【作者】 徐玉英；

【导师】 臧卫东； 张宏伟；

【作者基本信息】 郑州大学 ， 人体解剖与组织胚胎学， 2007， 硕士

【摘要】 目的神经病理性疼痛的治疗是临床工作中日益受到重视而又棘手的问题，目前实施的各种治疗方法都存在一定的局限性。转基因镇痛是将外源性基因转移至动物体内表达镇痛物质，从而发挥镇痛效应的一种方法，而脑啡肽作为一种内源性阿片肽类镇痛物质日益受到关注，许多动物实验已证明其有明显的镇痛效应。目前，转脑啡肽基因治疗的关键问题之一在于开发安全、高效的基因转导系统。非病毒载体具有使用方便、可大规模生产以及无免疫原性等优点。其中裸DNA作为最简单的非病毒载体方法是将目的基因连接在表达的质粒上直接注射动物体内，本实验利用基因重组技术将人前脑啡肽原基因(human preproenkephalin gene，hPPE)与真核表达载体pcDNA3.1(+)连接，构建重组质粒pcDNA3.1(+)-hPPE，然后将其直接注射到坐骨神经慢性压迫(chronic constriction injury of the sciatic nerve，CCI)模型大鼠的蛛网膜下腔，观察其对大鼠神经病理性疼痛的镇痛效应。该实验还利用基因重组技术将增强型绿色荧光蛋白(Enhanced green fluorescence protein，EGFP)与真核表达载体pcDNA3.1(+)连接，构建重组质粒pcDNA3.1(+)-EGFP后，将其直接注射到正常大鼠的蛛网膜下腔，观察裸DNA在大鼠体内转染后的时空效应。方法1．蛛网膜下腔注射重组质粒pcDNA3.1(+)-EGFP，观察其在大鼠体内转染后的时空效应。雄性SD大鼠54只被随机分成2组：实验组(n=48)又分8个亚组(24小时、1、2、3、4、5、6、7周组各6只)。空白对照组6只(不做任何处理)。实验组鞘内注射30μg/100μl重组质粒pcDNA3.1(+)-EGFP 100μl。2．建立神经病理性疼痛模型，即坐骨神经慢性压迫(CCI)模型。雄性SD大鼠20只被随机分成2组：CCI模型组(6/0丝线结扎右侧坐骨神经)和CCI模型假手术组(仅暴露出右侧坐骨神经，不做结扎)。CCI术前连续测量3天双侧后爪热刺激缩足反射潜伏期(Paw withdrawal thermal latency，PWTL)，术后第5天开始测量，隔日1次，连续测量6周。3．观察重组质粒pcDNA3.1(+)-hPPE的镇痛效应。20只CCI模型鼠被随机分成2组：实验组(CCI模型鼠蛛网膜下腔移入30μg/100μl重组质粒pcDNA3.1(+)-hPPE 100μl)和空质粒组(CCI模型鼠蛛网膜下腔移入30μg/100μl空质粒pcDNA3.1(+)100μl)；鞘内注射后第2到第18天，检测双侧后爪对热刺激缩足反射潜伏期，第8天进行腹腔注射纳洛酮(0.08mg/kg)实验，用药前测量一次，用药后10分钟开始测量，观察用药后1小时内动物双后肢热痛阈变化情况。第18天进行脑脊液灌流检测脑脊液中亮氨酸脑啡肽的含量和采用免疫组化技术测定两组大鼠脊髓背角N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor，NMDAR)亚单位NR2B表达的变化。结果1．蛛网膜下腔注射重组质粒pcDNA3.1(+)-EGFP在大鼠体内转染后的时空效应。实验组：鞘内注射重组质粒pcDNA3.1(+)-EGFP 24小时后在大鼠脑脊膜及脊神经的外膜上发现绿色荧光，在大鼠的心、肝、脾、肺、肾及股四头肌上没见到pcDNA3.1(+)-EGFP的表达。对照组：与实验组比较，没发现绿色荧光。重组质粒pcDNA3.1(+)-EGFP在实验组大鼠的表达，7到14天达到高峰，4周后明显下降，第7周消失。并且未发现脊髓神经损伤和动物行为学的改变。2．CCI模型动物后肢热痛阈的变化规律。CCI组结扎侧后爪术后第5天出现自发抬起损伤肢体、时而舔足、咬足或甩足等自发性疼痛；术后第7天出现明显的热痛敏感现象(PTWL降低，P<0.01)，持续33天。假手术组手术前后PTWL无明显变化(P>0.05)。3．裸质粒移植镇痛效应。空质粒组在给予pcDNA3.1(+)前后PTWL无明显变化(P<0.05)；实验组基因治疗第3天产生明显的抗伤害效应(PTWL升高，P>0.05)，持续镇痛长达18天，且镇痛效应能被纳洛酮翻转。实验组脑脊液灌流液L-ENK含量明显高于空质粒组，具有显著性差异，149.52±17.83 vs 45.55±12.58pg/ml，t=15.581，P=0.001<0.01。正常组大鼠脊髓背角有少量NR2B免疫阳性蛋白表达；与正常组比较，空质粒组大鼠脊髓背角有大量NR2B免疫阳性蛋白表达(P<0.01)，实验组大鼠脊髓背角NR2B阳性蛋白表达明显受到抑制(P<0.01)。结论1、裸DNA通过鞘内直接注射法可以被转染至脑脊膜和脊神经外膜，且外源基因得到表达。2、蛛网膜下腔注射重组质粒pcDNA3.1(+)-hPPE能够减轻大鼠神经病理性疼痛的热痛敏行为。更多还原

【Abstract】 ObjectiveTreatment of neuropathic pain is extremely difficult, current analgesic agents may be limited with regard to their analgesic effects or side effects. By targeting a specific receptor or other specific protein targets, gene therapy approach to the treatment of neuropathic pain may provide great analgesic efficacy without the limitations associated with current pharmacotherapy.Enkephalin(Enk),as a type of endogenous opioid peptides,has been tested to have analgesic effect by numerous investigations using animal models. So the possibility of using Enk gene therapy as a potential novel treatment for neuropathic pain is taken into consideration. The success of gene therapy rests on the development of a vector that can selectively and efficiently deliver a gene to the target cells. Advantages of non-viral vectors include their nonimmunogenicity and feasibility to be produced