NR2B在小鼠骨癌痛中的作用及机制研究
摘要
目的建立新的小鼠骨癌痛模型,观察NMDA受体2B亚基(NR2B)在骨癌痛小鼠前脑扣带回和脊髓中的表达及分布情况；明确NR2B与癌痛产生之间的关系；探讨NR2B在小鼠骨癌痛中的作用机制。
方法
1.小鼠骨癌痛新模型的制备及评价：选用成年雄性C57BL／6小鼠30只,随机分为4组,模型组(15只)：接种Lewis肺癌细胞2×106个；热灭活组(5只)：接种相同数目加热灭活的Lewis肺癌细胞；PBS组(5只)：接种等容积的PBS液；正常对照组(5只)：不予任何处理。接种后7天始隔日观察小鼠自发痛行为及测定机械性触诱发痛。第7、15、23天,将小鼠麻醉后行双侧后肢X线摄片,评估肿瘤诱发的骨组织破坏程度。模型组小鼠每次摄片后任选5只取术侧后肢,各对照组小鼠最后一次摄片后取术侧后肢,分别作苏木素-伊红(HE)染色观察骨质破坏情况。
2.NR2B在骨癌痛小鼠前扣带回和脊髓中的分布与表达：选用成年雄性C57BL/6小鼠81只,随机分为3组：癌痛组,假手术组和正常对照组。观察三组小鼠自发痛行为及测定机械性触诱发痛。分别于术后第6、10、14、18、22天各取3只,实时定量PCR检测前扣带回和脊髓NR2B mRNA的表达。依据PCR结果,每组分别于术后第14、18天各取3只,免疫组化检测前扣带回和脊髓NR2B的分布和表达；同时每组另各取3只,于术后第14、18天行Western Blot检测脊髓NR2B蛋白的表达。
3.脊髓NR2B基因沉默对小鼠骨癌痛的影响：选用成年雄性C57BL／6小鼠72只,随机分为3组：癌痛组,干扰组和干扰对照组。观察各组小鼠自发痛行为及测定机械性触诱发痛。每组分别于接种后第13、14、15、16、18、22天各取3只,实时定量PCR检测脊髓NR2B mRNA的表达。依据PCR的结果,于接种后第14和18天每组各取3只,Western Blot检测脊髓NR2B蛋白的表达。
4.MAPK信号通路介导的NR2B在小鼠骨癌痛中的作用机制：选用成年雄性C57BL／6小鼠48只,随机分为4组：正常组,癌痛组,干扰组和干扰对照组。观察各组小鼠自发痛行为及测定机械性触诱发痛。每组分别于接种后第14和18天各取3只,实时定量PCR检测脊髓NR2B、SP、c-fos和GFAP基因表达水平。选取接种后第14和18天的每组小鼠各3只,Western Blot检测脊髓NR2B、p-ERK、p-p38和GFAP蛋白表达水平。
结果
1.小鼠骨癌痛新模型的评价：模型组接种后第11天左右出现明显自发痛行为,表现为自发抬足时间延长；接种后第13天左右出现明显的触诱发痛,表现为50%缩足阈值明显下降,且持续整个观察期。接种后第23天放射学结果显示,术侧股骨下段骨髓腔消失,骨皮质中断、大块缺损。同时组织学可见肿瘤细胞充满骨髓腔,且穿破骨皮质向外生长,侵犯周围肌肉组织。行为学、放射学、组织学三方面证实建模成功。
2.NR2B在骨癌痛小鼠的前扣带回和脊髓中的分布与表达：
(1)前扣带回NR2B的实时荧光定量PCR结果显示：接种后第6天、10天,癌痛组NR2B的表达与假手术组比较无显著差异,而癌痛组和假手术组NR2B的表达比正常组明显增高；第14天、18天,癌痛组NR2B的表达明显增加,与假手术组和正常组比较,有显著差异(p<0.05),而假手术组与正常组比较,无显著差异(p>0.05)；第22天,癌痛组NR2B的表达下降,与假手术组和正常组比较无统计学意义。L4-6段脊髓NR2B的实时荧光定量PCR示：接种后第6天,癌痛组NR2B的表达与假手术组比较无显著差异,而癌痛组和假手术组NR2B的表达比正常组明显增高；第10、14、18、22天,癌痛组NR2B的表达明显增加,与假手术组和正常组比较,有显著差异(p<0.05)；而假手术组与正常组比较,无显著差异(p>0.05)。
