| In the brain of patient with Parkinson's disease(PD), the degeneration of dopamine(DA) neurons is prominently in the substantia nigra(SN), which results in the decrease of DA in the striatum. The tyrosine hydroxylase (TH)is the key enzyme of DA synthesis, and the glial cell line-derived neurotrophic factor(GDNF)is the most potent neurotrophic factor for dopamine neurons described so far, simultaneous delivery of GDNF and TH genes into the striatum is considered one of the most ideal therapeutic protocols for PD. The Long-lasting and constant expression of TH and GDNF gene in the brain after genes delivery may produce adverse effect, so the ragulation and control of TH and GDNF genes expression is needed according to the clinical manifestation of PD patients.The Tet-on system of lentiviral vector is specialized in regulating the expression of gene in the mammalian cell and exhibits very good practical future on human diseases. However, the relatively high basal expression of the system limits its use. In this experiment, we applied the improved Tet-on system, in which the reverse tet- transactivator is rtTA2s-M2, and the promoter of tetracycline response elemen(tTRE)is albumin gene promoter(Palb)of mice, In the presence of tetracycline antibiotics, rtTA2s-M2 interacts with TRE located in the lentiviral vector to activate transcription. Objective To construct the lentiviral vector carrying rat TH and GDNF genes on the basis of improved Tet-on system, then package them into lentivirus(Lv-TH-GDNF). To investigate the regulation of TH and GDNF genes expression in the presence of tetracycline antibiotics in vitro or in vivo. Furthermore, to study the protective and therapeutic and protective effects of the Lv-TH-GDNF intracerebral transfer on a rat PD model. Methods 1. The rat GDNF and TH genes were obtained respectively from the cerebellum of a P0 SD rat and the adrenal glands of a mature SD rat with Polymerase Chain Reaction (PCR). The GDNF and TH genes were cloned into the lentiviral transfer vector pRRL-Palb-Luciferase-Ires-RFP containing TRE to construct pRRL-Palb-TH-Ires-GDNF, which could be regulated by the improved Tet-on system. Then, the lentivirus vector (Lv-TH-GDNF)were packaged through lentiviral four plasmids co-transfected into human embryonic kidney cell line-293T by Lipofectamine 2000. The titer was determined by real-time fluorescence quantitative PCR ( real-time qPCR). Hela cells were infected by obtained Lv-TH-GDNF and rtTA2S-M2 virus in the identical multiplicity of infection (MOI). The expression regulation of TH and GDNF mRNA and protein were examined by real-time qPCR and Western blot in different concentration of doxycycline(DOX). We compared the effect among Hela group, Hela+Lv-TH-GDNF group, Hela+ Lv-TH-GDNF+ rtTA2S-M2 + DOX(0,5 mg·L-1)group. 2. To explore the protective and therapeutic and protective effect of Lv-TH-GDNF gene transfer on a rat PD model. For the observation of the protective effect, a single left intrastriatal injection of Lv-TH-GDNF together with rtTA2s-M2 viruses was carried out one week before the injection of 6-OHDA. The expression of TH and GDNF genes was induced by DOX. We compared the effect among non-injected-treated rats, PBS-pre-treated rats and Lv-TH-GDNF+rtTA2S-M2+DOX-pre-treated rats. For the observation of therapeutic effect, the Lv-TH-GDNF together with rtTA2s-M2 viruses were injected into the right striatum five weeks after the 6-OHDA delivery at medial forebrain bundle(MFB)in right substantia nigra(SN). The expression of GDNF and TH genes was induced by DOX. We compared the effect among non-injected-treated rats, PBS-treated rats, Lv-TH-GDNF+rtTA2S-M2-treated rats and Lv-TH-GDNF+rtTA2S-M2+DOX-treated rats. For the observation of the protective effect, a single left intrastriatal injection of Lv-TH-GDNF together with rtTA2s-M2 viruses was carried out one week before the injection of 6-OHDA. The expression of TH and GDNF genes was induced by DOX. We compared the effect among non-injected-treated rats, PBS-pre-treated rats and Lv-TH-GDNF+rtTA2S-M2+DOX-pre-treated rats.The effects of Lv-TH-GDNF were evaluated by apomorphine-induced rotational behavior, TH immunohistochemistry assay in the SN,measurement of DA and DOPAC level in the transplanted striatum by high performance liquid chromatography-electric chemical discharge (HPLC-ECD) . Immunofluorescence fluorescence double staining, RT-PCR and Western blotting were performed to check the expression of TH and GDNF in the transplanted striatum. Results 1. Tests showed that the recombinant lentiviral transfer vector plasmids (pRRL-Palb-TH-Ires-GDNF) were constructed correctly, the titer of lentiviral vector particles(Lv-TH-GDNF) was 2.1×1011 TU·L-1. The real-time PCR and western blot showed increased expression of mRNA and clear protein bands of TH and GDNF in the Dox-positive group respectively, but not in the Dox-negative group. 