Analysis of TGF-β1 Gene Expression in Contused Rat Spinal Cord Using Quantitative RT-PCR

Abstract

We have used northern blot analysis and quantitative reverse transcription polymerase chain reaction (RT-PCR) to determine the postinjury expression profile of the transforming growth factorial (TGF-β1) gene in the contused rat spinal cord. Spectrophotometric estimates of total sample RNA and quantitative analyses of cyclophilin mRNA using RT-PCR served as controls for comparisons between samples. No changes in cyclophilin gene expression were found at any postinjury survival times. The results of the TGF-β1 analyses, which were carried out on spinal cord samples taken at postinjury intervals ranging from 6 h to 10 days, show that the amount of TGF-β1 mRNA present in spinal cord increases rapidly following injury, reaching maximum levels 7 days postinjury. Unoperated control samples contained approximately 2 × 10⁸ molecules of TGF-β1 mRNA/0.5 μg total RNA. By 1 day postinjury, the amount of TGF-β1 mRNA in the cord had increased by a factor of 2.5 to 5 × 10⁸ molecules/0.5 μg total RNA. At 7 days postinjury, there were approximately 15 × 10⁸ molecules of TGF-β1 mRNA/0.5 μg total RNA. By 10 days postinjury the amount of TGF-β1 mRNA present in the spinal cord had declined to 8 × 10⁸ molecules of TGF-β1 mRNA/0.5 μg total RNA, a value similar to that observed at 3 days postinjury. The roles that TGF-β1 might play in modifying cellular responses in injured spinal cord are discussed.

Key words:

cytokine; cyclophilin; inflammation; reactive gliosis; regeneration; spinal cord injury; transforming growth factor beta