(2)L4-6段脊髓和前扣带回NR2B免疫组化测定结果：癌痛组NR2B主要分布在脊髓背角浅层、中央导水管、前角以及小鼠的前扣带回,脊髓各层NR2B表达量在中央导水管、背角浅层、前角依次增多。癌痛组于术后第14、18天,脊髓和手术对侧的前扣带回区NR2B阳性细胞光密度(OD)值与假手术组和正常组比较有统计学差异(P<0.05)；而假手术组与正常组比较,无显著差异(p>0.05)。
(3)L4-6段脊髓NR2B表达的Western Blot检测结果：癌痛组于接种后第14天、18天,脊髓NR2B的表达明显增高,与假手术组和正常组比较,有显著差异(p<0.05)；而假手术组与正常组比较,无显著差异(p>0.05)。
3.脊髓NR2B基因沉默对小鼠骨癌痛的影响：
(1)实时定量PCR检测小鼠脊髓NR2B mRNA的表达：接种后第14天和第18天干扰组小鼠L4-6段脊髓的NR2B mRNA表达水平比癌痛组明显降低,其中第14天下调最明显,干扰组比癌痛组NR2B的表达下调了74%；而干扰对照组与癌痛组比较,无显著差异(p>0.05)。
(2)Western Blot检测小鼠脊髓NR2B蛋白的表达：接种后第14和18天干扰组小鼠L4-6段脊髓的NR2B表达水平比癌痛组明显降低,其中第18天下调最明显,干扰组比癌痛组NR2B表达下调了67%；而干扰对照组与癌痛组比较,无显著差异(p>0.05)。
(3)干扰组与其他两组相比,自发抬足时间明显缩短,触诱发痛阈值明显增加,有显著差异(p<0.05)。
4.MAPK信号通路介导的NR2B在小鼠骨癌痛中的作用机制：
(1)实时定量PCR检测各组小鼠L4-6段脊髓的NR2B、SP、c-fos和GFAP基因mRNA的表达：接种术后第14、18天癌痛组小鼠脊髓的NR2B、SP、c-fos和GFAP基因表达比正常组明显上调(p<0.05)；干扰组NR2B、SP、c-fos和GFAP基因表达比癌痛组显著降低(p<0.05)；而干扰对照组与癌痛组比较,无显著差异(p>0.05)。
(2)Western Blot检测各组小鼠L4-6段脊髓的NR2B、p-ERK、p-p38和GFAP蛋白的表达：接种后第14、18天癌痛组小鼠脊髓的NR2B、p-ERK、p-p38和GFAP的蛋白表达比正常组明显上调(p<0.05)；干扰组NR2B、p-ERK、p-p38和GFAP的蛋白表达比癌痛组显著降低(p<0.05)；而干扰对照组与癌痛组比较,无显著差异(p>0.05)。
结论：
1.C57BL／6小鼠股骨骨髓腔接种Lewis肺癌细胞能成功制备小鼠骨癌痛新模型,此模型与人类骨癌痛具有相似性；
2.NR2B在小鼠骨癌痛模型中呈高表达,且同一癌痛模型中观察到NR2B在L4-L6段脊髓和前扣带回同时高表达,提示在癌痛的产生和维持中不仅激活了脊髓低位中枢,而且有脑部中枢参与；
3.本研究初次发现NR2B在癌痛模型的L4-6段脊髓前角细胞表达呈强阳性,为下一步研究提供了新的思路；
4.PEI/NR2B-siRNA复合物能特异性的使骨癌痛小鼠脊髓NR2B的表达明显下调；其表达下调与小鼠疼痛行为学减轻相一致,进一步证明NR2B在骨癌痛发生过程中起重要作用；
5.MAPK信号转导通路(ERK通路和p38通路)在骨癌痛小鼠模型中被激活；NR2B参与了MAPK信号通路介导的骨癌痛的中枢敏化；
6.NR2B参与介导的骨癌痛可能与GFAP的过表达相关,其机制有待进一步研究。
Objective:In order to investigate the mechanisms of bone cancer pain, a mouse bone cancer pain model is established to study the role of NR2B in the initiation and maintenance of bone cancer pain by observing the expression and distribution of NR2B in the anterior cingulate cortex and L4-6 spinal cord of the animal.
