慢病毒导入型人白细胞介素10基因表达质粒的构建及其对CCI大鼠的镇痛作用

The Construction of Lentiviral-delivery Human Interleukin-10 Expression Vector and the Abirritative Effect of Pain on CCI Rats

目的:构建含人白细胞介素10基因的慢病毒载体LV-hIL-10,观察鞘内注射LV-hIL-10对坐骨神经松结扎模型(chronic constriction injury,CCI)大鼠的镇痛作用。方法:应用PCR从pCYIL-10质粒上扩增hIL-10基因,把hIL-10基因亚克隆至pWPXL质粒上得到重组质粒pWPXL-hIL-10,将pWPXL-hIL-10与psPAX2、pMD2.G共转293T,收集上清浓缩后制备LV-hIL-10。同时将空质粒pWPXL-GFP与psPAX2、pMD2.G共转293T,收集上清浓缩后作实验对照。纯种健康清洁级成年雄性SD大鼠135只,随机分为9组:CCI疼痛模型4组(C0、C1、C2、C3),假手术4组(S0、S1、S2、S3)和正常对照组(N组)。给药组在蛛网膜下腔分别注射LV-hIL-10(C1组、S1组)、LV-GFP(C2组、S2组)、生理盐水(C3组、S3组),对照组C0组、S0组不做鞘内置管,不给药。各组在手术造模成功实施鞘内注射后观察LV-hIL-10组不同时间点痛阈的改变及脊髓、脑皮质、海马中的hIL-10 mRNA和蛋白表达。结果:获得IL-10基因片段,成功重组pWPXL-hIL-10载体,经序列验证无误。质粒共转染293T细胞后,获得了高滴度(2×1010)、高纯度的LV-hIL-10病毒颗粒。CCI大鼠鞘内注射LV-hIL-10后痛觉异常明显缓解,脊髓、脑皮质、海马中的IL-10表达上调,脊髓中IL-10表达上调最显著。结论:鞘内注射慢病毒感染导入型人IL-10表达质粒对CCI大鼠有明显镇痛效应。

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 × 10^10） 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.