中脑导水管周围灰质立体定向注射单纯疱疹病毒Ⅰ型扩增子载体介导人前脑啡肽原基因对甲醛炎性痛大鼠的镇痛效应

Periaqueductal gray administration of HSV-Ⅰ amplicon vector-mediated HPPE gene therapy of nocicepion in rats with formalin-induced pain

目的:观察中脑导水管周围灰质(PAG)立体定向注射介导人前脑啡肽原基因(HPPE)的单纯疱疹病毒Ⅰ型(HSV-Ⅰ)扩增子载体对甲醛炎性痛大鼠的镇痛效应。方法:成年雄性SD大鼠随机分为pHSVIRES-HPPE-LacZ载体SHPZ组,pHSVIRES-LacZ空白载体SHZ组和生理盐水NS组(n=51)。3组大鼠PAG立体定向注射SHPZ,SHZ和NS后于第3天、1周、2周、3周、4周、5周和6周时进行福尔马林实验观察镇痛效应。对注射后1周组(每组取9只)进行福尔马林实验后处死大鼠取出PAG标本观察HPPE转染表达情况,通过X-gal染色原位检测报告基因LacZ的表达,逆转录聚合酶链式反应(RT-PCR)方法检测HPPEmRNA的表达,放射免疫法检测PAG组织中L-脑啡肽(L-EK)浓度。结果:PAG立体定向注射SHPZ后,X-gal原位染色、RT-PCR和放射免疫法检测均显示外源HPPE能有效表达。对甲醛诱发的炎性疼痛中,疼痛加权评分(PIS)于第1周、2周、3周、4周、5周时在第1相和第2相与空白载体对照组或生理盐水组比较差异有统计学意义(P<0.05),于第6周时第2相PIS与空白载体对照组或生理盐水组比较差异有统计学意义(P<0.05),对甲醛的镇痛效应可被腹腔注射纳洛酮拮抗。结论:SHPZ能成功地将外源HPPE基因转染到PAG神经元细胞中,并使之有效表达,HPPE的表达产物对甲醛炎性痛大鼠产生镇痛效应。

Objective To investigate the antinociceptive effect of periaqueductal gray（PAG）administration of herpes simplex virus type-1（HSV-I）amplicon vector-mediated human preproenkephalin gene（HPPE）.Methods Sprague-Dawley rats weighting 260-320 g were randomly divided into pHSVIRES-HPPE-LacZ（SHPZ）group,pHSVIRES-LacZ（SHZ）group,and saline（NS）group which included 3 d,1 week,2 week,3 week,4 week,5 week,and 6 week groups（n=51）.The rats were anesthetized with intraperitoneal chloral hydrate（300～350）mg/kg.Rats were PAG delivered with recombinant HSV-Ⅰ amplicon vector SHPZ,SHZ or NS.One week after PAG administration 9 rats in each group were scacrificed and lumber segment of the spinal cord was removed for determination of expression of LacZ by X-gal staining and HPPE mRNA expression by reverse transcription-polymerase chain reaction and L-enkephalin content by radioimmunoassay in PAG.Formalin 50 μL（5%）was injected into the left hindpaw,and pain intensity scoring（PIS）was used to assess the antinociceptive effect.Results After in vivo transferring,neurocyte demonstrated strong positive sigals with X-gal immunohistochemical staining.The expression of HPPE mRNA was detected in PAG after administration of SHPZ.PAG delivery of SHPZ showed antinociceptive effect on formalin-induced pain for 6 weeks compared with SHZ group.Conclusion This amplicon virus can transfer HPPE into rat PAG neural cells and make it express efficiently.PAG administration of SHPZ can produce significant analgesic effect on formalin-induced pain in rats for 5 weeks.