【作者】 贾晓晶；

【导师】 龚守良；

【作者基本信息】 吉林大学 ， 放射医学， 2004， 博士

【摘要】 帕金森氏病(PD)是一种常见的中枢神经系统进行性退行性病变，其特征性病理改变为中脑黑质多巴胺能神经元的退行性变性和丢失，临床上表现为肌肉强直、肢体震颤和运动减少。虽然，近年来对PD的研究的不断深入，然而，人们对其病因机制尚未明确，目前的治疗方法亦仅限于对症支持治疗，不能从根本上阻止病变的进展。因此，体外PD细胞实验模型的建立及多巴胺能神经元保护性药物的研究，对PD等神经退行性病变防治及病因机制的探讨具有重要的理论意义。本研究采用孕14 d小鼠胚胎中脑组织进行原代细胞培养，探讨了1-甲基4-苯基-四氢吡啶离子(1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine，Mpp~+)和谷氨酸对多巴胺能神经元的损伤作用及其机制；并且，对胞二磷胆碱(citicoline)的多巴胺能神经元神经保护作用进行了探讨。实验结果表明，建立的小鼠胚胎中脑组织进行原代细胞培养的方法为PD实验研究提供了一个可靠的、有效的方法。胞二磷胆碱可有效地防止MPP~+及谷氨酸引起的多巴胺能神经元的损伤和死亡，为PD的防治提供一崭新有效的治疗途径。 1 PD体外实验细胞模型的建立 1．1 MPP~+对小鼠胚胎中脑组织原代细胞培养中多巴胺能神经元的损伤作用 1．1．1 MPP~+诱导多巴胺能神经元损伤的形态学改变 我们采用孕14 d小鼠胚胎中脑组织进行体外原代细胞培养，在培养第10 d，于培养基中加入不同浓度的MPP~+持续作用48 h后，采用多聚甲醛固定，酪氨酸羟化酶(TH)免疫染色以鉴别多巴胺能神经元。结果显示，0.1～15 μmol/L MPP~+可引起多巴胺能神经元数量明显减少，神经突起数量及长度明显减少，有的甚至完全丢失。另外，神经 吉林大学博士学位论文突起的表面变得粗糙，不光滑，扭曲，局部有串珠样肿胀。 10腼ol/L柳P+引起两种不同的多巴胺能神经元的损伤和死亡方式.其中，绝大多数的神经元的损伤表现为相对慢性的损伤，即为神经突起变短、甚至丢失，也就是说，神经元的损伤从远端开始:而另一损伤表现为神经元胞体丢失，而突起尚存，这种神经元的损伤和死亡方式占极少数。1.1.2即P+诱导多巴胺能神经元死亡的时程效应 本文探讨了10阳ol/L MPP+分别作用不同时间(2，4，8，24，48，72和96h)，引起多巴胺能神经元丢失的损伤效应。结果显示，在对照组中，每培养孔TH免疫染色阳性的神经元数量为840土15.5，而在2、4和8 h MPP‘药物组中分别为791土21.9、849士31.7和790土31.7，与对照组无显著差异。MPP+作用24h后，神经元的数量明显减少，在24、48、72和96h淤P‘药物组中神经元的数量分别为677土6.12， 412土26.6，229土20，3 and 191士15.2，明显少于对照组水平(只0.05)。在24、48和72 h MPP‘作用组中，任意两组之间神经元数量存在显著差别。因此，MPP+诱导的多巴胺能神经元的损伤和丢失与药物作用时间密切相关。M即+持续作用24、48和72h可分别引起20%、50%和75%神经元的丢失。1.1.3 MPP+对多巴胺能神经元丢失的剂量效应 本实验检测了0.1、1、10和巧枷o1/L4个不同MPP+药物浓度，持续作用48h对多巴胺能神经元丢失的影响.结果显示，在这4个浓度MPP+药物组中，神经元的数量分别为70.3土6.38%，67.4土4.92%，51.7土2.95%and 50.9土5.60%对照组水平。与对照组相比，均明显减少(只0.05)。并且，随着M即+药物浓度的增加，神经元数量减少，其中而l腼ol/L和10伽o1/L MPP+药物组之间存在统计学差异，故MPP+引起的多巴胺能神经元的丢失是呈剂量依赖关系的。1.2 MPP+对人神经纤维瘤细胞(S TAwe朋一3细胞株)的毒性作用 本研究在体外应用M竹分析法，探讨了不同浓度MPP’(62.5、125、250、500 and1000阳。1/L)分别作用不同时间(24、48、72和96h)对人神经纤维瘤细胞细胞活性的影响。结果显示，500阳o1/L和1000腼ol/L沙P+作用24h，细胞活性分别降至82.6土5.38%和75.1土5.80%对照组水平，明显低于对照组(只0.05)。62.5阳01/L淤P‘持续作用48h可降低细胞活性28%，125、250、500和1000枷01/L MPP‘作用48h，)乡6 吉林大学博士学位论文细胞活性分别减少为36%，53%67%和78%的对照组水平。500腼01/L柳P‘作用72h使细胞活性减低85%，在1000腼o1/L药物组中95%细胞活性丧失.500腼o1/L和I000P口01/L持续作用96h时，可分别降低细胞活性达9既and98%。因此，MPP+降低人神经纤维瘤细胞细胞活性呈时间和剂量依赖关系。1.3谷氨酸对小鼠胚胎中脑组织原代细胞培养中多巴胺能神经元的损伤作用1 . 3.1谷氨酸引起多巴胺能神经元损伤的时程变化 于体外培养第10d，我们将500腼ol/L谷氨酸加入细胞培养液中，持续作用15 min后，更换新的培养液。为明确谷氨酸引起多巴胺能神经元损伤的时程变化，于谷氨酸分别作用后2、4、6、24和48h后，采用TH免疫染色方法，对多巴胺能神经元的数量及形态学改变进行了探讨。结果表明，在对照组中，平均每孔中多巴胺能神经元的数量为778土18.6个，谷氨酸作用后Zh，多?更多还原

