论文标题:神经生长因子脂质体的制备及药代动力学初步研究
Production and Pharmacokinetic Research of NGF-liposome
论文作者 吕天润
论文导师 曹晓建；张宁；金正帅,论文学位 硕士,论文专业 骨外科
论文单位 南京医科大学,点击次数 83,论文页数 73页File Size3775k
2004-04-01论文网 http://www.lw23.com/lunwen_257566572/ NGF脂质体;同位素示踪;SPECT;药代动力学
NGF, liposome, isotopic tracing, ELISA, 3p97, pharmacokinetics
目的 提纯神经生长因子(NGF)，并使用自制脂质体包裹，初步研究神经生长因子脂质体的药代动力学。 方法 上部为神经生长因子脂质体的制备。取小鼠颌下腺组织，经过离心、CM52上柱分离，得到NGF，电泳法检测纯度，Bradford法测定NGF含量，鸡胚背根神经节培养鉴定NGF活性。改进传统的反向蒸发法制备脂质体，并使用磁力搅拌法包裹神经生长因子，采用DEAE Sephadex A50微型柱离心法测定脂质体对NGF的包封率。 下部为静脉用NGF脂质体在家兔体内血药浓度检测及药代动力学参数的分析，并初步观察其组织分布，使用~(99m)Tc标记NGF，测定~(99m)Tc放射性计数与NGF含量的线性关系，从而设计检测家兔血浆中NGF浓度的检测方法，将家兔分为静脉注射单纯NGF组和脂质体包裹NGF组，通过TCA沉淀法处理血样，测定不同时间的血药浓度，使用3P97软件分析药代动力学参数及房室模型。使用SPECT检测NGF的全身分布，初步观察其代谢，搜集尿液分析代谢情况，另外使用ELISA法检测脑脊液中NGF，初步分析其在脑部分布。 结果 上部结果：提纯的NGF得率为125μg／g，通过电泳法显示提取的蛋白质为2.5S NGF，无明显杂质条带，鸡胚背根神经节培养显示活性良好，Bradford法测定NGF含量为1mg／ml。成功制备NGF脂质体静脉注射剂。 下部结果：通过同位素示踪法检测NGF含量与放射性计数成线 」il)洲卜人分一‘吹卜L片训t仑又性关系，在剂量为40冬、g/kg时·脂质体包裹NGF的药代动力学参数为:t，:。，一o.2338h，t，:j厂1 .322911，Al一;C一1 06.57(ng/l二I)*h，单纯NG「的药代动力学参数为t.:《，一o.077h，t}:l于0.88h，AUC一98.656(ng/ml)*h，SPECT显示脂质体包裹后的99mTc一NGF在脑部有浓聚，而未包裹则无明显脑分布迹象，脑脊液ELISA检测显示只有脂质体NGF在用药2，J、时左右检测到NGF，尿液及粪便放射性计数显示脂质体包裹NGF为肾脏、胆汁排泄，而单纯NGF通过肾脏排泄，尿液沉淀实验显示NGF肾脏排泄以小分子片段形式，而不是原型或大分子多肤片段形式排出。 结论 自制脂质体包裹NGF后可以对NGF的吸收、分布、排泄、代谢产生一定的变化，初步结果显示使用脂质体包裹后NGF代谢时间延长并可能分布于脑组织。
ObjectiveTo pack nerve growth factor with liposome and to study its pharmacokinetic parameters.MethodsIn first part, NGF-liposome was produced. Tissue of mice"s submaxillary glands was centrifuged and separated in CM52 column, thus 2.5S NGF was produced. Electrophoresis was used to measure the purity and Bradford way to detect the content. To identify activity of NGF, cultivating DPG of chick embryo was performed. NGF was packed with liposome to produce NGF-liposome and correlative parameters were detected.In second part, pharmacokinetics of NGF-liposome in vivo was studied. NGF was labeled with 99mTc and isotopic tracing was utilized to detect concentration of NGF in rabbit blood. Compared with packed group, unpacked NGF group were detected. Data were inputted into computer to be analyzed of pharmacokinetic parameters, compartment model etc. by software (3p97). From SPECT distribution of lipo-99mTc-NGF in rabbit body was shown as well 99mTc-NGF to be compared with. Urine and dejecta was collected for the analysis of excretion in rabbit body. ELISA was the method to detect concentration of NGF in CSF and TCA method was used to detect radioactivity of deposit of urine.ResultsIn first part, it was indicated that the abstracted protein was NGF of which the content was ling/ml and biologic activity was fine. NGF was packed with liposome and NGF-liposome was produced.In second part, we have found that the relationship of concentration of NGF and count of -ray was nearly linear. From the test of recovery and precision, it was suggested that labeling NGF with 99mTc to detect concentration of NGF was feasible. Pharmacokinetic parameters of NGF packed with liposome injected in vivo in rabbit body were: t1/2=0.2338h, t1/2=1.3229h, AUC=106.57(ng/ml)*h. Parameters of unpacked NGF were: t1/2 =0.077h, t1/2 =0.88h,AUC=98.656(ng/ml)*h. It was shown in pictures of SPECT, if NGF was packed with liposome there was NGF distributing in brain. Analysis of urine and dejecta indicated that main excretion route of unpacked NGF was urine and that of NGF packed with liposome was urine as well bile. Detection of NGF in CSF pointed that if NGF was packed peak of concentration in CSF was at 2hours whereas if NGF was unpacked NGF was undetectable. From SPECT, it was shown that there was radiological concentration in brain if 99mTc-NGF was packed with liposome. Radioactivity of urine deposit was undetectable in TCA method.ConclusionWhen NGF is packed with liposome, absorption, distribution, metabolism and excretion of NGF will change. T1/2 and T1/2p are prolonged and NGF can pass BBB.
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