RNA干扰胶质细胞源性的神经营养因子表达对大鼠乳腺癌致骨癌痛的作用  

Analgesic effect of lentivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor gene on bone cancer pain

作　　者：孟馥芬[1] 维拉[1] 徐扬[2] 王廷华[2] 

机构地区：[1]新疆医科大学附属肿瘤医院麻醉科,乌鲁木齐830011 [2]四川大学华西医院神经科学转化中心

出　　处：《中华实验外科杂志》2014年第5期956-959,F0003,共5页Chinese Journal of Experimental Surgery

摘　　要：目的观察慢病毒介导的RNA干扰胶质细胞源性神经营养因子（GDNF）表达对乳腺癌致大鼠骨癌痛的影响。方法构建慢病毒包装的RNA干扰GDNF重组体（psiHIV—GDNF—mU6）。复制Medhurst乳腺癌致骨癌痛大鼠模型随机分为4组，分别给予蛛网膜下腔注射：生理盐水（Sa）、psiHIV—GDNF—mU6（siGDNF）、慢病毒空载体（psiHIV—U6，P＋NC）、吗啡（Mor）。比较造模前、给药后7、14d时的大鼠痛阈变化。应用Westernblot和免疫荧光技术检测大鼠脊髓GDNF、Fos及星形胶质细胞的变化。结果（1）构建psiHIV—GDNF—mU6成功，GDNF的最有效干扰片段为F3（0．045±0．034），与转染组（0．113±0．072）、对照组（0．117±0．060）比较差异有统计学意义（P〈0．05）。293Ta及HT1080细胞包装接种病毒成功，重组体转染效率为0．87，病毒滴度为3．48×10^8pfu／ml。（2）各组注药后7d时机械痛比较差异无统计学意义（P〉0．05）。siGDNF组注药后7d时热痛阈值（3．80±0．69）和注药后14d时机械痛阈值（2．58±1．93）、热痛阈值（3．71±1．10）均较Sa组（1．38±1．01、1．11±1．02、1．53±0．69）及P—NC组（1．39±1．18、0．67±0．27、1．21±1．06）延长（P〈0．05）。siGDNF组与Mor组比较差异无统计学意义（P〉0．05）。（3）GDNF表达水平在siGDNF组（0．07±0．00）显著低于其余各组（Sa：0．43±0．08；P—NC：0．28±0．10；Mor：0．38±0．11，P〈0．05）。（4）鞘内给药后，Fos，胶质纤维酸性蛋白（GFAP）在siGDNF组、Mor组表达较Sa组和P—NC组下降（P〈0．05）。星形胶质细胞阳性数呈现一致性变化。结论siGDNF对大鼠骨癌痛有类似吗啡的效果，单次鞘内给药镇痛可持续2周。抑制星形胶质细胞的活化，削弱痛敏形成的分子基础可能是siGDNF实现镇痛的原因。Objective To observe the analgesic effects of ientivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor （GDNF） on bone cancer pain. Methods （ 1 ） Lvs-siGDNF was constructed and bone cancer pain models were established by intra-tibial injection of MRMT-1 cells. （2） The rats were randomly divided into 4 groups according to different drugs which were infused into subarachnoid space through the intrathecal tubes： psiHIV-U6, psiHIV-GDNF-mU6, morphine, and normal saline. （3） The pain threshold at different time points （normal rats, 7 days and 14 days after dosing） was measured. （4） The changes of pain threshold were observed from preoperative day to 7 days and 14 days after administration. The levels of GDNF, glial fibrillary acidic protein （GFAP） , and Fos were detected by immunofluorescent staining technique and Western blotting. Results （ 1 ） As compared with control group （0. 117 ±0. 060） and transfection group （0. 113 ±0. 072）, GDNF siRNA sequence 3 （0. 045 ± 0. 034 ） was the most effective interference segment confirmed by Q-PCR （ P 〈 0. 05 ）. The percentage of fluorescent cells to the brightfield was 0. 87. TLvs-siGDNF was 3.48 × 10^5 pfu/ml. （2） Thermal pain threshold at 7th day （3. 80 ±0. 69） and 14th day （3.71 ± 1.10）, and mechanical pain thresh- old at 14th day （2. 58 ± 1.93） were all increased in siGDNF group than normal saline group （ 1.38 ± 1.01, 1.11 ± 1.02, and 1.53 ±0. 69） and psiHIV-U6 group （ 1.39 ± 1.18, 0. 67 ±0. 27, and 1.21 ± 1.06） （ P 〈 0. 05 ）, while there was no significant difference in mechanical pain threshold at 7 day after administration （ P 〉 0. 05 ）. （3） RNA interference effectively decreased the expression level of GDNF in siGDNF group （P 〈0. 05）. （4） As compared with other groups, the expression levels of Fos and GFAP in the spinal dorsal horn were decreased in morphine group and siGDNF group （ P 〈 0. 05 ）. Conclusion The

关 键 词：骨癌痛 胶质细胞源性神经营养因子 RNA干扰 慢病毒 

分 类 号：R737.9[医药卫生—肿瘤] R738.1[医药卫生—临床医学]