多巴胺受体显像无创评价帕金森病的实验研究

发布时间：2018-03-11 07:17

本文选题：帕金森病　切入点：Micro-PET　出处：《苏州大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分 18F-fallypride的合成及质量控制目的分别建立TLC法和HPLC法对18 F-fallypride显像剂及其前体进行质量控制并测定各自的放化纯度。方法 HPLC检测18F-fallypride显像剂前体的条件为Sep-Pak C18色谱柱(5μm,4.6×250 mm,Waters,USA);流动相:乙腈:水=60:40(含0.1%三乙胺);检测波长:295 nm;流速:0.8 m L/min;进样量10μL；Mini-scanTLC检测18F-fallypride显像剂，以二氯甲烷:甲醇(V/V)=9:1为展开剂上行展开。结果 HPLC检测18F-fallypride前体的出峰时间23.66 min放化纯度98%；TLC测定的Rf值约为0.5，，放化纯度96%。结论 18F-fallypride合成方法简便，放化纯度高，保证了临床使用的要求。第二部分 18 F-fallypride在小鼠体内生物分布研究目的考察18 F-fallypride在小鼠体内生物分布以及与脑内多巴胺受体特异性结合能力。方法小鼠尾静脉注射18 F-fallypride后于5、15、30、60、90、120 min处死,取出心、肝、脾、肺、肾、肠、骨等组织,脑内组织分区(小脑、纹状体、丘脑、海马、额叶、皮层、余脑),称重后测量放射性计数,计算每克组织百分注射剂量率(%ID/g)；同样小鼠尾静脉注射18 F-fallypride的Micro-PET结合2DROI技术计算纹状体以及小脑区域的放射性计数，显像结束后取出脑组织，γ计数仪测定纹状体和小脑的放射性值(%ID/g),并计算纹状体/小脑特异性比值；使用19F-fallypride与纹状体多巴胺受体结合竞争抑制测定18 F-fallypride的特异性。结果小鼠尾静脉注射18 F-fallypride后迅速在各组织分布，并以心、肝、脾、肾等组织的清除较快,骨组织则是随着时间的延长18F-fallypride摄取值增加,脑内分布结果显示18F-fallypride主要均匀集中在两侧纹状体内，在其他脑区如小脑内摄取量低，具有高度的与多巴胺受体结合特异性。结论 18F-fallypride可以作为多巴胺受体显像剂用于帕金森动物模型的显像与评价。第三部分传统方法及18F-fallypride Micro-PET 法对PD鼠模型的评价目的建立帕金森病小鼠模型,比较传统方法与无创Micro-PET 法对于帕金森病动物模型的评价差异。方法小鼠腹腔注射 MPTP(25 mg/kg),建立小鼠帕金森病模型;采用一般行为学、游泳实验、自主活动实验、免疫组织化学实验、氧化应激法、蛋白印迹实验等传统方法,以及通过无创Micro-PET 扫描后通过兴趣区技术手动选取小鼠冠状面,计算18F-fallypride 显像剂的摄取值来评价帕金森病小鼠模型。结果腹腔注射 MPTP(25 mg/kg)后,模型组小鼠一般行为学症状与帕金森病患者临床症状相似,游泳时间,移动格子数以及站立次数均显著性的少于空白对照组(P 0.001),免疫组化结果显示 MPTP 模型组动物的 TH 阳性神经元数、TH 蛋白表达量、DAT 数、以及 DAT 蛋白表达量均显著少于空白对照组;与空白对照组相比,MPTP 模型组小鼠纹状体内 MDA 含量显著增加,而 GSH-PX 以及 SOD 含量则显著降低;对于 Micro-PET 扫描图,模型组小鼠的18F-fallypride 摄取值显著性的低于空白对照组。结论传统方法与无创Micro-PET法结果均表明帕金森病小鼠模型建立成功,而与传统方法相比,Micro-PET 法可以更加直观的观察到纹状体内多巴胺受体含量的变化。第四部分左旋多巴对 PD 鼠模型的干预作用目的考察左旋多巴对帕金森病模型小鼠的干预作用。方法腹腔注射 MPTP(25 mg/kg)建立帕金森病小鼠模型,结合传统方法与 Micro-PET 法探讨左旋多巴治疗帕金森病模型小鼠的机理。结果左旋多巴组小鼠游泳时间,移动格子数以及站立次数与空白对照组均无显著性差异;透射电镜显示给予左旋多巴后可以减少由 MPTP 导致的黑质致密部细胞核和细胞质的形态学变化,如细胞质中空泡数减少,线粒体粒径大小及形态恢复正常等,还显著性的增加了 TH 阳性细胞的数目。此外,给予左旋多巴后,DOPAC/DA 和 HVA/DA 水平明显减少,多巴胺的代谢速率显著降低;左旋多巴组小鼠纹状体内18F-fallypride 摄取值与模型组小鼠相比也有明显的增加。结论给予左旋多巴治疗可能是通过改善 MPTP 诱导的小鼠黑质致密部损伤,增加小鼠 TH 阳性细胞数目和多巴胺受体含量,并降低多巴胺的代谢速率,从而达到减轻帕金森病症状的目的。
[Abstract]:The first part is the synthesis and quality control of 18F-fallypride TLC method and HPLC method were established for the quality control of 18 F-fallypride imaging agents and their precursors were determined respectively. The radiochemical purity HPLC detection method 18F-fallypride imaging agent precursor Sep-Pak C18 column (5 m, 4.6 x 250 mm, Waters, USA); mobile phase: acetonitrile: water =60:40 (containing 0.1% triethylamine three); detection wavelength: 295 nm; flow rate: 0.8 m L/min; the injection volume was 10 ~ L; Mini-scanTLC detection 18F-fallypride imaging agent, dichloromethane: methanol (V/V) as the agent of =9:1. The result of HPLC detection of 18F-fallypride uplink precursor peak time 23.66 min 98% TLC radiochemical purity; Determination of the Rf value is about 0.5, the radiochemical purity of 96%. conclusion 18F-fallypride synthesis method is simple, high radiochemical purity, ensure the requirements for clinical use. The second part 18 F-fallypride distribution in vivo biological small rat Study objective to investigate 18 F-fallypride in biodistribution in mice and binding capacity and dopamine receptor specificity. Methods the tail vein of mice after injection of 18 F-fallypride 5,15,30,60,90120 min to death, take out the heart, liver, spleen, lung, kidney, intestine, bone, brain tissue (cerebellum, striatum, thalamus partition, hippocampus the frontal cortex, brain, and Yu, radioactivity measurement) after weighing, calculation of percent injected dose per gram of tissue ratio (%ID/g); also the tail vein injection of 18 F-fallypride Micro-PET combined with 2DROI technology and the calculation of striatal radioactivity counts cerebellar regions, brain tissue imaging after determination of striatum and cerebellum gamma counting radioactive value (%ID/g), and calculate the striatum / cerebellum specific ratio; using 19F-fallypride and striatal dopamine receptor binding specificity