超顺磁性及胶体金偶联生物分子的性能,螯合抗podoplanin抗体合成靶向podoplanin的MRI金磁微粒对比剂(PodAb-GoldMag),螯合非特异性抗体IgG合成含IgG的MRI纳米粒(IgG-GoldMag)为对照,测量螯合前后两种抗体在280nm处吸光值,计算抗体在GoldMag表面的偶联效率;ELISA双抗体夹心法测定纳米粒的免疫活性。(2)用激光粒度仪分别对GoldMag核心微粒超小超顺磁性氧化铁(USPIO)、GoldMag、IgG-GoldMag和PodoAb-GoldMag进行Zeta电位测定;透射电子显微镜观察USPIO、GoldMag和PodoAb-GoldMag纳米粒形态变化并测定其粒径变化。(3)以磷酸盐缓冲液(PBS)为稀释剂将USPIO和GoldMag分别稀释为0.5～1000μg/ml的47个不同的浓度,以PBS液为对照,共获得48个浓度,行FSE T1WI、FSE T2WI和GRE T2*WI磁共振成像。观察不同浓度USPIO和GoldMag在3种序列中的信号强度变化规律,计算信号强度变化比率,绘制浓度-信号强度曲线图。(4)Wistar雌性大鼠80只,二甲基苯蒽(DMBA)灌胃诱发乳腺肿瘤,75只大鼠成功诱发乳腺肿块,随机分为A、B、C、D 4组,A组(PodoAb-GoldMag组)30只大鼠,B组(IgG- GoldMag组)、C组(GoldMag组)和D组(Gd-DTPA组)各15只大鼠。用1.5 T磁共振成像系统分别行FSE T1WI、T2WI、压脂序列、弥散加权成像,A、B、C组分别于注射对比剂前、注射对比剂后即刻、5 min、15 min、30 min、1 h和2 h行乳腺肿块GRE T2*WI增强扫描,D组于相同的时相点行FSE T1WI增强扫描,测量信号强度,绘制动态增强扫描时间-信号强度曲线。(5)扫描完毕后,灌注法固定标本,同一乳腺肿块分别于中央区和周围区取材,HE染色和podoplanin相关抗原免疫组化染色、免疫荧光染色以及普鲁士蓝铁染色,观察乳腺肿瘤病理类型和淋巴管生成情况,同一肿瘤分别观察中央区和周围区2个不同部位淋巴管生成情况。(6)乳腺肿瘤HE染色、免疫组化染色和LVD检查结果与MRI图像对照分析,分析乳腺肿瘤MRI表现的病理学基础,探讨podoplanin分子靶向MRI活体显像评价乳腺癌淋巴管生成的效果。结果(1)成功螯合抗podoplanin抗体与GoldMag,合成靶向podoplanin的PodAb- GoldMag,抗podoplanin抗体与GoldMag的平均结合率为64.81%,抗podoplanin抗体的免疫活性为43%。(2)USPIO的Zeta电位为12.0±18.9 mv,粒径分布范围15~30 nm;GoldMag的Zeta电位为25.2±95.2 mv,粒径分布范围40~80 nm;IgG-GoldMag的Zeta电位为18.2±1.6 mv,PodAb-GoldMag的Zeta电位为25.5±20.8 mv,粒径分布范围80~100 nm。(3)GoldMag与USPIO的MRI信号变化规律基本一致,不同成像序列信号强度比较,随浓度增加,FSE T1WI信号强度呈升高趋势,FSE T2WI信号强度略有下降,GRE T2*WI信号强度明显降低。两种对比剂信号强度比较,在FSE T1WI,浓度高于30μg/ml时信号强度有显著性差异(P <0.05),在FSE T2WI,浓度为40～90μg/ml时信号强度有显著性差异(P <0.05),在GRE T2*WI,两种对比剂信号强度无显著性差异(P >0.05)。(4)75只大鼠成功诱发乳腺肿块,68只大鼠诱发1个乳腺肿块,7只大鼠诱发2个乳腺肿块。75只大鼠中,乳腺增生性疾病21只,炎性肉芽肿2只,乳腺癌52只,乳腺肿块诱发成功率为93.8%,乳腺癌诱发成功率为69.3%。(5)在MRI平扫T1WI,乳腺良、恶性病变均表现为低信号或等信号;在T2WI,乳腺良性病变表现为高信号和等信号为主的混杂信号,乳腺恶性肿瘤中,42只表现为高信号和等信号为主的混杂信号,其余10只表现为低信号为主的混杂信号;在压脂序列T2WI,乳腺脂肪高信号被抑制,良、恶性病变均表现为高信号。(6)GoldMag靶向增强扫描,A、B、C 3组乳腺癌信号强度均在注射对比剂即刻显著下降(P<0.01),并随时间推移逐渐回升,注射后1 h信号强度不再变化,此时A组信号强度较注射前低,其差异有显著性(P<0.05),B、C两组信号强度基本回复到注射前水平,差异无显著性(P>0.05)。不同组织类型乳腺癌信号强度在注射对比剂前后无显著性差异(P>0.05),不同分化程度乳腺癌信号强度在注射对比剂即刻至5min内差异有显著性(P<0.05),其它时间点无显著差异。(7)LVD随乳腺癌分化程度的降低而显著增高(P<0.05),LVD与A组乳腺肿瘤信号降低程度呈显著负相关(r =-0.814,P = 0.000),与B、C两组乳腺癌信号强度降低程度无显著相关性(P>0.05),与D组乳腺肿瘤信号强度增高程度呈显著正相关(r = 0.972,P = 0.000)。结论(1) GoldMag作为一种核壳结构的超顺磁性复合微粒,其核心为USPIO,USPIO与GoldMag MRI信号特点变化相似,随浓度增加,FSE T1WI信号强度呈升高趋势,FSE T2WI信号强度略有下降,GRE T2*WI信号强度明显降低。GRE T2*WI为USPIO和GoldMag的最佳MRI成像序列。(2)成功螯合靶向PodAb-GoldMag纳米粒对比剂,抗podoplanin抗体与GoldMag的平均结合率为64.81%,抗podoplanin抗体的免疫活性为43%,PodAb-GoldMag的Zeta电位为25.5±20.8 mv,粒径分布范围80~100 nm。(3)大鼠诱发性乳腺癌MRI平扫T1WI表现为低信号或等信号;T2WI多数表现为高信号和等信号为主的混杂信号;压脂序列T2WI病变均表现为高信号,与人乳腺癌MRI表现相似。(4)乳腺增生性疾病仅可见少许新生淋巴管和少量较大、较成熟淋巴管。乳腺癌新生淋巴管多位于肿瘤边缘部位癌巢周围的间质中,LVD随乳腺癌分化程度的降低而升高。(5) PodAb-GoldMag增强扫描乳腺癌周边区信号强度降低较中央区明显;不同分化程度和不同组织类型的乳腺癌,PodAb-GoldMag增强扫描信号强度变化基本相似。(6) PodAb-GoldMag靶向增强MRI与肿瘤微淋巴管密度相关,根据大鼠乳腺肿瘤PodAb-GoldMag增强扫描,可初步鉴别其淋巴管生成情况,可活体评价乳腺癌淋巴管生成。更多还原

【Abstract】 Objective(1) To observe pathological features and lymphangiogenesis of rat breast cancer growth and differentiation; (2) synthesis podoplanin targeted MRI (magnetic resonance imaging) contrast agent, investigate the physical and chemical characteristics of the nano-scale contrast agent in vitro; (3) podoplanin targeted MR imaging in vivo to evaluatebreast cancer lymphangiogenesis. Methods(1) Goldmag was applied to synthesis molecular probe by its superparamagnetic characteristic and the feature of binding biomolecule by colloidal gold. Goldmag was used to bind with anti-podoplanin antibody to synthesis