The Effect Of Rho/ROCK-2and ShROCK-2on Apoptosis Of Spinal Cord Neurons In Rats Induced By Hypoxia

Part1. The Expression of ROCK-2in Neuron Apoptosis Induced by HypoxiaObjectiveTo assay the expression of the Rho kinase gene ROCK-2in the process of neuronal apoptosis induced by hypoxia during different phases and investigate their relationships.MethodsSpinal cord neuron cells of neonatal S-D rats were obtained by primary cell culture technique in vitro and then these cultured cells were identified with NeuN immunohistochemistry. Neurons were processed to complete hypoxia for24,48,72and96hrs. At the above-mentioned four phases, ROCK-2and apoptosis genes (caspase-3, caspase-9) were detected with qRT-PCR. Neurons apoptosis was counted by flow cytometry.ResultsThe number of primary spinal cord cells tended to be stable after one month in vitro. Then these cells were identified as spinal neurons by NeuN immunohistochemistry. The apoptosis rate of neurons were5.4%,15.4%,24.9%and36.6%at24h,48h,72h and96h post-hypoxia respectively. Correspondingly, the relative expression of gene ROCK-2were1.02,2.35,7.87and4.41. The relative expression of gene caspase-3were2.15、6.42、3.68and0.89and caspase-9were1.45、1.89、1.42and1.44.Conclusion Rho kinase genes took an important part in the process of neuron apoptosis induced by hypoxia. Moreover, ROCK-2might induce neuron apoptosis by the caspase pathway.Part2. Inhibitory Effect of siRNA on Neuronal Apoptosis Induced by HypoxiaObjectiveTo assay the effect of ROCK-2specific siRNA on apoptosis of spinal cord neuronsMethodsSpinal cord cells of neonatal S-D rats were obtained by primary cell culture technique in vitro. Searching the rat ROCK2gene sequences in GeneBank, constructing the shROCK2carriers, screening the higher inhibition efficiency shROCK2plasmid, then transfecting spinal cord neurons. Total RNA was extracted from the cells, corresponding expression changes of ROCK2and caspase3and caspase-9genes was detected by Real-time qPCR. Total protein was extracted from the cells, corresponding expression changes of ROCK2and caspase3and caspase-9protein was detected by Western blot.Average OD490was detected by MTT,then the cell growth curve was drawed. Flow cytometry technology was applied to detect spinal cord neurons’apoptosis.ResultsIn blank control group, the negative control group, hypoxia group and shROCK2inhibition group of ROCK2relative expression of gene transcription level respectively was1.00,0.94,1.39,0.94. In the corresponding set of relative expression of caspase3was1.00,1.02,1.63.0.90, respectively. In the corresponding set of caspase9the relative expression was1.00,0.98,1.85,0.78, respectively. In blank control group, the negative control group, hypoxia group and shROCK2inhibitory group of ROCK2protein relative expression quantity respectively was:1.00±0.102、1.05±0.061、2.77±0.160、0.79±0.044. In the corresponding set of caspase3protein relative expression was1.00±0.105、0.86±0.075、1.55±0.239、0.79±0.070. In the corresponding set of caspase9protein relative expression was1.00±0.090、 0.96±0.090、2.60±0.250、0.67±0.092. In blank control group, the negative control group, hypoxia group and shROCK2inhibition group of48h cell proliferation rate respectively was:2.24±0.161、2.03±0.051、1.31±0.023、2.61±0.101. In the corresponding set of G2/M phase cells proportion respectively was:15.84±0.66%、12.14±0.44%、8.26±0.44%、20.25±0.66%. Proportion of early apoptosis cells in the corresponding group respectively was1.95%,2.30%,29.81%,0.69%.ConclusionThe above results showed that the ROCK-2specific siRNA significantily inhibited the ROCK-2, caspase-3, caspase-9gene transcription level and protein expression, inhibited the spinal cord neurons apoptosis, promoted the cell proliferation. ROCK-2and caspase3, caspase9changes in the gene transcription level and protein expression level were in parallel relationship, ROCK-2play a role of promoting apoptosis by activating caspase gene way.