非酒精性脂肪肝炎枯否细胞MR成像的实验研究

【作者】 焦志云；

【导师】 李澄； 马占龙；

【作者基本信息】 扬州大学 ， 影像医学与核医学， 2010， 硕士

【摘要】 本研究以大鼠为研究对象,在建立非酒精性脂肪肝炎（NASH）的动物模型基础上,采用自行研制的壳聚糖-g-聚丙烯酸（CS-g-PAA）纳米Fe₃O₄作为磁共振成像的对比剂,经大鼠尾静脉注射CS-g-PAA/ Fe₃O₄后行1.5T MR增强扫描来初步评估大鼠NASH枯否细胞（KC）的吞噬功能。共分为三个部分。第一部分:壳聚糖-g-聚丙烯酸（CS-g-PAA）纳米Fe₃O₄的制备目的:用壳聚糖-g-聚丙烯酸（CS-g-PAA）对纳米Fe₃O₄进行表面改性,使其在中性溶液中仍具备水分散稳定性能和超顺磁性。方法:采用接枝聚合的方法制备了壳聚糖-g-聚丙烯酸（CS-g-PAA）表面改性Fe₃O₄并得到其水分散稳定体系。用傅立叶红外光谱仪（FTIR）表征了复合纳米粒子的结构,扫描式电子显微镜（SEM）、投射电子显微镜（TEM）表征了复合纳米粒子的尺寸与形貌,X射线衍射（XRD）、振荡样品磁强计（VSM）分别表征了复合纳米粒子的晶型结构与超顺磁性。并研究了表面改性与聚合物层的组成对Fe₃O₄在水体系中的分散稳定性的影响,动态光散射（DLS）表征了复合粒子的水力半径,表明粒子的水力半径随体系PH值的变化而变化。结果:用壳聚糖及聚丙烯酸表面修饰可以成功地获得Fe₃O₄纳米粒子的中性（PH=7.4）水悬浮分散胶体。CS-g-PAA包裹后的Fe₃O₄粒子直径在15.0-60.0nm之间呈现稳定的单体分散状态,单个粒子呈现球形或椭圆形,纳米粒子的粒径分布均匀,并且其晶体结构没有改变,具有稳定的超顺磁性。结论:通过CS-g-PAA表面改性的Fe₃O₄纳米粒子,在中性环境下具备水分散稳定性能。CS-g-PAA/Fe₃O₄粒子粒径分布在15.0-60.0nm之间,具有与Fe₃O₄类似的尖晶石结构,超顺磁性稍有降低,但仍可以满足磁共振对比剂的要求。第二部分:壳聚糖-g-聚丙烯酸（CS-g-PAA）纳米Fe₃O₄磁共振体外信号检测目的:研究磁共振检测壳聚糖-g-聚丙烯酸（CS-g-PAA）纳米Fe₃O₄（PH=7.4）的稳定性及其在不同浓度下体外磁共振信号的变化规律。方法:将CS-g-PAA/Fe₃O₄磷酸盐溶液按浓度梯度排列,MRI检测其稳定性及其信号变化;所用MRI序列为:FSE T1WI、FRFSE T2WI及FIESTA T2*WI。结果:CS-g-PAA/Fe₃O₄在PH=7.4时MRI各序列信号均匀（P>0.05）;CS-g-PAA/Fe₃O₄在T1WI上信号强度随铁质量浓度的增加先升高后降低,在T2WI及T2*WI上随质量浓度的增加信号降低;浓度越低,T1WI及T2*WI较T2WI越敏感,以T2*WI最敏感（P<0.05）;在浓度≤20mg/L时,各序列上信号变化均较明显。