podoplanin targeted MRI contrast agent (PodAb-GoldMag), and bind with non-specific IgG to synthesis IgG-GoldMag as contrast. Measured before and after chelation of two antibodies absorbance at 280nm, and calculated the surface of the antibody coupling efficiency in GoldMag. ELISA double antibody sandwich method was performed to detect the immunoreactivity of molecular probe. (2) Laser particle size analyzer was used to detect the Zeta electric potential of ultra-small superparamagnetic iron oxide (USPIO), GoldMag, IgG-GoldMag, PodoAb- GoldMag. Transmission electron microscopy (TEM) was applied to observe the size and morphology of USPIO, GoldMag and PodoAb-GoldMag. (3) USPIO and GoldMag was diluted to different concentration (0.5～1000μg/ml) by PBS. And PBS was taken as contrast, so totally got of 48 concentrations. Each sample was performed FSE T1WI, FSE T2WI and GRE T2*WI MR imaging to investigate signal intensity variation of different concentration of USPIO and GoldMag. And calculated the signal intensity ratio, rendered concentration-intensity curve. (4) 80 Wister female rats were administrated intragastrically with DMBA to induce breast tumor. 75 rats were successfully induced breast tumor, and randomly divided into 4 groups, included A (PodoAb-GoldMag, 30 rats), B (IgG-GoldMag, 15 rats), C (GoldMag, 15 rats) and D (Gd-DTPA, 15 rats). 1.5 T MR imaging system was applied to perform FSE T1WI, FSE T2WI, pressure grease sequence and diffusion-weighted imaging. Group A, B and C was taken GRE T2*WI enhanced scanning the time before and after (0, 5, 15, 30min, 1h and 2h) injection of contrast agent. Group D was taken FSE T1WI enhanced scanning in the same time points. Detected signal intensity and rendered dynamic enhanced scan time-signal intensity curve. (5) After MRI examination and perfusion-fixation procedure, the same tissue of breast tumor was drawn in the central and around area. Breast tumor was performed HE stain and podoplanin related antigen immunohistochemisty, immunofluorescence and prussian blue iron stain to observe the pathological type and lymphangiogenesis of breast tumors. (6) HE stain, immunohistochemisty stain, LVD results and MRI images were contrast to analysis pathological basic of breast tumor MRI perform and investigate the value of podoplanin targeted MRI evaluation of breast cancer lymphangiogenesis.Results(1) Anti-podoplanin antibody was successfully bound with GoldMag to synthesis PodAb-GoldMag, and the mean binding rate was 64.81%, immunoreactivity of PodAb-GoldMag was 43% of original. (2) The Zeta electric potential of USPIO, GoldMag, IgG-GoldMag and PodAb-GoldMag was 12.0±18.9mv, 25.2±95.2mv, 18.2±1.6mv, 25.5±20.8 mv respectively. And the particle size distribution of USPIO, GoldMag and PodAb-GoldMag was 15~30 nm, 40~80 nm, 80~100 nm respectively. (3) MRI signal intensity variation of USPIO and GoldMag was similar. As the concentration increased, FSE T1WI signal intensity stepped-up, FSE T2WI signal intensity dropped a little, and GRE T2*WI signal intensity dropped quickly. Took two contrast agent signal intensity to compare, the signal intensity had significant differences under FSE T1WI when the concentration above 30μg/ml (P<0.05), the signal intensity had significant differences under FSE T2WI when the concentration between 40~90μg/ml (P<0.05), the signal intensity was similar under GRE T2*WI (P>0.05). (4) 75 rats breast tumor were