中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
5期
956-959,封3
,共5页
孟馥芬%维拉%徐扬%王廷华
孟馥芬%維拉%徐颺%王廷華
맹복분%유랍%서양%왕정화
骨癌痛%胶质细胞源性神经营养因子%RNA干扰%慢病毒
骨癌痛%膠質細胞源性神經營養因子%RNA榦擾%慢病毒
골암통%효질세포원성신경영양인자%RNA간우%만병독
Bone cancer pain%Glial cell line-derived neurotrophic factor%RNA interference%Lentiviral
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Western blot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3.48×108 pfu/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14 d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.
目的 觀察慢病毒介導的RNA榦擾膠質細胞源性神經營養因子(GDNF)錶達對乳腺癌緻大鼠骨癌痛的影響.方法 構建慢病毒包裝的RNA榦擾GDNF重組體(psiHIV-GDNF-mU6).複製Medhurst乳腺癌緻骨癌痛大鼠模型隨機分為4組,分彆給予蛛網膜下腔註射:生理鹽水(Sa)、psiHIV-GDNF-mU6 (siGDNF)、慢病毒空載體(psiHIV-U6,P+NC)、嗎啡(Mor).比較造模前、給藥後7、14d時的大鼠痛閾變化.應用Western blot和免疫熒光技術檢測大鼠脊髓GDNF、Fos及星形膠質細胞的變化.結果 (1)構建psiHIV-GDNF-mU6成功,GDNF的最有效榦擾片段為F3(0.045±0.034),與轉染組(0.113 ±0.072)、對照組(0.117 ±0.060)比較差異有統計學意義(P＜0.05).293Ta及HT1080細胞包裝接種病毒成功,重組體轉染效率為0.87,病毒滴度為3.48×108 pfu/ml.(2)各組註藥後7d時機械痛比較差異無統計學意義(P＞0.05).siGDNF組註藥後7d時熱痛閾值(3.80±0.69)和註藥後14 d時機械痛閾值(2.58±1.93)、熱痛閾值(3.71±1.10)均較Sa組(1.38±1.01、1.11 ±1.02、1.53 ±0.69)及P-NC組(1.39±1.18、0.67±0.27、1.21 ±1.06)延長(P＜0.05).siGDNF組與Mor組比較差異無統計學意義(P＞0.05).(3) GDNF錶達水平在siGDNF組(0.07±0.00)顯著低于其餘各組(Sa:0.43 ±0.08;P-NC:0.28 ±0.10;Mor:0.38 ±0.11,P＜0.05).(4)鞘內給藥後,Fos,膠質纖維痠性蛋白(GFAP)在siGDNF組、Mor組錶達較Sa組和P-NC組下降(P＜0.05).星形膠質細胞暘性數呈現一緻性變化.結論 siGDNF對大鼠骨癌痛有類似嗎啡的效果,單次鞘內給藥鎮痛可持續2週.抑製星形膠質細胞的活化,削弱痛敏形成的分子基礎可能是siGDNF實現鎮痛的原因.
목적 관찰만병독개도적RNA간우효질세포원성신경영양인자(GDNF)표체대유선암치대서골암통적영향.방법 구건만병독포장적RNA간우GDNF중조체(psiHIV-GDNF-mU6).복제Medhurst유선암치골암통대서모형수궤분위4조,분별급여주망막하강주사:생리염수(Sa)、psiHIV-GDNF-mU6 (siGDNF)、만병독공재체(psiHIV-U6,P+NC)、마배(Mor).비교조모전、급약후7、14d시적대서통역변화.응용Western blot화면역형광기술검측대서척수GDNF、Fos급성형효질세포적변화.결과 (1)구건psiHIV-GDNF-mU6성공,GDNF적최유효간우편단위F3(0.045±0.034),여전염조(0.113 ±0.072)、대조조(0.117 ±0.060)비교차이유통계학의의(P＜0.05).293Ta급HT1080세포포장접충병독성공,중조체전염효솔위0.87,병독적도위3.48×108 pfu/ml.(2)각조주약후7d시궤계통비교차이무통계학의의(P＞0.05).siGDNF조주약후7d시열통역치(3.80±0.69)화주약후14 d시궤계통역치(2.58±1.93)、열통역치(3.71±1.10)균교Sa조(1.38±1.01、1.11 ±1.02、1.53 ±0.69)급P-NC조(1.39±1.18、0.67±0.27、1.21 ±1.06)연장(P＜0.05).siGDNF조여Mor조비교차이무통계학의의(P＞0.05).(3) GDNF표체수평재siGDNF조(0.07±0.00)현저저우기여각조(Sa:0.43 ±0.08;P-NC:0.28 ±0.10;Mor:0.38 ±0.11,P＜0.05).(4)초내급약후,Fos,효질섬유산성단백(GFAP)재siGDNF조、Mor조표체교Sa조화P-NC조하강(P＜0.05).성형효질세포양성수정현일치성변화.결론 siGDNF대대서골암통유유사마배적효과,단차초내급약진통가지속2주.억제성형효질세포적활화,삭약통민형성적분자기출가능시siGDNF실현진통적원인.
Objective To observe the analgesic effects of lentivirus-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,mor-phine,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 × 108 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 analgesic effect of siGDNF is similar to that of morphine in bone cancer rats,and siGDNF worked effectively more than 2 weeks.SiGDNF realized analgesic effect probably by inhibiting the astrocyte activation.
