Experimental Study On Gene Silence Of IL-1 Beta By Adenovirus-mediated In Protecting Rats Spinal Cord Injury

Purpose1.To structure recombinant adenovirus vector expressing shRNA targeting IL-1? gene.2.To explore the protective effect and mechanism of adenovirus vector expressing IL-1? small hairpin RNA(shRNA)on spinal cord injury in rats.Methods1.The siRNA was designed based on the nucleotide sequence of IL-1?queried from National Center for Biotechnology Information.And then the shRNA were synthesized.The annealed shRNA product and adenovirus vector were connected to structure the recombinant adenovirus vector shuttle plasmid expressing shRNA targeting IL-1?.Then,the shuttle plasmid and skeleton vector were co-transfected with HEK 293 cells,and the recombinant adenovirus vector expressing shRNA were packaged and amplified.Using the structured recombinant adenovirus vector to infect H9C2 cells.The fluorescence microscope was used to observe the infection.The Western blot assay was used to detect the expression of recombinant adenovirus vector expressing shRNA targeting IL-1? gene.2.Seventy-two adult male SD rats were randomly assigned to 4 groups incluing the Sham,the Vehicle,the Ad-GFP and the Ad-shIL-1?groups.In the Sham group,a laminectomy was performed only.In the Vehicle group,3 ?l normal saline was injected into the cord at T12 using a 5-?l micro-syringe with a 33-gauge needle at a rate of0.2 ?l /min 7 days before SCI.In the Ad-GFP group,3 ?l Ad-GFP was injected 7 days before SCI.In the Ad-shIL-1? group,3 ?l Ad-sh IL-1?was injected 7 days before SCI.The fluorescence microscope was used to observe the expression of green fluorescence.The Basso-Beattie-Bresnahan(BBB)score was used to evaluate motor function of hind limbs.The immunofluorescence staining was used to observe the expression of NeuN-positive cell.The expression of IL-1? in spinal cord tissues was detected by Western blot and the expressions of TNF-?and IL-6 in spinal cord tissues was detected by Enzyme-linked immunosorbent assay(ELISA).The changes of apoptosis index(AI)were detected by TUNEL assay.Results1.The sequences of shRNA adenovirus vector shuttle plasmid were consistent with the sequences of target shRNA based on the sequencing results.2.It was observed the expression of green fluorescence in injured spinal cord tissue in the Ad-GFP and Ad-shIL-1? groups at one day after spinal cord injury.The motor function was improvement in the Ad-shIL-1? group compared to the Vehicle and Ad-GFP group at 7,14 and 21 days after spinal cord injury.The NeuN-positive cells increased in the Ad-shIL-1? group compared to that of the Vehicle and Ad-GFP group at 21 days after spinal cord injury.What's more,the down regulation of IL-1? decreased the levels of IL-1?,TNF-? and IL-6 one day after spinal cord injury.The level of AI were significantly decreased in the Vehicle,Ad-GFP and Ad-shIL-1? groups compared to that of the Sham group,the level of AI in Ad-shIL-1? group was lower than that of Vehicle group and Ad-GFP group.Conclusions1.Recombinant adenovirus vector expressing shRNA were constructed successfully and could down-regulate the expression of IL-1?.2.Down regulation of IL-1?could promote the survival and motor function recovery of SCI in rats,and had a protective effect on SCI.3.Down regulation of IL-1?could protect the spinal cord through the anti-inflammatory effect.4.Down regulation of IL-1?could protect the spinal cord through the anti-apoptotic effect.