【作者】 刘天蔚；

【导师】 谢俊霞；

【作者基本信息】 青岛大学 ， 生理学， 2007， 博士

【摘要】 帕金森病(Parkinson’s disease，PD)是一种常见于中老年人的中枢神经系统变性疾病，其主要病理学改变是黑质多巴胺(dopamine，DA)能神经元变性坏死，纹状体内DA能神经递质匮乏。PD的发病机制目前仍不清楚，因此至今尚无满意的治疗方法。理想的PD治疗方法应该不仅能纠正脑内DA含量的不足，还要保护残存的DA能神经元。目前认为，干细胞移植结合基因治疗可能是实现这一目标的最佳途径之一。胚胎干(embryonic stem，ES)细胞因具有在体外无限或较长期增殖和多向分化潜能而成为早期胚胎发育研究和细胞治疗的良好来源。传统的PD基因治疗主要有两条途径：通过将促进DA合成的相应酶基因如酪氨酸羟化酶(tyrosine hydroxylase，TH)引入纹状体，以提高纹状体内DA含量；或者通过将神经营养因子基因如胶质细胞源性神经营养因子(glial cell line derived neurotrophic factor，GDNF)引入黑质-纹状体系统，阻止DA能神经元凋亡，维持其存活和促进其功能修复，使受损的DA能神经元有可能重建和恢复功能。若同时给予GDNF基因和TH基因，可能达到防治兼备的目的，应该是目前PD基因治疗的理想策略。本研究采用了分子克隆、细胞培养和基因转染等研究方法，分别将小鼠GDNF基因和人源性TH基因克隆到真核表达质粒载体pIRES的上下游，构建出pIRES-TH-GDNF。将此功能载体转染分化的大鼠肾上腺嗜铬细胞瘤细胞(differentiated PC12，dPC12)，检测了转染该基因的dPC12拮抗MPP~+损伤的作用。然后将此功能载体pIRES-TH-GDNF转染小鼠ES细胞，并将携带TH和GDNF基因的ES(TH-GDNF-ES)细胞和ES细胞分别植入6-羟基多巴胺(6-hydoxydopamine，6-OHDA)损伤的PD模型大鼠纹状体、黑质和同时植入黑质及纹状体(双移植)。通过观察大鼠行为学改变和移植细胞分化成TH阳性神经元的情况，以了解ES细胞或TH-GDNF-ES细胞对PD大鼠的治疗作用。并将逆行追踪剂荧光金(Fluo-Gold，FG)注射入损毁侧纹状体，观察DA神经通路的重建，以寻找PD细胞移植及基因治疗的最佳途径。实验结果如下：1、构建了双基因表达的pIRES-TH-GDNF载体，酶切鉴定及测序结果证实其是正确的。2、将pIRES-TH-GDNF转染dPC12和ES细胞，经G418筛选后，得到了稳定表达TH和GDNF的dPC12和ES细胞。用RT-PCR和细胞免疫荧光等方法能检测出TH和GDNF在细胞中转录和表达。3、MTT法检测细胞存活率显示：未转染pIRES-TH-GDNF的dPC12细胞在25μM的MPP~+作用下，细胞存活率明显下降；而转染TH-GDNF基因的细胞在100μM浓度的MPP~+作用下，细胞存活率才出现明显下降(P＜0.05)，表明转染后的dPC12能很好的对抗MPP~+毒性作用。4、免疫组织化学结果显示：ES细胞及TH-GDNF-ES细胞植入6-OHDA损伤侧的纹状体及黑质后细胞存活状态较好，部分细胞分化为TH阳性神经元。与ES细胞移植组相比，TH-GDNF-ES细胞移植组生成的TH阳性神经元数目明显增多(P＜0.05)。5、行为检测结果显示：移植后4～8w，ES细胞移植(纹状体、黑质、纹状体+黑质)组及TH-GDNF-ES细胞移植(黑质)组PD大鼠的旋转行为与对照组相比无明显改善(P＞0.05)。而TH-GDNF-ES细胞移植(纹状体、纹状体+黑质)组PD大鼠的旋转行为与对照组相比明显改善(P＜0.05)，但TH-GDNF-ES细胞移植(纹状体)组与TH-GDNF-ES细胞移植(纹状体+黑质)组相比差别无统计学意义(P＞0.05)。6、HPLC检测结果显示，移植后8w，与对照组相比，ES细胞移植(纹状体、黑质、纹状体+黑质)组及TH-GDNF-ES细胞移植(黑质)组PD大鼠纹状体内DA及其代谢产物3，4二羟基苯乙酸(3，4-dihydroxyphenylacetic acid，DOPAC)含量无明显增高(P＞0.05)。而TH-GDNF-ES细胞移植(纹状体、纹状体+黑质)组PD大鼠纹状体内DA及DOPAC含量明显增高(P＜0.05)。但TH-GDNF-ES细胞移植(纹状体)组与TH-GDNF-ES细胞移植(纹状体+黑质)组相差别无统计学意义(P＞0.05)。7、FG注射纹状体2w后，在TH-GDNF-ES细胞移植(纹状体+黑质)组PD大鼠同侧的黑质中观察到有FG逆行标记的TH阳性神经元。上述结果表明，我们成功构建了双基因表达的pIRES-TH-GDNF载体，其携带的功能基因(TH和GDNF)导入到dPC12和ES细胞内，随细胞分裂而进入子代细胞并能高效表达，且在dPC12细胞内起到对抗MPP~+毒性的作用；ES细胞及TH-GDNF-ES细胞移植到6-OHDA制备的PD大鼠纹状体及黑质后细胞存活状态较好，并分化成TH阳性神经元；TH-GDNF-ES细胞移植(纹状体、纹状体+黑质)可增加PD大鼠纹状体的DA及DOPAC含量，有效改善PD大鼠旋转行为；TH-GDNF-ES细胞移植(纹状体+黑质)有助于脑内黑质-纹状体通路的重建。本实验证实TH-GDNF基因修饰的ES细胞在对PD模型大鼠的治疗中发挥了积极的作用，为PD细胞移植及基因治疗提供了相应的实验依据。更多还原

