Cloning And Eukaryotic Expression Of Rat TH Gene And Experimental Study On Rotational Behavior Of Parkinson Disease Rat Model After Transplanting TH Engineered NIH-3T3 Cells

Objective: l.To obtain the re cDNA sequence of rat tyrosine hydroxylase(TH) and construct an eukaryotic vector-plasmid pcDNAS. 1/TH, Synthesizing THmRNA in vitro. To observe the expression of pcDNAS. 1/TH and THmRNA in eukaryotic li. 2. To prepare 6-hydroxydopamine (6-OHDA) induced Parkinson disease (PD) rat model and observe the expression of plasmid pcDNAS. 1/TH in PD rat model.Methods: l.The full ength of cDNA from rat TH was used as template and the specific oilgonucleotide primers were designed for Polymerase Chain Reaction(PCR). An eukaryotic vector, plasmid pcDNAS. 1/TH was then constructed. After DNA sequence analysis, the newly constructed pcDNAS. 1/TH and THmRNA was transfected in vitro to the cultured NIH-3T3 cells. The expression of TH was tested by using a method of immunocytochemistry. 2. By mircroinjecting 6-OHDA into rat unilateral medial forebrain bundle to induce the damage of unilateral mesencephalic dopamine neurons, a well-known PD rat model was established. After transplanting NIH-3T3-TH engineered cells into the lesioned striatum by stereotaxic technique, the rats were tested for rotational behavior induced by apomorphine.Results: 1. The re cDNA seguence of rat TH was harvested. An eukaryotic vector, plasmid pcDNAS. 1/TH was then proved correct.DNA sequence analysis indicated that the inserted fragment was consistent with that of rat THmRNA(genebank 012740). The expression of TH was shown in NIH-3T3 by immunocytochemistry staining. There exists some differences between the expression of pcDNAS. 1/Th and THmRNA in NIH-3T3 cells. The expression of pcDNAS. 1/TH seemed more sready and long. 2.After transplanting NIH-3T3-TH engineered cells into the lesioned striatum , the engineered NIH-3T3-TH cells can steadily express TH for 4 weeks which was proved by immunohistochemistry staining. The rotational behavior of PD rat model was significantly reduced by 40.3 % ~ 70.2 % after transplanting NIH-3T3-TH engineered cells.Conclusion: The recombined eukaryotic vector, plasmid pcDNAS. 1/TH is a valuable tool to the treatment for PD. By using ex vivo method, NIH-3T3-TH engineered cells can significantly improve the rotational behavior of PD rat model. These results offered helpful evidences in the basic research and clinical utility of gene therapy on Parkinson disease in the future.