| ObjectiveTo investigate the regulation of expression of CRMPs mRNA by Rho kinase in cultured rat hippocampal neurons.Materials and methodsPrimarily cultured neurons from hippocampus of postnatal rats were treated with Rho kinase activator LPA and inhibitor Y27632 to change Rho kinase's activity.The expression of CRMPs mRNA in neurons was determined by real-time quantitative PCR.Then the neurites were extracted and the change in outgrowth of neurites was measured by Neurite Outgrowth Quantification Assay Kit.Results(1) Establishment of real-time fluorescent quantitative PCR for CRMPs mRNA determination:cDNA reversely transcribed from total RNA was used as standard product,which showed good amplification,standard and melting curves:equal distances between the consecutive amplification curves,well linear regression of the standard curves(correlation coefficient for all genes r~2＞0.98),near 100%of the amplification efficiencies for all genes, about 5%difference between every target gene and the reference gene.The melting cures and gel electrophoresis specifically demonstrated the products of each of the genes,while no primer-dimers and non-specific amplications were observed during the real-time PCR amplification of 43 cycles.(2) Expression of CRMPs mRNA:All genes were detected in the cultured hippocampal neurons of rats.The transcript levels of CRMP1,2,4,5 were evidently high and close to each other.However,CRMP3 showed significantly lower expression level than others.The expression of CRMP1,2,3,4 mRNA was found to be increased in the LPA group,yet displayed a decreased tendency in Y27632 group,and significant difference in mRNA level was found in CRMP1 and CRMP3 when compared with control group.The transcript level of CRMP5 in LPA group was found to be decreased but showed no change in Y27632 group.When cultured neurons were treated with Y27632 after LPA incubation,the expression levels of CRMP1-5 mRNA were observed to be down-regulated,and a significant difference was detected in CRMP1 and CRMP2 when compared with the control and LPA group.The same phenomenon was observed in CRMP3 and in CRMP4 when compared with LPA group.And the same change was found in CRMP5 when compared with the control.When cultured neurons were treated with LPA after incubation with Y27632,the transcript levels of CRMP1-5 were also found to be down-regulated.Among these changes,CRMP1-4 showed the most remarkable changes when compared with the control or LPA group,and CRMP5 had the greatest alteration when compared with LPA group.(3) Determination of neuritis extracts:The optical density was measured to be 0.0426±0.0062 in the control group.When the cells were treated with LPA,the absorbance was determined to be significantly lower than the control,but significantly higher when treated with Y27632.However,when cultured neurons treated with Y27632 after LPA incubation,or treated with LPA after Y27632 incubation,the optical density in the two groups showed no significant changes compared with the control,though they were found to be significantly higher than the LPA group.Conclusions(1) The real-time PCR method was successfully established by using SYBR Green I fluorescence dye to determine the expression levels of CRMPs mRNA.(2) All CRMPs genes were detected in the cultured hippocampal neurons of rats,the transcript levels of CRMP1,2,4,5 were found to be remarkably high and close to each other,but CRMP3 mRNA was found to be lower than others.(3) Activation of Rho kinase induced neurites collapsing,accompanied with increased expression of CRMP1 and CRMP3 mRNA and decreased expression of CRMP5 mRNA;Inhibition of Rho kinase resulted in neurites outgrowth,accompanied with decreased expression of CRMP1 and CRMP3 mRNA;Activation and then inhibition of Rho kinase,or inhibition followed by activation of Rho kinase did not show changes in the neurites outgrowth but decreased in transcription of CRMP1-4 genes.(4) Rho kinase mediated neurites growth by regulating the expression of CRIMPs mRNA.
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