was determined by competitive inhibition F-fallypride. Results 18 The intravenous injection of 18 F-fallypride rapidly in all tissues, and in the heart, liver, spleen, kidney and other tissues of the bone tissue is cleared rapidly, with the extension of time 18F-fallypride uptake value increased, brain distribution showed that 18F-fallypride mainly concentrated in uniform on both sides of the striatum, in other brain regions such as cerebellum uptake low volume, high and dopamine receptor binding specificity. Conclusion 18F-fallypride can be used as a dopamine receptor imaging agent for imaging and evaluation of Parkinson's animal model. The evaluation of PD rat model of the third part of the traditional method and 18F-fallypride Micro-PET method to establish a mouse model of Parkinson's disease, compared with the traditional method and Micro-PET method for noninvasive evaluation of animal disease Parkinson model differences. Methods the mice by intraperitoneal injection of MPTP (25 mg/kg), the establishment of mouse model of Parkinson's disease; the general behavior of swimming The experiment, independent activity test, immunohistochemistry, Western blot method of oxidative stress, and other traditional methods, as well as by noninvasive Micro-PET scan technology by region of interest manually select mice coronal, calculate 18F-fallypride imaging agent uptake value to evaluate the mouse model of Parkinson's disease. The results of intraperitoneal injection of MPTP (25 mg/kg), mice in model group, the general behavior of patients with Parkinson's disease symptoms and clinical symptoms are similar, swimming time, mobile number and lattice standing times were significantly less than the control group (P 0.001), immunohistochemistry results showed that MPTP animal model group the number of TH positive neurons, the expression of TH protein, DAT, and DAT protein expression the amount was significantly less than the control group; compared with the control group, MPTP model mice striatum MDA content increased significantly, while GSH-PX and SOD were significantly To reduce; Micro-PET scans, significantly lower than the blank control group value model mice 18F-fallypride uptake. Conclusion the traditional methods and non-invasive Micro-PET method results show that Parkinson's disease mouse model was established successfully, and compared with the traditional method, can be more intuitive to observe the content of dopamine receptors in striatum induced changes of Micro-PET intervention method. Fourth: effect of levodopa on PD rat model to investigate levodopa in Parkinson s disease mice. Methods the intervention effect of intraperitoneal injection of MPTP (25 mg/kg) to establish a mouse model of Parkinson's disease, combined with traditional method and Micro-PET method to investigate the mechanism of levodopa in treatment of Parkinson s disease mice. Results the levodopa group mice swimming time, there were no significant differences the mobile number and the number of standing lattice and the blank control group; transmission electron microscopy showed that treated with multi Pakistan can be reduced after morphological changes caused by MPTP in substantia nigra in nucleus and cytoplasm, such as cytoplasmic void number, particle size and morphology of mitochondria recovered, also significantly increased the number of TH positive cells. In addition, DOPAC/DA and HVA/DA after administration of levodopa, dopamine levels significantly reduced metabolic rate the levodopa group decreased significantly; mouse striatum 18F-fallypride uptake value compared with the model group mice also increased significantly. Conclusion levodopa treatment may be given by mouse substantia nigra induced impairment induced by MPTP, increase in the number of TH positive cells and dopamine receptor content, and reduce the metabolic rate of dopamine, so as to alleviate the symptoms Parkinson's disease.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R742.5

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