Equivalent effects, in animals handled with DA neurotoxin methyl phenyl , tetrahydropyridine , have been also obtained: DA neurons, containing CaBP, had better resistance against MPTP . The experimental scientific studies of excitatory neurotoxicity in vitro have also shown that CaBP has some significant neuroprotective effects on DA neurons . Nevertheless, the neuroprotective mechanism of CaBP in DA neurons continues to be unclear. Our former studies concerning the neuroprotective mechanism from the glial cell line derived neurotrophic issue in DA neurons have demonstrated that GDNF can activate the PI kinase Akt pathway when also promoting the expression of CaBP . Thus, we hypothesized the neuroprotective mechanism of CaBP in DA neurons may be related to the activation in the PI K Akt pathway. The cell line MND, a fusion of embryonic ventral mesencephalic and neuroblastoma cells, is extensively utilized being a model of DA neurons because it expresses tyrosine hydroxylase and synthesizes and releases DA. These cells are also put to use to test mechanisms and prospective therapeutics pertinent on the loss of DA neurons in PD.
So, to check our hypothesis, we constructed a recombinant plasmid, pcDNA CB, and transfected the MND cells with it to boost the expression of CaBP selectively. Then, we examined the activation of PI K Akt pathway. Concurrently, we examined the activation within the nuclear factor kappa light Procaine chain enhancer of activated B cells non classical pathway to investigate the downstream signaling molecules of Akt. EXPERIMENTAL Method Cell culture The MND cells had been derived from your fusion of rostral mesencephalic neurons with all the NTG neuroblastoma cells. The MND cells were maintained at C, with CO in the humidified incubator to increase in poly D lysine coated culture flask, containing Dulbecco?s modified eagle?s medium ham?s nutrient mixture F culture medium supplemented with fetal bovine serum, U ml penicillin, and g ml streptomycin. Cell transfection When the MND cells grew to confluence, they have been plated on very well culture plates and seeded at cells per nicely. Then, the recombinant plasmids were launched into the cells .
The MND cells transfected with all the recombinant plasmid containing CaBP cDNA had been labeled as the pcDNA CB group, the MND cells transfected using the recombinant plasmid Sodium Monofluorophosphate containing the green fluorescent protein cDNA because the pcDNA GFP group, and non transfected MND cells were utilised as the manage. Neurotoxin treatment method At h following cell transfection, the MND cells have been exposed to M hydroxydopamine for min and then cultured for h constantly. MND cells not treated with OHDA served since the manage group.