發(fā)布時間：2018-03-11 07:17

  本文選題：帕金森病　切入點：Micro-PET　出處：《蘇州大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分 18F-fallypride的合成及質(zhì)量控制目的 分別建立TLC法和HPLC法對18 F-fallypride顯像劑及其前體進行質(zhì)量控制并測定各自的放化純度。 方法 HPLC檢測18F-fallypride顯像劑前體的條件為Sep-Pak C18色譜柱(5μm,4.6×250 mm,Waters,USA);流動相:乙腈:水=60:40(含0.1%三乙 胺);檢測波長:295 nm;流速:0.8 m L/min;進樣量10μL；Mini-scanTLC檢測18F-fallypride顯像劑，以二氯甲烷:甲醇(V/V)=9:1為展開劑上行展開。結(jié)果 HPLC檢測18F-fallypride前體的出峰時間23.66 min放化純度98%；TLC測定的Rf值約為0.5，，放化純度96%。結(jié)論 18F-fallypride合成方法簡便，放化純度高，保證了臨床使用的要求。第二部分 18 F-fallypride在小鼠體內(nèi)生物分布研究目的 考察18 F-fallypride在小鼠體內(nèi)生物分布以及與腦內(nèi)多巴胺受體特異性結(jié)合能力。方法 小鼠尾靜脈注射18 F-fallypride后于5、15、30、60、90、120 min處死,取出心、肝、脾、肺、腎、腸、骨等組織,腦內(nèi)組織分區(qū)(小腦、紋狀體、丘腦、海馬、額葉、皮層、余腦),稱重后測量放射性計數(shù),計算每克組織百分注射劑量率(%ID/g)；同樣小鼠尾靜脈注射18 F-fallypride的Micro-PET結(jié)合2DROI技術(shù)計算紋狀體以及小腦區(qū)域的放射性計數(shù)，顯像結(jié)束后取出腦組織，γ計數(shù)儀測定紋狀體和小腦的放射性值(%ID/g),并計算紋狀體/小腦特異性比值；使用19F-fallypride與紋狀體多巴胺受體結(jié)合競爭抑制測定18 F-fallypride的特異性。結(jié)果 小鼠尾靜脈注射18 F-fallypride后迅速在各組織分布，并以心、肝、脾、腎等組織的清除較快,骨組織則是隨著時間的延長18F-fallypride攝取值增加,腦內(nèi)分布結(jié)果顯示18F-fallypride主要均勻集中在兩側(cè)紋狀體內(nèi)，在其他腦區(qū)如小腦內(nèi)攝取量低，具有高度的與多巴胺受體結(jié)合特異性。結(jié)論 18F-fallypride可以作為多巴胺受體顯像劑用于帕金森動物模型的顯像與評價。第三部分 傳統(tǒng)方法及18F-fallypride Micro-PET 法對PD鼠模型的評價目的 建立帕金森病小鼠模型,比較傳統(tǒng)方法與無創(chuàng)Micro-PET 法對于帕金森病動物模型的評價差異。 方法 小鼠腹腔注射 MPTP(25 mg/kg),建立小鼠帕金森病模型;采用一般行為學(xué)、游泳實驗、自主活動實驗、免疫組織化學(xué)實驗、氧化應(yīng)激法、蛋白印跡實驗等傳統(tǒng)方法,以及通過無創(chuàng)Micro-PET 掃描后通過興趣區(qū)技術(shù)手動選取小鼠冠狀面,計算18F-fallypride 顯像劑的攝取值來評價帕金森病小鼠模型。 結(jié)果 腹腔注射 MPTP(25 mg/kg)后,模型組小鼠一般行為學(xué)癥狀與帕金森病患者臨床癥狀相似,游泳時間,移動格子數(shù)以及站立次數(shù)均顯著性的少于空白對照組(P 0.001),免疫組化結(jié)果顯示 MPTP 模型組動物的 TH 陽性神經(jīng)元數(shù)、TH 蛋白表達量、DAT 數(shù)、以及 DAT 蛋白表達量均顯著少于空白對照組;與空白對照組相比,MPTP 模型組小鼠紋狀體內(nèi) MDA 含量顯著增加,而 GSH-PX 以及 SOD 含量則顯著降低;對于 Micro-PET 掃描圖,模型組小鼠的18F-fallypride 攝取值顯著性的低于空白對照組。 結(jié)論 傳統(tǒng)方法與無創(chuàng)Micro-PET法結(jié)果均表明帕金森病小鼠模型建立成功,而與傳統(tǒng)方法相比,Micro-PET 法可以更加直觀的觀察到紋狀體內(nèi)多巴胺受體含量的變化。 第四部分 左旋多巴對 PD 鼠模型的干預(yù)作用 目的 考察左旋多巴對帕金森病模型小鼠的干預(yù)作用。 方法 腹腔注射 MPTP(25 mg/kg)建立帕金森病小鼠模型,結(jié)合傳統(tǒng)方法與 Micro-PET 法探討左旋多巴治療帕金森病模型小鼠的機理。 結(jié)果 左旋多巴組小鼠游泳時間,移動格子數(shù)以及站立次數(shù)與空白對照組均無顯著性差異;透射電鏡顯示給予左旋多巴后可以減少由 MPTP 導(dǎo)致的黑質(zhì)致密部細胞核和細胞質(zhì)的形態(tài)學(xué)變化,如細胞質(zhì)中空泡數(shù)減少,線粒體粒徑大小及形態(tài)恢復(fù)正常等,還顯著性的增加了 TH 陽性細胞的數(shù)目。此外,給予左旋多巴后,DOPAC/DA 和 HVA/DA 水平明顯減少,多巴胺的代謝速率顯著降低;左旋多巴組小鼠紋狀體內(nèi)18F-fallypride 攝取值與模型組小鼠相比也有明顯的增加。 結(jié)論 給予左旋多巴治療可能是通過改善 MPTP 誘導(dǎo)的小鼠黑質(zhì)致密部損傷,增加小鼠 TH 陽性細胞數(shù)目和多巴胺受體含量,并降低多巴胺的代謝速率,從而達到減輕帕金森病癥狀的目的。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R742.5

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