转脑啡肽基因移植细胞微囊化对大鼠慢性周围神经损伤的镇痛作用

Analgesic effect of transplantation of microcapsulated enkephalin on rat chronic neuropathic pain

目的:观察微囊化转大鼠脑啡肽原基因(pENK)细胞移植对慢性脊神经压榨性损伤大鼠的镇痛效应。方法:实验于2006-03/2007-04在郑州大学医学重点实验室完成。①实验方法:通过反转录-聚合酶链反应技术可获得大鼠pENK基因,HindⅢ,ClaⅠ双酶切后,同相应双酶切的pLNCX2载体大片段连接,构建成pENK基因反转录病毒载体pLNCX2-Enk,然后用脂质体法将该载体转染PT67细胞,G418筛选,获得携带pENK基因高滴度反转录病毒产毒细胞系。用海藻酸钠-多聚赖氨酸-海藻酸钠微囊包埋后,进行体外培养,定期检测微囊化细胞活性和pENK分泌量变化。同时,将转基因细胞移植于慢性脊神经压榨性损伤大鼠的蛛网膜下腔。②实验分组:SD大鼠60只,其中53只按照Bennett和Xie法制作大鼠慢性左侧坐骨神经压迫性损伤模型,其中42只造模成功。术后1周,将动物按随机数字表法分为3组,每组16只:微囊化转基因细胞移植组、空囊移植组和阴性对照组。微囊化转基因细胞移植组、空囊移植组分别植入微囊化细胞悬液80μL(约300个微囊)、空微囊悬液80μL(约300个空囊),阴性对照组不注射任何悬液。③实验评估:术后2周和8周行甲醛实验,在大鼠一侧后爪掌侧皮下注射体积分数为0.05的甲醛50μL,观察其注射后1h内的痛反应。采用Abbott等所推荐的以缩腿及舔爪时间之和作为行为学反应的指标。注射甲醛后,立即记录大鼠1h内每5min的缩腿及舔爪时间。结果:①术后2周甲醛实验:空囊移植组第一时相的急性痛阶段(注射后5min)和第二时相的慢性痛阶段(注射后41～45min)大鼠的缩腿及舔爪时间都少于微囊化转基因细胞移植组(P<0.01)。而在静息期,3组大鼠的缩腿及舔爪时间差异无显著性意义(P>0.05)。②术后8周甲醛实验:3组大鼠的痛觉行为都呈明显的双时相反应。除了静息期(注射后5～15min),微囊化转基因细胞移植组在各时间点上大鼠的缩腿及舔爪时间均低于空囊移植组(P<0.05)。微囊化转基因细胞移植组大鼠的缩腿及舔爪时间在第一时相的急性痛阶段和第二时相的慢性痛阶段都低于空囊移植组(P<0.05)。结论:微囊化转pENK基因细胞移植对慢性神经痛大鼠有一定的镇痛作用,有望成为运动员慢性疼痛治疗的新方法。

AIM： To investigate the analgesic effect on the neuropathic pain induced by chronic constriction injury （CCI） to rat spinal nerves through the transplantation of microcapsulated rat proenkephalin gene （pENK）-expressing cells. METHODS： The experiment was carded out in the Key Laboratory of Medical Science at Zhengzhou University from March 2006 to April 2007. ①The rat pENK was cloned by reverse transcription-polymerase chain reaction, double digested by restriction enzymes Hind HI and Cla Ⅰ, then inserted into retrovirus vector pLNCX2. The recombinant plasmid pLNCX2-pENKwas transfected into packaging cell PT67 by lipofectamine and the transfectants were selected by G418. pLNCX2-pENK was constructed and a stable virus producing packaging cell line with high titer was established successfully. The genetically modified cells expressing pENK gene were enclosed within the alginate-polylysine-alginate （APA） microcapsules and then cultured in vitro. Microcapsulated cell activity and pENK expression were determined periodically. At the same time, these transgenic cells were transplanted into the subarachnoid space of CCI rats.②Sixty SD rats were used in this study, 53 of them were induced for CCI to left sciatic nerves according to Bennett and Xie＇s method, and 42 models were established successfully. One week later the animals were divided into three groups at random （n =16）： group A was implanted with 80 μL microcapsulated cell suspension （300 APA microcapsules）; group B was implanted with 80 μL microcapsulated suspension （300 empty microcapsules）; group C receiving no injection was taken as positive control group. ③At the 2^nd and the 8^th weeks postoperatively, unilateral hind-paws of rats were subcutaneously injected with 5% formaldehyde （50 μL） to observe the pain reaction within 1 hour. Recommended by Abbott et al＇s method, behavior index was defined as the addition of flinching and licking time every 5 minutes for 1 hour. RESULTS： ①Formaldehyde test at the 2^nd week postoperatively： At acute pain stage of the first phase （5 minutes after injection） and chronic pain stage of the second phase （41-45 minutes after injection）, the flinching and licking time of group B was shorter than that of group A （P 〈 0.01）. In static phase （5-15 minutes after injection）, there was no significant difference among three groups （P 〉 0.05）. ②Formaldehyde test at the 8^th week postoperatively： The pain reaction occurred in all the rats with obvious double phase manner. Except static phase, the flinching and licking time of group A was shorter than that of group B at other points of time （P 〈 0.05）. The similar condition also appeared at acute pain stage of the first phase and chronic pain stage of the second phase （P 〈 0.05）. CONCLUSION： The microcapsulated pENK-expressing cells can reduce chronic neuropathic pain on rats, and may serve as a new tool of gene therapy for chronic pain in athletes.