Experimental Studies On TH-GDNF Gene Modified Embryonic Stem Cells For The Treatment Of Parkinson's Disease Rats

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.