【Abstract】 In the thesis, we set up cell models of degeneration of dopaminergic neurons by using mice mesencephalic dissociated culture in vitro. The toxicity of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPP*) and excititory animo acid (glutamate) were investigated and evaluated. The neuroprotective effect and possible mechanism of citicoline on the dopaminergic neurons were studied. It will provide a theoric base for its further application in the treatment of Parkinson’s disease (PD) and to a better understanding of the possible mechanism of PD. 1 Cell models for PD in vitro1.1 The toxicity of MPP+ on dopaminergic neurons in mouse mesencephalic dissociated culture1.1.1 The morphological change of dopaminergic neurons induced with MPP*Our results showed that after incubation with different concentrations of MPP+ (0.1-15 umol/L), the density of the dopaminergic neurons was significantly reduced, and the procesesses became shorter and even lost. The numbers of the processes reduced significantly. Most of the processes were distort and swollen. 10 umol/L MPP+ induced the death of dopaminergic neurons in two different ways. The loss and degeneration of dopaminergic neurons in MPP+-treated groups were mainly in a relative chronic way that the loss and shortening of the processes were found first and then subsequently the cell bodies were lost. Only few dopaminergic neurons underwent death through another pathway in which the dopaminergic neurons bodies were lost before the processe lossing.1.1.2 The time-dependent effect of MPP+ on the loss of dopaminergic neuronsThe time course of dopaminergic neurons death induced with MPP+ was investigated.The numbers of TH-ir neurons in the control group, 2-, 4-, and 8-hour MPP+-treated group were 840+15.5, 791 +21.9, 849 + 31.7 and 790 + 31.7 respectively. The numbers of TH-ir cells in the latter three groups didn’t decrease as compared with those in the control. After 24 hours incubation of MPP+, the loss of TH-ir neurons became appearant. The TH-ir cell numbers per well were 677 + 6.12,412 + 26.60,229 +20.31 and 191 + 15.23 in 24-, 48-, 72-and 96-hour MPP+-treated groups respectively. So the loss of dopaminergic neurons was related to the incubation time of MPP+. And there was a significant difference between any two groups which have been treated with MPP+ for 24, 48 and 72 hours. The numbers of dopaminergic neurons reduced by about 20% after 24-hour incubation with 10umol/L MPP+, 50% after 48 hours, and 75% after 3 days or 4 days. 1.1.3 the dose-dependent effect of MPP+ on the loss of dopaminergic neuronsIn order to clarify the dose effect of MPP+ on the loss of dopaminergic neurons, MPP+ of different final concentrations (0.1, 1, 10and 15 umol/L) were added into the cultures on the 10th DIV, and incubated for 48 hours. Our datas showed that the dopaminergic neuron loss induced with MPP+ was in a dose-dependant pattern. The numbers of dopaminergic neurons were 100% in the control group, while 70.3 + 6.38 %, 67.4 + 4.92 %, 51.7 + 2.95 % and 50.9 + 5.60 % in the 0.1,1,10, and 15 umol/L MPP+-treated groups respectively. The numbers of TH-ir cells were significantly lower than that in the control (p<0.05). The toxicity of MPP+ on the degeneration of dopaminergic neurons was dose-dependent. But there was no statistical significance between the 0.1umol/L group and 1 umol/L group, as well as 10 umol/L and 15 umol/L. But there was statistical difference between 1 umol/L and 10 umol/L. 1.2 The toxicity of MPP+ on the human neuroblastoma cell line (STA-NB-3)The cultures of human neuroblastoma cell line (STA-NB-3) were treated with different concentrations of MPP+ (62.5, 125, 250, 500 and 1000umol/L) and incubated for different days (1, 2, 3 and 4 days). Then the cell viability was determined by MTT assay. Our data showed that after treatment with 500 umol/L and 1000 umol/L of MPP+ for 1 day reduced the cell viability to 82.6 + 5.38% and 75.1 + 5.80% of the control respecrively (P<0.05).The cell viability decreased by 28% after treatment with 62.5 umol/L of MPP+ for 2 days. T更多还原