| Objective To design and screen an effective sequence of Vimentin silence by small interfering RNA(si RNA). To observe the inhibition of Glial Fibrillary Acid Protein(GFAP) and Chondroitin Sulfate Proteoglycans(CSPG) caused by si RNA-Vimentin. Meanwhile, hind limb motor and neuroelectricity function of SD rats after spinal cord injury were observed. Furthermore, the expression of GFAP, CSPG and Vimentin as well as the influence of reactive astrogliosis and glial scar formation in spinal cord tissue after trauma were researched.Methods Three si RNA sequence were designed on internet and screened by Western Blot and RT-PCR. The best one was picked out correspondingly. Cortex astrocytes were isolated from new born SD rats, purified and cultured. Astrocytes were identified by GFAP immunochemistry. Astrocytes were divided normal astrocytes group, zero load group and si RNA-Vimentin group. Astrocytes multiplication ability was evaluated by scratch adhesion test and MTT. Influence of reactive astrogliosis and glial scar formation in spinal cord tissue after trauma were assessed by immunochemistry and RT-PCR. The interaction of GFAP, CSPG and Vimentin also was studyed. Simultaneously, 90 adult SD rats were prepared as spinal cord trauma models and randomly divided into three groups: sham operated group(30) was injected 5μl DMEM/F 12, control group was injected 5μl DMEM/F 12 and zero load plasmid(30), si RNA-Vimentin group was injected 5μl DMEM/F12 and si RNA-Vimentin plasmid. Tested the BBB score at 24 h, 1w, 2w, 4w, 8w and 12 w after operation. Tested SEP and MEP at 24 h and 12 w after operation. The expression of GFAP, CSPG and Vimentin in spinal cord tissue were tested at 4w after operation by RT-PCR. To calculate the volume of glial scar at 12 w after trauma. To collect all kinds of data and to carry out statistic analysis.Results si RNA-Vimentin-1073 is the best one among three kinds of designed sequence of Vimentin silence. The transfection efficiency of si RNA-Vimentin-1073 was over 80%, which was highest at 24 h after transfection. Cortex astrocytes were isolated from new born SD rats were identified by GFAP immunochemistry and DAPI stain. Positive cells rate of GFAP was(96.14±1.03)%. Scratch adhesion test results showed that the rate of 2d/0d breadth was(0.7379±0.17), which higher than other groups(P <0.05). At the same time, MTT result showed that si RNA induced Vimintin low expression could inhibit reactive astrogliosis. The expression of GFAP, CSPG and Vimentin in astrocytes that transfected by si RNA-Vimintin were down regulated concurrently. The Western Blot and IOD value of si RNA-Vimintin group were significantly different from others(P<0.05). It was shown that the BBB score was higher in si RNA-Vimentin group at 8w and 12w(P<0.05). The SEP and MEP were also better than other groups at 12 w after operation(P<0.05). The volume of glial scar in si RNA-Vimentin injected group was smaller than other groups at 12 w after trauma(P<0.05). The expression of GFAP, CSPG and Vimentin in spinal cord tissue at 4w after operation were strikingly down regulated at the same time.Conclusion Small interfering RNA sequence of Vimentin was designed successfully. It could inhibit the reactive astrogliosis by down regulated the expression of GFAP, CSPG and Vimentin. The expression of them could be down regulated by local injected si RNA-Vimentin plasmid in spinal cord tissue after trauma. si RNA induced Vimentin silence could cause down regulation of GFAP and CSPG expression. All of them together played a key role in suppressing reactive astrogliosis and glial scar formation after spinal cord trauma. It was provided a better surroundings for axons regeneration and nerval function recovery. |
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