The Effects Of NR2B Gene Silencing In Spinal Cord Of Bone Cancer Pain Mouse

Objective:In order to investigate the mechanisms of bone cancer pain, a mouse bone cancer pain model is established to study the effects of NR2B gene silencing by intrathecal injection of PEI/NR2B-siRNA complex in the bone cancer mouse. To explore the feasibility of cancer pain treatment by using NR2B/SiRNA to interfere the expression of NR2B gene in the vivo.Method:72 adult male C57BL/6 mice were randomly divided into three groups:cancer pain group (N=24):Lewis lung cancer cells 2×106/10ul were inoculated as stated above; interference group (N=24): PEI/NR2B siRNA complex was intrathecally injected 12 days later after cancer cell inoculation; control group of the interference (N=24):the PEI /Negative control siRNA complex was used in intrathecal injection. Spontaneous lifting time and mechanical allodynia were measured pre-operation and on the 6th,10th,12th,13th,14th,15th,16th,18th and 22nd day after inoculation. L4-6 spinal cord segments were isolated for fluorescence quantitative RT-PCR on the 13th,14th,15th,16th,18nd and 22nd day after inoculation. According to the results of the PCR to do Western Blot to detect the expression levels of NR2B.Results(1) Real-time quantitative PCR detection:the expression of NR2B in L4-6 spinal cord in the interference group was reduced significantly as compared to the cancer pain group and the control group of the interference group on the 14th and 18th day after inoculation (P<0.05). The most obvious change in the expression of NR2B in the interference group was observed on the 14th day after inoculation, showing 74% lower expression in the test group as compared to that in the cancer pain group and the control group of the interference. There was no significant difference between the later two groups (P>0.05).(2) The results of Western Blot:the expression of NR2B in the L4-6 spinal cord in the interference group was reduced significantly as compared to that in the cancer pain group on the 14th and 18th day after inoculation (P<0.05). The most obvious change in the expression of NR2B was observed on the 18th day after inoculation, demonstrating 67% lower expression in the interference group than that in the cancer pain group. There was no significant difference between the later two groups (P>0.05).(3) Spontaneous lifting time was decreased and mechanical allodynia was increased obviously in the interference group as compared to the cancer pain group and the control group of the interference.Conclusion:1. Inoculation of Lewis lung cancer cells into the marrow cavity of distal femur of C57BL/6 mice can effectively establish a mouse bone cancer pain model. There was much similarity between human cancer pain and this model. The observation that NR2B was expressed in large quantity in the L4-6 spinal cord of the bone cancer pain:2. The expression of NR2B was reduced in the L4-6 spinal cord after intrathecal injection of PEI/NR2B-siRNA complex in the mouse with cancer pain, which was consistent with the behavior changes of the animals, and provided further evidence showing that NR2B might play an important role in the initiation of bone cancer pain.