2.①In the protective effect of Lv-TH-GDNF on PD in rat , the results showed that , 4 weeks post the injection of 6-OHDA(or 5 weeks post transplantation), the apomorphine-induced contralateral turning effect was significantly improved in Lv-TH-GDNF+ rtTA2s-M2+DOX -pre-treated rats as compared with PBS-pre-treated rats ( p<0.01 ) , the number of TH-positive cells in the lesion side SNpc and DA levels in the lesion side striatum were significantly higher in Lv-TH-GDNF+ rtTA2s-M2+DOX -pre-treated rats than that in PBS-pre-treated rats ( p<0.01 ). The results of RT-PCR and western blotting showed that the expression of exogenous TH and GDNF genes was significantly enhanced in the lesion side striatum in Lv-TH-GDNF+ rtTA2s-M2+DOX-pre-treated rats as compared with those of PBS-pre-treated rats( p<0.01 ).②①In the therapeutic effect of Lv-TH-GDNF on PD in rat , the results showed that, 9 weeks post the injection of 6-OHDA(or 4 weeks post transplantation), the apomorphine-induced contralateral turning effect was significantly improved in Lv-TH-GDNF+rtTA2s-M2+DOX-treated rats as compared with PBS-treated rats ( p<0.01 ),the number of TH-positive cells in the lesion side SNpc and the content of DA and DOPAC in the lesion side striatum were significantly heightened in Lv-TH-GDNF+ rtTA2s-M2+DOX-treated rats as compared with PBS-treated rats ( p<0.01 ) , which were not in Lv-TH-GDNF+ rtTA2s-M2-treated rats( p>0.05 ). The result of immunofluorescence fluorescence double staining showed that cells having the coexpression of TH and GDNF in the lesion side striatum had a lot in Lv-TH-GDNF+rtTA2s-M2+DOX-treated rats, which were rare in Lv-TH-GDNF+rtTA2s-M2-treated rats, nothing was founded in PBS-treated rats. The result of Western blotting showed that the expression of GDNF and TH was significantly raised in the striatum of Lv-TH-GDNF+rtTA2s-M2+DOX-treated rats as compared with those of Lv-TH-GDNF+rtTA2s-M2-treated rats and PBS-treated rats( p<0.01 ), the expression in Lv-TH-GDNF+rtTA2s-M2-treated rats had the increased tendency, but not significant , as compared with that of PBS-treated rats ( p>0.05 ).there weren't significant difference in Lv-TH-GDNF+rtTA2s-M2-treated rats between PBS-treated rats ( p>0.05 ).②In the protective effect of Lv-TH-GDNF on PD in rat , the results showed that , 4 weeks post the injection of 6-OHDA(or 5 weeks post transplantation), the apomorphine-induced contralateral turning effect was significantly improved in Lv-TH-GDNF+ rtTA2s-M2+DOX -pre-treated rats as compared with PBS-pre-treated rats ( p<0.01 ) , the number of TH-positive cells in the lesion side SNpc and DA levels in the lesion side striatum were significantly higher in Lv-TH-GDNF+ rtTA2s-M2+DOX -pre-treated rats than that in PBS-pre-treated rats ( p<0.01 ). The results of RT-PCR and western blotting showed that the expression of exogenous TH and GDNF genes was significantly enhanced in the lesion side striatum in Lv-TH-GDNF+ rtTA2s-M2+DOX-pre-treated rats as compared with those of PBS-pre-treated rats( p<0.01 ). Conclusion The plasmid of recombinant lentiviral transfer vector(pRRL-Palb-TH-Ires-GDNF)regulated by the improved Tet-on system carrying TH and GDNF genes had been constructed successfully and lentiviral vector ( Lv-TH-GDNF ) was packaged successfully, the expression of TH and GDNF in Lv-TH-GDNF was effectively regulated by tetracycline antibiotics without basal expression in vitro. The expression of TH and GDNF genes could be induced by DOX after the Lv-TH-GDNF was intrastriatal transferred in a rat PD model, which could significantly retard progressive degeneration of dopaminergic neurons of nigrostriatal system from 6-OHDA-induced injury and significantly remit the apomorphine-induced contralateral turning effect in the rat PD model , it suggests that the transfer of Lv-TH-GDNF has the neuroprotective and therapeutic effect on PD rat. Otherwise, there was a weak basal activity in the expression of TH and GDNF regulated by the improved Tet-on system in vivo. |
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