successfully induced, 21 were breast proliferative disease, 2 were inflammatory granuloma, and 52 were breast cancer. The total achievement ratio of induced breast lump was 93.8%, and the achievement ratio of induced breast cancer was 69.3%. (5) Breast benign and malignant disease showed hypointensity or isointensity under T1WI; benign mainly showed hyperintensity and isointensity under T2WI, 42 malignant showed hyperintensity and isointensity and 10 malignant showed hypointensity under T2WI; benign and malignant both showed hyperintensity under pressure grease sequence. (6) After injection of PodAb-GoldMag, signal intensity of group A, B and C breast cancer dropped down immediately (P<0.01), and gradually recovered over time. 1h after injection, the signal strength was stable, then signal intensity of group A was lower than before injection, the difference was significant (P <0.05); signal intensity of group B, C was back to levels before injection, no significant difference (P> 0.05). Signal intensity in different histological types of breast cancer before and after contrast agent injection was no significant difference (P> 0.05), the signal intensity of different degree of differentiation of breast cancer in the immediate injection of contrast agent to within 5min significant difference (P <0.05), other time points no significant difference. (7) LVD with the decrease of the degree of differentiation of breast cancer was significantly higher (P <0.05), LVD and group A signal change were significantly negatively correlated (r =-0.814,P = 0.000), and no significant difference in correlation with group B and C. LVD and group D signal intensity was significantly correlated (r = 0.972,P = 0.000).Conclusion(1) GoldMag was a core-shell superparamagnetic composite particles, and the core of GoldMag was USPIO. USPIO and GoldMag MRI signal characteristics were similar, as concentration increased, FSE T1WI signal intensity tended to increase, FSE T2WI signal intensity decreased slightly, GRE T2 * WI signal intensity decreased significantly. GRE T2*WI was the most appropriate MRI imaging sequences for USPIO and GoldMag.(2) PodAb-GoldMag nanos-scale contrast agent was successfully synthesized. And the mean binding rate was 64.81%. Immunoreactivity of PodAb-GoldMag was 43% of original. The Zeta electric potential of PodAb-GoldMag was 25.5±20.8 mv. Particle size distribution of PodAb-GoldMag was 80~100 nm.(3) MRI T1WI scan of rat induced breast cancer showed hypointensity or isointensity; T2WI showed hyperintensity and isointensity; pressure grease sequence T2WI scan appeared hyperintensity, and similar to human breast cancer MRI performance.(4) Proliferative breast disease can be seen only a little lymphangiogenesis and a bit larger, more mature lymphatic vessels. Lymphangiogenesis always showed in stroma around the edges of tumor nests. LVD with the decrease of the degree of differentiation of breast cancer increased.(5) PodAb-GoldMag enhanced breast cancer MRI signal intensity reduction in surrounding area was more obvious than central area. Different differentiation and histological types of breast cancer, PodAb-GoldMag enhanced MRI signal intensity changes were similar.(6) PodAb-GoldMag targeted enhanced MRI correlated with tumor lymphatic vessel density. Rat breast cancer PodAb-GoldMag enhanced scan can help to initially identify the lymphangiogenesis of breast cancer in vivo.更多还原