鞘内注射miR-365反义锁核酸对小鼠骨癌痛行为的影响

Effects of intrathecal injection miR-365 antisense locked nucleic acid on the pain behavior in mice with bone cancer pain

目的研究小鼠脊髓中miR-365表达水平的变化规律与骨癌痛发生和发展的关系,及连续鞘内注射miR-365反义锁核酸(LNA-anti-miR-365)对骨癌痛小鼠疼痛行为的影响。方法(1)将48只SPF级C3H/HeJ雄性小鼠,按照随机数字表法将其随机分为假手术组(Sham组)和肿瘤组(T组),每组24例。两组小鼠分别于右侧股骨远端骨髓腔内注射不含肿瘤细胞的α-MEM和纤维肉瘤细胞NCTC 2472。采用Real-time-RTPCR方法在建模前1 d,建模后4、7、10、14、21 d分别检测Sham组和T组小鼠脊髓miR-365的表达情况。(2)另取24只SPF级C3H/HeJ雄性小鼠随机分为4组:L组(鞘内注射LNA-anti-miR-365),L’组(鞘内注射LNA’-negative control),C组(鞘内注射溶媒无核酸酶水)和S组(假手术处理),每组6只。建模前l d,建模后4、7、10、14 d监测小鼠疼痛行为指标,包括自发抬足次数和机械缩足阈值(PWMT)。建模后14 d,每日分别给L组、L’组、C组小鼠鞘内注射LNA-anti-miR-365、LNA’-negative control和无核酸酶水各5μl,连续注射7 d,每日测小鼠疼痛行为指标,至建模后2l d。结果与基础值比较,Sham组建模后4~21 d脊髓水平miR-365差异均无统计学意义(P均>0.05);与基础值和Sham组相比,T组小鼠脊髓水平miR-365在建模后7 d(3.45±0.58)、10 d(4.11±0.23)、14 d(3.21±0.67)、21 d(4.82±0.55)均明显升高,差异均有统计学意义(P均<0.05)。从建模后19~21 d,与L’组和C组比较,L组小鼠PWMT明显升高[19 d(1.11±0.14)g,20 d(1.15±0.20)g,21 d(1.29±0.23)g](P均<0.05),自发抬足次数明显降低[19 d(8.6±1.1)次,20 d(8.6±1.4)次,21 d(8.5±1.1)次](P均<0.05)。相比给药前,L组小鼠建模后19~21 d的PWMT明显升高(P均<0.05),自发抬足次数明显降低(P均<0.05)。结论骨癌痛发生发展过程中,miR-365脊髓表达量有所升高,连续鞘内注射LNA-anti-miR-365可缓解小鼠骨癌痛。

Objective To investigate the change of miR-365 expression during the development and maintenance of bone cancer pain and the effects of repeated intrathecal injection of miR-365 antisense locked nucleic acid(LNA)on pain behaviors in a mouse model of bone cancer pain.Methods Forty-eight male C3H/HeJ mice were randomly divided into group Sham and group Tumor(group T),in accordance with the random number table,24 mice in each group.The mouse model of bone cancer pain was developed by intra-femur inoculation ofα-minimal essence media(α-MEM)with NCTC 2472 osteosarcoma cells in group T.The mice were inoculated byα-MEM without any cells in group Sham.Using Real-time-RTPCR to detect the expression of miR-365 on 1 d before the inoculation and 4,7,10,14,21 d after the inoculation.Another twenty-four male C3H/HeJ mice which were inoculated with NCTC 2472 osteosarcoma cells were randomly divided into group L(intrathecal injection with LNA-anti-miR-365),group L'(intrathecal injection with LNA'-negative control),group C(intrathecal injection with RNase-free water)and group S(sham operation),6 mice in each group.The pain behaviors were observed on 1 d before the inoculation and 4,7,10,14 d after the inoculation,including paw withdrawal mechanical threshold(PWMT)and the number of spontaneous lifting behavior.On 14 d after the inoculation,the mice in group L,group L'and group C were intrathecal injected the drugs respectively.Pain behaviors were observed every day from 14 d to 21 d after the inoculation.Results Compared with the base level,there was no significant difference in the level of mir-365 from 4 to 21 d in group Sham(all P>0.05);Compared with the base level and group Sham,the level of mir-365 was significantly increased at 7 d(3.45±0.58)and 10 d(4.11±0.23),14 d(3.21±0.67),21 d(4.82±0.55)respectively(all P<0.05).From 19 to 21 d,PWMT was significantly increased[19 d(1.11±0.14)g,20 d(1.15±0.20)g,21 d(1.29±0.23)g](all P<0.05),and the number of spontaneous lifting behavior of group L[19 d(8.6±1.1)times,20 d(8.6±1.4)times,21 d(8.5±1.1)times]was significantly decreased compared with those of group L'and group C(all P<0.05).The number of spontaneous lifting behavior and PWMT were significantly different in group L compared with those of group L'and group C(all P<0.05).Conclusion The high expression of miR-365 was related to the development and maintenance of bone cancer pain,and repeated intrathcal injection of LNA-anti-miR-365 can alleviate the pain behaviors in the mouse model of bone cancer pain.