The Study Of Parkinson Disease's Relating Genes In Rat

Part I Creation of rat model for Parkinson Disease Objective: To create successfully the rat model of for Parkinson's desease Method: Forty male healthy SD rats with body weight of 240-270g are adopted. The single side PD animal model is created with stero-orientationly injecting 6-OHDA into the right ventral tegmental area (VTA) and the right median forebrain bundle (MFP). From the second week on, the Apomorphine induced rotation behavior is detected once a week in the second, third, and forth week after the operation.Result: All rats survive the 6-OHDA injections. Thirty-two of the 40 rats develop deflexion of head, retardation of movement, rigor of tail and spontaneous rotation in the early stage after injection. 1-10 minutes after the abdominal injection of APO, the rats develop rotation behavior, with the manifestation of quick movement toward the normal side, or rotating around the outer circle (>7 turns/min), excitation and rigor of tail. Conclusion: To evaluate whether the rat model is successful or not, the drug inducing test, which means the success of animal model for PD when the rat turns toward the healthy side with 7 or more turns per min, is often used. The present experiment adopts the method of stereo-orientation injection of 6-OHDA to VTA+MFP. The rate of successful animal model in the study is 80%.Part II: Screen for the PD related genesObjective: To select the known and unknown gene fragments and to screenfor the PD related genes using Northern Blotting.Method: The total RNA from brain tissue of substantia nigra of PD rats isextracted at the 1st, 2nd, 4th and 8th week and comparison is made between itand that from normal rats. The total RNA, after RNA formaldehydedegenerated sepharose electrophoresis and separation and Northern transfer, isadsorbed unto colloxylin membrane. Then ECL labeled Selenoprotein P andan unknown gene fragment are used as probes to hybridize with the RNA onthe colloxylin membrane. And the hybridized result is obtained withenzyme-linked immunosorbent assay. Finally, analysis of OD is made usingONE-Dscan software.Result: At the 1st, 2nd, 4th and 8th week, in the substantia nigra of PD rats, theexpression of Selenoprotein P and the unknown gene increases. At the 8thweek, the expression of Selenoprotein P increases significantly in the PD rats.Conclusion: Selenoprotein P and the unknown gene seem to be the generelating to PD.PartIII: The construction of cDNA library of PD ratObjective: To construct the cDNA library of PD rat; to obtain the full-lengthgene relating to the disease from the cDNA library and to provide basis forfurther studyMethod: The cDNA library is constructed using the method of the longdistance PCR (LD-PCR). The followings are the main steps: l.the synthesis of single chain cDNA; 2. the synthesis of the second chain using the LD-PCR; 3.the digestion of proteinase K; 4. Sfi I digestion; 5. cDNA size fractionation by CHROMA SPIN-400 and collection of the fragments greater than 400bp, 6.ligate cDNA to phage carrier; T.package library and titer the library; S.the amplification of cDNA library; 9. the analysis of the inserted fragments in cDNA; lO.the release of cDNA library and titer of phage plasmid. Conclusion: 1. the volume of library is 1.3 106;2. the titer of phage is detected to be 1.6 X 1010pfu/ml;3.the mean size of the inserted fragments is 800bp with a rangeof500bpto2kb.