on a large scale. Theoretically, non-viral gene transfer systems using naked plasmid DNA has the characteristics of safety、reliablility and simplicity,up to now, plasmid DNA has been the predominant non-viral vectors utilized in research.To find more convenient, effective, economic and safe antinocicepive method, the the recombinant plasmid pcDNA3.1(+)-hPPE was constructed using gene recombination technology and transfected into the subarachnoid space of CCI model rats via direct intrathecal injection and the antinocicepive effects were evaluated in CCI model rats. Then,to investigating the feasibility of delivering exogenous genes into the subarachnoid space using naked plasmid DNA, the recombinant plasmid pcDNA3.1(+)-EGFP was constructed and was transfected into the subarachnoid space of normal rats via direct intrathecal injection.Methods1. The expression of pcDNA3.1(+)-EGFP in normal rat. We investigated the feasibility of delivering exogenous genes into the subarachnoid space using naked plasmid DNA. 20 Spague-Dawley male rats weighing 220-250g were randomly distributed into two groups:control group (n =6) and experimental group(n =48) which was further divided into 8 subgroups (24-hour subgroup, 1-week subgroup, 2-week subgroup, 3-week subgroup,4-week subgroup, 5-week subgroup, 6-week subgroup and 7-week subgroup). The rats of experimental group were injected into the spinal subarachnoid with 30μg/100μl of the pcDNA3.1(+)-Enhanced green fluorescence protein(EGFP) recombinant plasmid.2.Results of the behavioral tests. The chronic constriction injury of the sciatic nerve (Bennett model; CCI model): 20 Spague-Dawley male rats weighing 220-250g were randomly distributed into two groups (n =10,for each group), ten CCI and ten sham-operated groups. In CCI group, right sciatic nerves were separated in the midthigh region and loosely tied four ligatures( 6-0, silk thread) so that they touched but barely constricted the nerve; In the sham-operated rats, the sciatic nerves were separated but not ligated. In all rats, the contralateral sides were not disturbed. The paw withdrawal thermal latency (PWTL) was assessed once daily for 3 days before the CCI operation and the PWTL were detected every other day from day 5 to 6 weeks after the operation in both group.3. To evaluate the efficacy of the pcDNA3.1(+)-hPPE recombinant plasmid on neuropathic pain after intrathecal administration,20 CCI model rats were randomly distributed into two groups (n =10), pcDNA3.1(+)-hPPE group and pcDNA3.1(+) group. In the pcDNA3.1(+)-hPPE group,the pcDNA3.1(+)-hPPE recombinant plasmid was intrathecally injected in a dose of 30μg/100μl;in the control group, the eukaryotic expression vector pcDNA3.1(+) was intrathecally injected in a dose of 30μg/100μl. PWTL of two groups was assessed from day 2 to day 18 after intrathecal administration. Naloxone (0.08mg/kg) was intraperitoneally injected in both groups at days 8 after intrathecal injection, PWTL was measured before and from 10-60 minutes after intraperitoneal