结论:临床应用型1.5T MRI可检测CS-g-PAA/Fe₃O₄的稳定性;在浓度≤20mg/L时MRI信号改变明显;最敏感MR检测序列为T2*WI。第三部分:壳聚糖-g-聚丙烯酸（CS-g-PAA）纳米Fe₃O₄磁共振成像评估大鼠非酒精性脂肪肝炎枯否细胞功能的实验研究目的:初步探讨CS-g-PAA/Fe₃O₄作为MRI对比剂评估大鼠非酒精性脂肪肝炎（NASH）枯否细胞（KC）功能的可行性。方法:20只雄性SD大鼠按完全随机法分为实验组和对照组,每组10只,实验组喂养高脂饲料,对照组喂养普通饲料。8周后对所有大鼠行肝脏MRI及CS-g-PAA/Fe₃O₄增强扫描,计算肝脏组织平均信号强度下降百分比（PSIL）和CS-g-PAA/Fe₃O₄增强前后肝脾对比信号强度比值（RSIR）,测定血清总胆固醇（TC）和甘油三酯（TG）值,并取肝脏标本行HE染色和普鲁士蓝染色分析病理表现。结果:实验组1只大鼠因麻醉过深死亡,其余9只血清TC及TG值分别为（6.58±1.25） mmol/L和（1.53±0.23） mmol/L ,较对照组[（1.64±0.22） mmol/L和（0.55±0.14） mmol/L]均明显升高（t值分别为11.716和11.558,P值均<0.01）。CS-g-PAA/Fe₃O₄增强扫描后两组肝实质信号强度在各序列明显下降,在PDWI及T1WI上实验组下降程度分别为（34.78±4.51）%和（60.38±3.49%）,较对照组[分别为（64.96±2.42）%和（81.08±1.66）% ]小,差异有统计学意义（t值分别为-18.415和-16.240,P值均<0.01）。PDWI、T2WI、T2*WI和T1WI序列实验组CS-g-PAA/Fe₃O₄增强前后肝脾RSIR分别为(1.002±0.141、5.000±0.516、20.004±1.490和2.601±0.077,较对照组（0.400±0.102、1.500±0.115、-0.3±0.058和0.503±0.105）大（t值分别为10.745、19.800、39.168和92.785,P值均<0.01）;实验组肝脏组织中普鲁士蓝染色阳性颗粒积分（2.33±0.50）分较对照组（4分）明显减少（t=-10.000,P<0.01）。结论:高脂饮食诱导的SD大鼠NASH模型接近人类发病情况且容易建立,临床应用型1.5T MRI通过CS-g-PAA/Fe₃O₄增强肝脏扫描可评估KC细胞的功能,提示NASH的发病机制与KC功能的下降有关。更多还原