1. The establishment and evaluation of the new model of mouse cancer pain:thirty adult male C57BL/6 mice were randomly assigned to four groups:the test group (n=15):10μl PBS containing 2×106 Lewis lung cancer cells were inoculated into the marrow cavity of distal femur of the animal; the heat-inactivated group (n=5):the same number of Lewis lung cancer cells that had been heat-inactivated were inoculated; PBS group (n=5):the same volume of PBS solution was inoculated; normal control group (n=5):no treatment. We observed the spontaneous lifting time and mechanical allodynia of mice on alternate days from the 7th day after inoculation. The structural damage of the operated femur induced by tumor was monitored by radiological analysis by comparing with the other femur bone of the animal and observed respectively on the 7th,15th,23rd day. In the test group, five femurs from operated animals that were randomly selected were stained with hematoxylin and eosin (HE) after radiography; bone destruction was observed on 7th,15th,23rd day, respectively. Other groups were observed only on the 23rd day.
2. The expression and distribution of NR2B in the anterior cingulate cortex and spinal cord in the test group:eighty-one adult male C57BL/6 mice were randomly assigned to three groups:cancer pain group (N=27); sham-operated group (N=27); normal control group (N=27).10μl of D-Hanks solution containing 2×106 Lewis lung cancer cells were inoculated in the cancer pain group; the same volume of solution without cancer cells was used in the sham-operated group; the normal control group:no treatment. Spontaneous lifting time and mechanical allodynia of mouse hind paw were measured on the 6th,10th,14th,18th,22nd day after inoculation. Three mice were taken to observe the expression of NR2B mRNA by using the qRT-PCR detection at the same time. According to the results of the PCR, three mice from each group were decapitated to extract the L4-6 spinal cord and anterior cingulate cortex to do fluorescence quantitative RT-PCR and immunohistochemical staining to observe the expression and distribution of NR2B, the remaining three mice were used to detect NR2B protein by Western Blot on the 14th and 18th day after inoculation.
3. The effects of NR2B gene silencing in bone cancer pain mouse: 72 adult male C57BL/6 mice were randomly divided into three groups: cancer pain group (N=21):Lewis lung cancer cells 2×106/10ul were inoculated as stated above; interference group (N=21):PEI/NR2B siRNA complex was intrathecally injected 12 days later after cancer cell inoculation; control group of the interference (N=21):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 and Western Blot to detect the expression levels of NR2B in each group.
4. The effects of NR2B were mediated by MAPK signaling pathway in the mouse bone cancer pain:forty-eight adult male C57BL/6 mice were randomly divided into four groups:normal group, cancer pain group, interference group and control of interference group. Spontaneous lifting time and mechanical allodynia in each group were measured pre-inoculation and on the 10th,12th,14th,16th and 18th day after inoculation. L4-6 spinal cord of the animals from each group was used to detect the expression levels of NR2B, SP, c-fos and GFAP gene by real-time quantitative PCR, and the expression levels of NR2B, p-ERK, p-p38 and GFAP protein were detected by Western Blot on the 14th and 18th day.
