Experimental Studies For Transfection Of SCs With PEGFP-N₁/GDNF,pcDNA3.1(+)/GDNF And Construction Bioactive Artifical Nerve Chamber

PART IA comparison of different techniques for SCs (Schwanncells) culture and purification in vitro.Objectives: To obtain an optimal technique of SCs culture for tissue engineering.Methods: SCs were harvested either from rat dorsal root ganglions or from brachial and sciatic nerves. Different exposure time of arabinoside cytarabine were used . The nerve growth factor or bovine pituitary extract (BPE) were added to the medium. The results of different techniques were compared.Results: The brachial and sciatic epineurium were gently stripped. SCs were exposed to arabinoside cytarabine for 16-24 hours. 20% of FCS (total name) and 50mg/L of BPE were added to the DMEM. The rapid low concentration trypsinization and differential adhension methods were used. The confluent time of SCs was shortened to 6 days and the final density was more than 2×10⁷SCs/ml. The harvested SCs were identified with rabbit anti-S100 protein antibody by SABC ICH and by Cy3 IFA methods. The purity of cells was more than 95%.Conclusion: Our technique was probably the optimalmethod to harvest SCs for tissue engineering.Part II Cloning of Rat GDNF gene and reconstructionofpEGFP-N₁/GDNF, pcDNA3.1(+)/GDNFObjectives: To clone rat glial cell line-derived neurotrophic factor (GDNF) gene and to reconstruct the pEGFP—N₁/GDNF, pcDNA3.1(+)/GDNF.Methods: Neural tissue of one-day old SD rat was used to extract total RNA. A 560bp positive cDNA band was obtained by RT-PCR. This fragment was cloned into the pEGFP—N₁ and pcDNA3.1(+). The cloned fragment was sequenced and identified by restricting enzymes.Results: RT-PCR product was a 560bp specific segment. Analysed by restricting enzymes digestion and DNA sequencing, the cDNA3.1(+)/GDNF recombination was 5.4kb and 560bp, while the pEGFP —N₁/GDNF recombination was 4.7kb and 560bp. The DNA sequencing revealed that GDNF cloning was successful.Conclusion: The pEGFP-N₁/GDNF and pcDNA3.1 (+)/GDNF recombinations could be used in further research.Part III Transfection of SCs with pEGFP—N₁/GDMnpcDNA3.1(+)/GDNFObjective: To examine the effectiveness of rat GDNF gene transfection to Schwann cells.Methods: The pEGFP—N,/GDNF,. pcDNA3.1 (+)/GDNF were transfected to SCs by Lipofectamine2000 and the positive SCs were selected by G418. The level of GDNF mRNA in GDNF-SCs and normal SCs was tested by RT-PCR. The level of GDNF protein was examined by Immunofluorescence assay.Result: (l)The GDNF cDNA band was detectable in Different SCs groups, but they were more prominent in GDNF-SCs groups than that of normal SCs. (2)Different SCs groups were immunol fluorence positive when examined with anti-GDNF by immunofluorescence assay, but the red Fluorescence in...