【Abstract】 This study used self-developed the Fe₃O₄ nanoparticles modified with chitosan-graft-poly （acryl acid） （CS-g-PAA） as a magnetic resonance imaging contrast agent, taking rat as experimental materials, to establish the animal model of non-alcoholic steatohepatitis （NASH） , to perform liver 1.5T MRI, were imaged before and after CS-g-PAA / Fe₃O₄ nanoparticles from the tail vein infusion, assessing rat NASH Kupffer cells （KC） phagocytic function, Which was divided into three parts.Part I: The preparation of Fe₃O₄ nanoparticles modified with chitosan-graft-poly （acryl acid） （CS-g-PAA）Objective: The well-dispersed Fe₃O₄ nanoparticles suspension was successfully prepared through surface modification with chitosan–poly （acrylic acid） （CS–PAA） copolymer by graft copolymerization technique.Methods: The graft polymerization was prepared by Chitosan–poly （acrylic acid） （CS–PAA） surface modification of Fe₃O₄ and get their water dispersed stable system. The structure of synthesized nanoparticles was characterized by Fourier transform infrared spectrometer （FTIR）. The size and morphology of the nanoparticles was investigated by field emission scanning electron microscopy （FE-SEM） and Transmission electron microscope （TEM）. The crystallization and superparamagnetism of the nanoparticles were characterized by X-ray diffraction （XRD） and vibrating sample magnetometer （VSM） respectively. The hydrodynamic radius of the modified particles was characterized by Dynamic light scattering （DLS）. The results indicated that the hydrodynamic radius was dependant on PH of the system.Results:The Fe₃O₄ nanoparticles successfully modified with chitosan-g-polyacrylic acid （CS-g-PAA）. CS-g-PAA/Fe₃O₄ nanoparticles were in water （PH=7.4）dispersion stability on globose or ellipse, diameter 15-60nm, uniform size distribution, and its crystal structure has not changed, with the stability of superparamagnetism.Conclusions: The well-dispersed Fe₃O₄ nanoparticle suspension was successfully prepared on PH=7.4 through surface modification with Chitosan–poly （acrylic acid） （CS–PAA） copolymer, the stability of its suspension mechanism is electrostatic repulsion and steric effects of synergy. CS-g-PAA/Fe₃O₄ nanoparticle size distribution of the 15.0-60.0nm, Which has the spinel structure, superparamagnetism slightly lower, but still meet the requirements of MRI contrast agent.Part II: MR imaging of CS-g-PAA/Fe₃O₄ nanoparticles in vitroObjective: To study the stability and the changing patterns of MR signals in differrnt concentration of CS-g-PAA/Fe₃O₄ nanoparticles （PH=7.4） in vitro magnetic resonance imaging.Methods: The CS-g-PAA/Fe₃O₄ nanoparticles phosphate solution were disposed by density, to observe the stability of CS-g-PAA/Fe₃O₄ and regular of MR signal; MR imaging was performed as following sequences: FSE T1WI, FRFSE T2WI and FIESTA T2*WI.Results: The CS-g-PAA/Fe₃O₄ nanoparticles were homogeneous MR signal （P>0.05）. The signal intensity of CS-g-PAA/Fe₃O₄ nanoparticles on T1WI was firstly increased, but decreased following the increase of the concentration of Fe₃O₄. The signal on T2WI and T2*WI decreased as the increase of the concentration of Fe₃O₄. The lower the concentration, the more sensitive on T1WI and T2*WI when compared with that of T2WI, with T2*WI the most significant （P<0.05）. The changes of signal intensity in all sequences were more distinct as the iron concentration≤20mg/l.Conclusion: The stability of CS-g-PAA/Fe₃O₄ nanoparticles could be examined by 1.5T MR scan using in clinical practice, the changes of signal intensity were remarked when iron concentrations≤20mg/l. T2*WI was the most sensitive sequence for detecting the CS-g-PAA/Fe₃O₄ nanoparticles in vitro.Part III: A preliminary study on the phagocytic activity of Kupffer cells with CS-g-PAA/Fe₃O₄ nanoparticles enhanced MR imaging in a rat nonalcoholic steatohepatitis modelObjective: To investigate the feasibility of using CS-g-PAA/Fe₃O₄ nanoparticles as MRI contrast agent to assess rat nonalcoholic steatohepatitis Kupffer cells （KC） function.Methods: Twenty male SD rats were randomly divided into experimental and control group, 10 rats in each group, experimental group was fed high fat diet, control group fed normal diet. After eight weeks, plain MR and CS-g-PAA/Fe₃O₄ enhanced were performed in all the rats; Blood lipids were measured, and HE and Perl’s blue staining in all livers specimen was done. The related result of the staining were analyzed.Results: Experimental group TC and TG levels [（6.58±1.25） mmol/L and （1.53±0.23） mmol/L respectively] were significantly higher than control group [（1.64±0.22） mmol/L and （0.55±0.14） mmol/L respectively] （t = 11.716 and 11.558, P <0. 01）; Experimental and control group hepatic signal intensity decreased in all sequences after CS-g-PAA/Fe₃O₄ enhanced, but in experimental group the level of decline [（34.78±4.51）% and （60.38±3.49%） respectively] was less than control group [（64.96±2.42）% and （81.08±1.66）% respectively] on PDWI and T1WI, and statistically significant difference （t = -18.415 and -16.240, P <0.01） were found. In experimental group the ratio of signal intensity of liver to spleen（1.002±0.141, 5.000±0.516, 20.004±1.490 and 2.601±0.077）was more than control group（0.400±0.102, 1.500±0.115, -0.3±0.058 and 0.503±0.105）before and after contrast enhancement on PDWI, T2WI, T2*WI and T1WI （t = 10.745, 19.800, 39.168 and 92.785, P <0.01）. Typical histological hepatic lesions of NASH were observed in experimental group. Perl’s staining-positive particles in experimental group（2.33±0.50）were fewer than in control group （4） （t= -10.000, P <0.01）.Conclusion: The high-fat diet-induced model of SD rats close to the human NASH and was easy to establish. Clinical application of CS-g-PAA/Fe₃O₄ enhanced MR successfully assessed the phagocytic activity of Kupffer cells in the study, and it suggested that the pathogenesis of NASH related to the decreased phagocytic activity of Kupffer cells.更多还原