1. Evaluation of bone cancer pain model:compared with the controls, the test group displayed a gradual development of spontaneous pain showing longer flinch time from the 11th day on; there was obvious mechanical allodynia from the 13th day on after inoculation as demonstrated by a 50% reduction of von Frey threshold. On the 23rd day after operation, X-ray showed that medullary cavity of lower end of femur on ipsilateral side had disappeared and some cortical bone had been lost. Pathological examination showed that tumor cells penetrated the medullary canal and invaded peripheral tissues. Therefore, the model was validated by data from the above three aspects.
2. The expression and distribution of NR2B in the anterior cingulate cortex and spinal cord of mouse with bone cancer pain:
Real-time quantitative florescence PCR detection in the anterior cingulate cortex:there was no significant difference between the cancer pain group and the sham operated group on the 6th and 10th day after inoculation, but the NR2B expression in both groups was higher than that in the control group; on the 14th and 18th day, the expression of NR2B in the cancer pain group increased significantly as compared to the groups of normal control and the sham operated (p<0.05), while there was no significant difference between the sham operated group and the normal control group. On the 22nd day, the expression of NR2B in the cancer pain group was reduced, but there was no statistical significance as compared to the other two groups. Real-time quantitative florescence PCR detection of NR2B expression in L4-6 spinal cord:on the 6th day after inoculation, there was no significant difference between the cancer pain group and the sham operated group, but the expression of NR2B in these two groups was increased significantly as compared to the normal control group. On the 10th,14th,18th and 22nd day, the expression of NR2B in the cancer pain group was increased significantly as compared to the groups of normal control and the sham operated (p<0.05), while there was no significant difference between the sham operated and the normal control group.
(2) The results of immunohistochemical examination in the anterior cingulate cortex and L4-6 spinal cord:the expression of NR2B in the cancer pain group was mainly located in the superficial dorsal horn, central aqueduct, anterior horn of the spinal cord and the anterior cingulate cortex, the expression level of NR2B was highest in the anterior horn, followed bysuperficial dorsal horn and lowest levels were observed in the central aqueduct. The optical density (OD) values of NR2B-positive cells in L4-6 spinal cord and area of contralateral anterior cingulate cortex in the cancer pain group were significantly different as compared to the sham-operated and the normal controls on the 14th and 18th day after inoculation (P<0.05), whereas there was no significant difference between the later two groups (P>0.05).
(3) The results of the Western Blot in L4-6 spinal cord:the expression of NR2B in the spinal cord of cancer pain group was significantly increased as compared to the groups of the normal control and the sham operated on day 14 and 18 after inoculation (p<0.05), while there was no significant difference between the later two groups (P>0.05).
3. The effects of NR2B gene silencing in cancer bone pain mouse:
(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.
4. The effects of NR2B were mediated by MAPK signaling pathway in bone cancer pain in mouse:
(1) Real-time quantitative PCR detection:the expression levels of NR2B, SP, c-fos and GFAP genes in the L4-6 spinal cord of cancer pain group was significantly increased as compared to the normal control group on the 14th and 18th day after inoculation (P<0.05); the expression of these genes in the L4-6 spinal cord in the interference group was significantly lower than that of the cancer pain group; while there was no significant difference between the cancer pain group and the control of interference group(p>0.05).
(2) Western Blot detection results:the expression of NR2B, p-ERK, p-p38 and GFAP protein in the spinal cord of cancer pain group was significantly increased as compared to that in the normal group on the 14th and 18th day after inoculation (P<0.05); the expression of these proteins in the L4-6 spinal cord of the interference group was significantly lower than that in the cancer pain group (P<0.05); while there was no significant difference between the cancer pain group and the control group of interference group(p>0.05).
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.
2. The observation that NR2B was expressed in large quantity in both the L4-6 spinal cord and anterior cingulate cortex suggests that the generation and maintenance of bone cancer pain not only can be activated in the spinal cord, but also is related to the anterior cingulate cortex.
3. In this study we found that the expression of NR2B was strongly positive in the anterior horn cells of spinal cord of the bone cancer pain model, which provided some new insights about the mechanism of cancer pain and might be useful in later research.
4. 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.
