The Construction of Lentiviral-delivery Human Interleukin-10 Expression Vector and the Abirritative Effect of Pain on CCI Rats / 中国生物工程杂志

ABSTRACT

Objective:To construct the lentiviral-vector encoding human interleukin-10 protein(LV-hIL-10) and to observe the effect of LV-hIL-10 on controlling neuropathic pain via intrathecal administration in CCI rats. Methods:hIL-10 gene fragment was isolated and amplified from pCYIL-10 plasmid by PCR, and was cloned into pWPXL. The recombinant plasmid pWPXL-IL-10,envelope plasmid pMD2.G and packaging plasmid psPAX2 were cotransfected into 293T cells, LV-hIL-10 is prepared by concentrating the collected supernatant .At the same time, the empty plasmid pWPXL-GFP,pMD2.G and psPAX2 were cotransfected into 293T cells, LV-GFP is prepared for contrast.135 sheer breed pathogen-free adult male Sprague-Dawley rats divided into 9 arrays at random CCI models 4 arrays (C0,C1,C2,C3), sham operatived rats 4 arrays (S0,S1,S2,S3) and a normal contrast array (N), each respectively intrathecal injection LV-hIL-10 (C1,S1)、LV-GFP (C2, S2),isotonic Nachloride (C3,S3) and control (no implanted catheters and no administration, C0,S0), the pain threshold of each array and the expression of mRNA and protein of IL-10 in spinal cord,pallium and hippocampus on different time were observed after intrathecal administration LV-hIL-10 in successful CCI model rats . Results:The hIL-10 gene fragment was obtained from pCYIL-10 plasmid, pWPXL-hIL-10 was recombinated successfully. the cloned gene segment was validated by DNA sequencing .High titer(2?1010)and highly purified LV-hIL-10 particles were obtained by three plasmids were cotransfected into 293T cells. The mechanical allodynia and thermal hyperalgesia were alleviated via intrathecal injection LV-hIL-10 in CCI rats. The overexpression of IL-10 were detected in spinal cord,pallium and hippocampus , especially in the spinal cord .Conclusions:The mechanical allodynia and thermal hyperalgesia can be relieved by intrathecal injection LV-hIL-10 in CCI rats.