【Abstract】 Parkinson’s disease (PD) is a neurodegenerative disorder characterized by theprogressive loss of dopaminergic neurons in the substantia nigra (SN) and a severedecrease of dopamine (DA) content in the striatum. Up to now, there is still no way toprevent the progress of the disorder because of the unknown pathogenesis. The idealtherapeutic method for PD should not only replenish the DA content, but also protect theremaining dopaminergic neurons. The combination of the stem cell transplantation andgene therapy may be the optimal pathway. Embryonic stem (ES) cells are pluripotent cellsthat can differentiate into a wide variety of cell types. This has made them an attractivesource of donor cells for developmental studies and cell therapy. Current gene therapymodels for PD are described in two parts: genetic transfer of the biosynthetic enzymes fordopamine synthesis (i.e. tyrosine hydroxylase, the key enzyme of dopamine synthesisTH), and genetic transfer of the genes encodingseveral neurotrophic factors (such as glialcell line derived neurotrophic factor (GDNF)) for protection and restoration ofdopaminergic neurons. We suppose that the combination of these two strategies may bemore efficient than each of then alone.In this study we construct a pIRES vector carrying glial cell line-derivedneurotrophic factor (GDNF) and tyrosine hydroxylase (TH). First, differentiated PC12(dPC12) cells were transfected with pIRES-TH-GDNF, and the transfected dPC12 cellsagainst neurotoxicity of MPP~+ were investigated. Then ES cells were transfected withpIRES-TH-GDNF and the transfected ES cells or ES cells were then collected andimplanted into the striatum, substantia nigra and substantia nigra plus striatumrespectively. The rotational behavior of rats, the differentiation of transplanted cells wereinvestigated to evaluate the therapeutic effect of the transplanted genes. Further,Fluo-Gold (FG), a fluorescent dye, was microinjected into the ipsilateral striatum to observe the reconstruction of the nigrostriatal pathway after transplantation. The resultswere as follows:1. A vector pIRES-TH-GDNF carrying TH and GDNF were constructed, restrictionanalysis and nucleotide sequencing showed that the two target gene fragments wereinserted into pIRES correctly and the size of recombinant plasmid is correct.2. After transfecting dPC12 and ES cell lines with pIRES-TH-GDNF, the co-expressionof TH and GDNF was detected by RT-PCR and immunocytochemisty under G418selection.3. MTT assay showed that the viability of naive differentiated PC12 (dPC12) cellsdecreased significantly with 25μM N-methyl-4-phenylpridinium iron (MPP~+)treatment. While the viability of these cells transfected by pIRES-TH-GDNFdecreased when treated with 100μM MPP~+.4. Immunohistochemistry results showed that after TH-GDNF-ES cells or ES cells wereimplanted into the striatum and the substantia nigra, the cells survived, and part ofthem differentiated into TH positive neurons. There were more TH positive neurons inTH-GDNF-ES transplanted groups compared with the ES transplanted groups(P＜0.05).5. Rotational behavior recording showed that 4～8w after transplantation, the rotationalbehavior of PD rats in ES transplanted (striatum, substantia nigra, striatum plussubstantia nigra) groups and TH-GDNF-ES transplanted (substantia nigra) group didnot change compared with the control. While the rotational behavior of PD rats inTH-GDNF-ES transplanted (striatum, striatum plus substantia nigra) groupssignificantly decreased compared with the control (P＜0.05), but there was nodifference between the two groups (P＞0.05).6. High performance liquid chromatography -electrochemical detection (HPLC-ECD) results showed that: 8w after transplantation, DA and DOPAC contents in the striatumof ES transplanted (striatum, substantia nigra, striatum plus substantia nigra) groupsand TH-GDNF-ES transplanted (substantia nigra) group did not change comparedwith the control. While DA and DOPAC contents in the striatum of TH-GDNF-EStransplanted (striatum, striatum plus substantia nigra) groups significantly increasedcompared with the control (P＜0.05), but there was no difference between the twogroups (P＞0.05).7. 2w after the injection of the retrograde tracer FG into the ipsilateral striatum,fluorescent-labeled cells within the ipsilateral substantia nigra were detected inTH-GDNF-ES transplanted (striatum plus substantia nigra) group.The results suggest that a vector pIRES-TH-GDNF carrying TH and GDNF weresuccessfully constructed and the functional genes TH/GDNF transfected into dPC12 andES cells could be highly expressed; The viability of pIRES-TH-GDNF transfected dPC12cells increased when treated with MPP~+; The transplanted ES cells and TH-GDNF-EScells in the striatum and the substantia nigra survived, and part of them differentiated intoTH positive neurons; The rotational behavior and the contents of DA/DOPAC in thestriatum ameliorated significantly in TH-GDNF-ES transplanted (striatum, striatum plussubstantia nigra) groups; Nigrostriatal pathway was reconstructed in TH-GDNF-EStransplanted (striatum plus substantia nigra) group. This work verifies that TH-GDNF-EStransplantation is effective in the treatment of PD, and provides a useful framework toadvance our knowledge of cell transplantation and gene therapy.更多还原