5. The MAPK signaling pathway was activated in the bone cancer pain model, suggesting that MAPK signal transduction pathway (ERK pathway and p38 pathway) is involved in the generation and maintenance of bone cancer pain.
6. Bone cancer pain is mediated by NR2B receptor system, and may be related to the over-expression of GFAP, yet the mechanisms involved need to be further explored.
方法
1.小鼠骨癌痛新模型的制备及评价：选用成年雄性C57BL／6小鼠30只,随机分为4组,模型组(15只)：接种Lewis肺癌细胞2×106个；热灭活组(5只)：接种相同数目加热灭活的Lewis肺癌细胞；PBS组(5只)：接种等容积的PBS液；正常对照组(5只)：不予任何处理。接种后7天始隔日观察小鼠自发痛行为及测定机械性触诱发痛。第7、15、23天,将小鼠麻醉后行双侧后肢X线摄片,评估肿瘤诱发的骨组织破坏程度。模型组小鼠每次摄片后任选5只取术侧后肢,各对照组小鼠最后一次摄片后取术侧后肢,分别作苏木素-伊红(HE)染色观察骨质破坏情况。
2.NR2B在骨癌痛小鼠前扣带回和脊髓中的分布与表达：选用成年雄性C57BL/6小鼠81只,随机分为3组：癌痛组,假手术组和正常对照组。观察三组小鼠自发痛行为及测定机械性触诱发痛。分别于术后第6、10、14、18、22天各取3只,实时定量PCR检测前扣带回和脊髓NR2B mRNA的表达。依据PCR结果,每组分别于术后第14、18天各取3只,免疫组化检测前扣带回和脊髓NR2B的分布和表达；同时每组另各取3只,于术后第14、18天行Western Blot检测脊髓NR2B蛋白的表达。
3.脊髓NR2B基因沉默对小鼠骨癌痛的影响：选用成年雄性C57BL／6小鼠72只,随机分为3组：癌痛组,干扰组和干扰对照组。观察各组小鼠自发痛行为及测定机械性触诱发痛。每组分别于接种后第13、14、15、16、18、22天各取3只,实时定量PCR检测脊髓NR2B mRNA的表达。依据PCR的结果,于接种后第14和18天每组各取3只,Western Blot检测脊髓NR2B蛋白的表达。
4.MAPK信号通路介导的NR2B在小鼠骨癌痛中的作用机制：选用成年雄性C57BL／6小鼠48只,随机分为4组：正常组,癌痛组,干扰组和干扰对照组。观察各组小鼠自发痛行为及测定机械性触诱发痛。每组分别于接种后第14和18天各取3只,实时定量PCR检测脊髓NR2B、SP、c-fos和GFAP基因表达水平。选取接种后第14和18天的每组小鼠各3只,Western Blot检测脊髓NR2B、p-ERK、p-p38和GFAP蛋白表达水平。
结果
1.小鼠骨癌痛新模型的评价：模型组接种后第11天左右出现明显自发痛行为,表现为自发抬足时间延长；接种后第13天左右出现明显的触诱发痛,表现为50%缩足阈值明显下降,且持续整个观察期。接种后第23天放射学结果显示,术侧股骨下段骨髓腔消失,骨皮质中断、大块缺损。同时组织学可见肿瘤细胞充满骨髓腔,且穿破骨皮质向外生长,侵犯周围肌肉组织。行为学、放射学、组织学三方面证实建模成功。
2.NR2B在骨癌痛小鼠的前扣带回和脊髓中的分布与表达：
