piresgfphpenk的构建及其鞘内注射的表达-construction of piresgfphpeng and expression of its intrathecal injection.docxVIP

河南科技大学硕士学位论文 河南科技大学硕士学位论文 摘要 摘要 PAGE PAGE IV III III 4 个亚组(1、2、3、4 周组各 3 只) ，对照组 12 只不做处理。实验组向蛛网膜下 腔注射相应质粒 100μl（浓度为 30μg/100ml）。采用免疫组化技术观察绿色荧光 蛋白和 ENK 的表达。 结果 1. 真核表达质粒 pIRES-GFP-hPENK 成功构建，测序结果与 GenBank 报 道的序列完全吻合，并转染至 NIH3T3 细胞得到表达。 2. 转染了新构建质粒的 NIH3T3 细胞分泌 ENK 至上清液中，膜片钳检测显 示 ENK 能引起神经元胞膜上 K 外流增加。 3. 鞘内注射重组质粒 pIRES-GFP-hPENK 24 小时后得到表达，在脑膜、脊 神经外膜上观察到绿色荧光，第 7~14 天达到高峰，第 4 周时下降明显，6 周时 消失。与实验组比较，对照组未发现绿色荧光。实验组也未发现大鼠脊髓神经损 伤和动物行为学的改变。 4. 免疫组化结果显示实验组各时间点 ENK 的表达量明显高于对照组（P＜ 0.01），且在 2 周时最高（P＜0.01），4 周时明显下降（P＜0.01）。 结论 1. pIRES-GFP-hPENK 转染细胞后的产物 ENK 可以引起神经元膜表面 K 离 子外流增加。 2. 鞘内注射重组质粒 pIRES-GFP-hPENK 可转染至脑膜和脊神经外膜，外 源基因调控的 ENK 表达长至四周。 关 键 词：镇痛；人前脑啡肽原基因；膜片钳；鞘内注射；绿色荧光蛋白 论文类型：基础研究 Subject: Construction of pIRES-GFP-hPENK and its expression of intrathecal injection Specialty: Human Anatomy and Embryologyh Name: Yong-hui LI Supervisor: Professor Xin-ming TU and Wei-dong ZANG ABSTRACT Objective Gene therapy of pain is considered as an effective and safe treatment,and it has become the treatment of hot spots. And its key aspect is to choose a valid target gene and the vector. Enkephalin (enkephalin, ENK) is a kind of endogenous opioid peptides. Compared with other opiates, the much superiority of its analgesic has been confirmed. Thus, the human preproenkephalin gene (human preproenkephalin gene, hPENK), as the controlling gene of enkephalin expression has become a relatively ideal selection of gene therapy for chronic pain. It is a commonly research tools that virus vector carrying PENK express ENK by transfecting cells or injecting directly, but its clinical application is limited because of the virus self-replication and human anti-viral immune response. To further study about the PENK and the ENK controlled and synthesised by PENK, we constructed the recombinant plasmid pIRES-GFP- hPENK,which was highly efficient expression, low toxicity and immunogenicity.And we studied it by two asp