Construction Of Recombinant Adenovirus Vector Expressing ShRNA And Over-expression Targeting IL-1β Gene In Rats

Objective:Neuropathic pain is a type of pain which is very difficult to curing in clinical. The mechanism for the occurrence and development of neuropathic pain and a number of cytokines and protein were involved. Interleukin-1 beta(IL-1β) is a very important cytokine among these, the increase for the expression of IL-1β is played a critical rule in the occurrence and development of neuropathic pain. After peripheral nervous injury, astrocytes are activated and followed by the release of Interleukin 1 beta(IL-1β). IL-1β is involved in the onset and maintenance of neuropathic pain through PKA and PKC cell signal transduction pathways and the specificity of down-regulation of IL-1β can alleviate the pain behaviors effectively. We can down-regulating the expression of IL-1β genetically by the RNA interference(RNAi) technique. The theory for this technique is that we can design and synthesize the small interfering RNA(si RNA) targeting IL-1β to suppress the expression of IL-1β at a posttranscriptional level. Short hairpin RNA(sh RNA) is a kind of RNA similar to double-stranded RNA and designed and synthesized on the basis of the sequence of small interference RNA(si RNA). Compared with si RNA, sh RNA could inhibit the expression of target gene more stably and efficiently. However, simplex sh RNA could not enter in cells to down-regulate target gene efficiently, it must rely on some vectors. Adenovirus vectors have wide host range, high infection efficiency and stable expression in host cells. In present study, recombinant adenovirus vector expressing sh RNA targeting IL-1β was constructed to regulate the expression of target gene specifically. Therefore, we constructed the recombinant adenovirus vector expressing sh RNA and over-expression recombinant adenovirus vector targeting IL-1β gene to searching for an efficient treatment. So the specific objectibe is to construct recombinant adenovirus vector expressing sh RNA and over-expression recombinant adenovirus vector targeting IL-1β gene and detect its effect on the expression of target gene. Methods:Construct the recombinant adenovirus vector expressing sh RNA targeting IL-1β gene:Three si RNAs were designed on the basis of the nucleotide sequence of IL-1β obtained from NCBI and then three sh RNAs(sh RNA1, sh RNA2 and sh RNA3) were synthesized. The annealed sh RNA product and adenovirus vector p HBAd/U6/GFP digested by Bam HⅠand Eco RⅠwere connected to construct the recombinant adenovirus vector shuttle plasmid expressing sh RNA targeting IL-1β. Then put the shuttle plasmid to transforming the rat competent cell DH5α. Put the bacterial liquid to Nissan Shanghai Biological Technology Co., Ltd for the sequencing. After sequencing, HEK 293 cells were co-transfected by the shuttle plasmid and skeleton vector and three r Ad/sh RNAs(r Ad/sh RNA1, r Ad/sh RNA2 and r Ad/sh RNA3) were packaged and amplified. Rats H9C2 cells were infected by r Ad/sh RNAs and fluorescence microscope was used to observe the infection efficiency. The effect of r Ad/sh RNAs on the expression of target gene was detected by Western Blot.Construct the over-expression recombinant adenovirus vector targeting IL-1β:The nucleotide sequence of IL-1β obtained from NCBI. The primer was designed and the amplification for IL-1β gene was completed by PCR. The adenovirus vector p HBAd/MCMV/RFP and the amplification products were digested by Eco R I and Not I. Then the gene fragments and vectors connected to construct the recombinant adenovirus vector shuttle plasmid overexpression IL-1β. Then put the shuttle plasmid to transforming the rat competent cell DH5α. Put the bacterial liquid to Nissan Shanghai Biological Technology Co., Ltd for the sequencing. After sequencing, HEK 293 cells were cotransfected by the shuttle plasmid and skeleton vector and the over-expression recombinant adenovirus vector targeting IL-1β(r Ad/IL-1β) were packaged and amplified. Rats H9C2 cells were infected by the r Ad/IL-1β and fluorescence microscope was used to observe the infection efficiency. The over-expression of IL-1β was proved by Western Blot. Results:The sequencing results showed that the sequences of three sh RNAs adenovirus vector shuttle plasmid were consistent with the sequences of three designed sh RNAs; The sequences of the recombinant adenovirus vector shuttle plasmid overexpression IL-1β was consistent with the sequences of the nucleotide sequence of IL-1β we obtained from NCBI. The r Ad/IL-1β, r Ad/sh RNA1, r Ad/sh RNA2 and r Ad/sh RNA3 were constructed successfully. The r Ad/sh RNA1, r Ad/sh RNA2 and r Ad/sh RNA3 could down-regulate the expression of IL-1β in rats H9C2 cells and the down-regulation effect of r Ad/sh RNA2 was the most significant. On the other hand, the r Ad/IL-1β could up-regulate the espression of IL-1β obviously. Conclusion:The recombinant adenovirus vector expressing sh RNA targeting IL-1β gene can provide an efficient treatment for the neuropathic pain. The application prospect of RNAi for the treatment of neuropathic pain is very broadness.