脊髓Caprin-1介导的CaMKⅡα表达与小鼠痛觉感知的关系

Relationship between spinal cord Caprin-1-mediated expression of CaMKⅡαand pain perception in mice

目的评价脊髓胞质活化/增殖相关蛋白1(Caprin-1)介导的钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡα)表达与小鼠痛觉感知的关系。方法通过Nestin^(CreERT2)小鼠与Caprin-1^(flox/flox)小鼠多代杂交,获得Nestin^(CreERT2);Caprin-1^(flox/flox)纯合子小鼠,采用随机数字表法分为2组(n=5):Nestin^(CreERT2);Caprin-1^(flox/flox)溶剂对照组(SC组)和Caprin-1敲除组(KO组)。KO组连续5 d腹腔注射他莫昔芬100 mg/kg,以诱导性敲除Caprin-1基因,SC组给予等容量溶剂。于连续给药前1 d和给药结束后5 d时测定机械缩足反应阈(MWT)。随后处死小鼠,取脊髓L_(4-6)节段,采用Western blot检测脊髓Caprin-1、CaMKⅡα表达,采用实时定量PCR法检测脊髓Caprin-1、CaMKⅡα的mRNA表达,采用免疫荧光双标染色检测Caprin-1与CaMKⅡα共表达情况。结果与SC组比较,KO组他莫昔分给药结束后5 d时MWT升高,脊髓Caprin-1及其mRNA、CaMKⅡα及其mRNA表达下调(P<0.05),Caprin-1与CaMKⅡα在脊髓背角共定位表达下调。结论脊髓Caprin-1可通过促进CaMKⅡα表达,参与小鼠痛觉感知的形成。

Objective To evaluate the relationship between spinal cord cytoplasmic activation/proliferation-associated protein-1(Caprin-1)-mediated expression of calmodulin-dependent protein kinaseⅡalpha(CaMKⅡα)and pain perception in mice.Methods Nestin^(CreERT2)mice and Caprin-1^(flox/flox)mice were hybridized to obtain Nestin^(CreERT2);Caprin-1^(flox/flox)homozygous mice.The mice were divided into 2 groups(n=5 each)using a random number table method:Nestin^(CreERT2);Caprin-1^(flox/flox)solventcontrol group(SC group)and Caprin-1 gene knockout group(KO group).Tamoxifen 100 mg/kg was intraperitoneally injected for 5 consecutive days to generate inducible knockout of the Caprin-1 gene in KO group.The mechanical paw withdrawal threshold was measured at day 1 before and day 5 after the end of administration.Then the mice were sacrificed,and L_(4-6)segments of the spinal cord were removed for determination of the expression of Caprin-1 and CaMKⅡαprotein and mRNA(by Western blot or real-time quantitative polymerase chain reaction)and co-expression of Caprin-1 with CaMKⅡα(by immunofluorescent double staining).Results Compared with SC group,the mechanical paw withdrawal threshold was significantly increased at 5 days after the end of tamoxifen administration,the expression of Caprin-1 and CaMKⅡαprotein and mRNA was down-regulated(P<0.05),and the co-localization expression of Caprin-1 and CaMKⅡαin the spinal dorsal horn was down-regulated in KO group.Conclusions Caprin-1 is involved in the development of pain perception by promoting CaMKⅡαexpression.