Naloxone injection at interval of 10 minutes. CSF samples were obtained for the determination of the concentration of Leucine enkephalin using radio-immunological assay, and the changes in NR2B protein expression in the spinal cord was detected by immunohistochemical techniques in both groups at 18 days intrathecal injection.Results1 .To determine the sites of cellular uptake and expression of the transfected gene, autofluorescence in nerve tissue,heart,liver,spleen,lung,kidney were detected at different time points (At 24 hours ,weeks 1, 2, 3, 4, 5, 6 and 7 following intrathecal injection). At 24 hours following the pcDNA3.1(+)-EGFP recombinant plasmid injection a clear EGFP expression mainly in meninges and the lumbar dorsal roots was detected. No EGFP was found in the other tissues. The most intensive autofluorescence was seen between 7 and 14 days after the pcDNA3.1(+)-EGFP recombinant plasmid injection and the intensity was decreased after 4 weeks. In control group ( without any treatment), no EGFP activity was noted at either time point.2. In CCI group,the animals tended to avoid weight-bearing on the affected foot, both at rest and while walking. The foot was often held in an everted position with the toes plantar-flexed. Sham-operated rats had normal posture and gait. Behavioural testing to detect signs of thermal hyperalgesia was carried out on all CCI and sham-operated rats,responses to thermal stimuli were tested with a Plantar Analgesia Instrument(Ugo-Basile, Italy).The right PTWL were significantly lower than the left PTWL in CCI group from 7 to 33 days after the operation (P < 0:01). There were no significant differences in withdrawal latencies between the paws left and right in the sham-operated rats at any of the time tested (P>0.05) . 3.Then; were no significant differences between withdrawal latencies of the two hindpaws in the pcDNA3.1(+) group after intrathecal injection at any of the time tested (P < 0.05); The right PTWL were significantly higher than the left PTWL in the pcDNA3.1(+)-hPPE group from 2 to 18 days after intrathecal injection (P > 0:05).The attenuation of the thermal hyperalgesia induced by CCI in rats can be blocked by naloxone:.The concentration of the leucine enkephalin in CSF of the pcDNA3.1 (+)-hPPE group significantly higher than those of the leucine enkephalin in CSF of the pcDNA3.1(+) group, 149.52±17.83vs.45.55±12.58 pg/ml, t= 15.581, P=0.001<0.01. The expression of NR2B protein in the spinal cord was minimal in normal group ;in the pcDNA3.1(+) group, the expression of NR2B protein has significantly increased, in the pcDNA3.1(+)-hPPE group, the expression of NR2B protein was significantly decreased in the spinal cord.Conclusions1. Naked DNA can be transfected into meninges and perilemma through the intrathecal injection and exogenous gene can be expressed.2. The pcDNA3.1(+)-hPPE recombinant plasmid transfected into the subarachnoid space of CCI model rats via intrathecal injection could significantly attenuate thermal hyperalgesia.更多还原