Regulation Of Stem Cell Differentiate Into Dopaminergic Nerve By Extracts From Plastrum Testudinis Promote

Objective @In Parkinson's disease(PD),it is precisely the TH+ dopaminergic cells of the subst-antia nigra whose degeneration paralles the symptoms of the disease.At present,PD is treated by drugs but drugs cann't step down the degeneration of substantia nigra ne-uron,and the effect of drugs decrease for long time use while untoward reaction enhanced.Stem cells are capable of self-renewal and differentiation into many cell types including TH+ cell.For Parkinson's,the goal of growing replacement tissue from stem cells is attractive because this approach may circumvent problems with tissue availability and immunological rejection.Many kinds of cells,including embryonic stem cells(ESCs),neuron stem cells(NSCs)and mesenchymal stem cells(MSCs),have been considered as candidates for transplantation therapy. @Tyrosine hydroxylase(TH)is the first and rate-limiting enzyme for dopamine biosynthesis,and the important molecular events for the determination of the catecholaminergic phenotype at the embryonic stage and the TH enzyme expression level in catecholaminergic cells at postnatal developmental and adult stages.The regulation of its expression appears to be complex and implies different mechanisms depending of the site of expression.Understanding the molecular basis of TH gene expression will enable us to rationally intervene to direct differentiation of cells toward the desired phenotype,as well as permitting promoter-driven selection of the TH+cells. @Methods @For the study of effect of PTE on the TH gene,we produced mTHpro-Luc which contains about 1.2kb(-1115/+3)of the mouse.TH gene and the region was ligaged to a firely luciferase reporter plasmid,PGL₃-basic,and sequenced before use.P19 cell were transfected with the calcium phosphate co-precipitation method.Transfected cells were harvested after drugs treatment of 6h,12h,24h or 36h for luciferase activity measurement. BMP4 plays a critical role in the early development of nerve differentiation.The effects of BMP4 on differentiation dopaminergic neuron were reported previously by other study groups.So we want to study the role of BMP4 in the effect of PTE on TH gene.To operate RT-PCR got cDNA of P19 cell,and clone the sequence of BMP4 mRNA then the region was ligaged to pEGFP plasmid,and sequenced before use.pEGFP-BMP4 and the TH promoter were transfected to P19 cell with the calcium phosphate co-precipitation method and the cells were treated by 30ug/ml PTE for different times after transfection. Plastrum Testudinis Extracted with ethyl acetate(PTE)could increase MSCs proliferation was previously described.NSCs from embryo of rat were cultured for study PTE'S effection on differentiation of NSCs.Passageâ…¡,â…¢andV NSCs were treated by Brdu for 24h and then change the culture medium with 30ug/ml PTE for another 5 days.After that Cells were harvested and incubated with TH and anti-Brdu antibody,flow cytometry(FeM) was used for cells phaenotype.At the same time TH immunocytochemistry was used for cells treated by 30ug/ml PTE for 5 days only. @Results @The luciferase activity stronger in P19 cells of TH promoter plasmid than PGL3-basic plasmid.The experimental results of the analysis suggest that stearic acid ethyl ester(S-6), (+)-4-Cholesten-3-one(Sg)increase the luciferase activity,The effect of S6 on TH promoter transcription increased by time.Luciferase activity was inhibited by Retinoic acid(RA)all the time.TH effect of PTE on luciferase activity was reduced by RA.BMP4 could increase the luciferase activity in earlier period(6h)but inhibit later(24h).BMP4 could increase the luciferase activity in earlier period(6h)but inhibit later(24h). @The NSC results suggest PTE obviously increases the numbers of TH positive cells, especially passageâ…¡andâ…¢,the percentages of TH-positive cell were about 45%. @Conclusions @The results demonstrate the TH promoter has activity in P19 cell.PTE has been demonstrated to up-regulate the TH promoter activity by dose-dependent and timedependent style in P19 cell.And it may caused by PTE were mixture,stearic acid ethyl ester and(+)-4-Cholesten-3-one dose positive regulation and RAR dose negative regulation in the style;BMP4 up-regulation promoter activity in short time,PTE and BMP4 use together to remove the inhibition of the TH promoter by BMP4 at 24h and may need new protein cooperation;NSC induced to TH+cells by PTE.