(1)前扣带回NR2B的实时荧光定量PCR结果显示：接种后第6天、10天,癌痛组NR2B的表达与假手术组比较无显著差异,而癌痛组和假手术组NR2B的表达比正常组明显增高；第14天、18天,癌痛组NR2B的表达明显增加,与假手术组和正常组比较,有显著差异(p<0.05),而假手术组与正常组比较,无显著差异(p>0.05)；第22天,癌痛组NR2B的表达下降,与假手术组和正常组比较无统计学意义。L4-6段脊髓NR2B的实时荧光定量PCR示：接种后第6天,癌痛组NR2B的表达与假手术组比较无显著差异,而癌痛组和假手术组NR2B的表达比正常组明显增高；第10、14、18、22天,癌痛组NR2B的表达明显增加,与假手术组和正常组比较,有显著差异(p<0.05)；而假手术组与正常组比较,无显著差异(p>0.05)。
(2)L4-6段脊髓和前扣带回NR2B免疫组化测定结果：癌痛组NR2B主要分布在脊髓背角浅层、中央导水管、前角以及小鼠的前扣带回,脊髓各层NR2B表达量在中央导水管、背角浅层、前角依次增多。癌痛组于术后第14、18天,脊髓和手术对侧的前扣带回区NR2B阳性细胞光密度(OD)值与假手术组和正常组比较有统计学差异(P<0.05)；而假手术组与正常组比较,无显著差异(p>0.05)。
(3)L4-6段脊髓NR2B表达的Western Blot检测结果：癌痛组于接种后第14天、18天,脊髓NR2B的表达明显增高,与假手术组和正常组比较,有显著差异(p<0.05)；而假手术组与正常组比较,无显著差异(p>0.05)。
3.脊髓NR2B基因沉默对小鼠骨癌痛的影响：
(1)实时定量PCR检测小鼠脊髓NR2B mRNA的表达：接种后第14天和第18天干扰组小鼠L4-6段脊髓的NR2B mRNA表达水平比癌痛组明显降低,其中第14天下调最明显,干扰组比癌痛组NR2B的表达下调了74%；而干扰对照组与癌痛组比较,无显著差异(p>0.05)。
(2)Western Blot检测小鼠脊髓NR2B蛋白的表达：接种后第14和18天干扰组小鼠L4-6段脊髓的NR2B表达水平比癌痛组明显降低,其中第18天下调最明显,干扰组比癌痛组NR2B表达下调了67%；而干扰对照组与癌痛组比较,无显著差异(p>0.05)。
(3)干扰组与其他两组相比,自发抬足时间明显缩短,触诱发痛阈值明显增加,有显著差异(p<0.05)。
4.MAPK信号通路介导的NR2B在小鼠骨癌痛中的作用机制：
(1)实时定量PCR检测各组小鼠L4-6段脊髓的NR2B、SP、c-fos和GFAP基因mRNA的表达：接种术后第14、18天癌痛组小鼠脊髓的NR2B、SP、c-fos和GFAP基因表达比正常组明显上调(p<0.05)；干扰组NR2B、SP、c-fos和GFAP基因表达比癌痛组显著降低(p<0.05)；而干扰对照组与癌痛组比较,无显著差异(p>0.05)。
(2)Western Blot检测各组小鼠L4-6段脊髓的NR2B、p-ERK、p-p38和GFAP蛋白的表达：接种后第14、18天癌痛组小鼠脊髓的NR2B、p-ERK、p-p38和GFAP的蛋白表达比正常组明显上调(p<0.05)；干扰组NR2B、p-ERK、p-p38和GFAP的蛋白表达比癌痛组显著降低(p<0.05)；而干扰对照组与癌痛组比较,无显著差异(p>0.05)。
结论：
1.C57BL／6小鼠股骨骨髓腔接种Lewis肺癌细胞能成功制备小鼠骨癌痛新模型,此模型与人类骨癌痛具有相似性；
2.NR2B在小鼠骨癌痛模型中呈高表达,且同一癌痛模型中观察到NR2B在L4-L6段脊髓和前扣带回同时高表达,提示在癌痛的产生和维持中不仅激活了脊髓低位中枢,而且有脑部中枢参与；
3.本研究初次发现NR2B在癌痛模型的L4-6段脊髓前角细胞表达呈强阳性,为下一步研究提供了新的思路；
4.PEI/NR2B-siRNA复合物能特异性的使骨癌痛小鼠脊髓NR2B的表达明显下调；其表达下调与小鼠疼痛行为学减轻相一致,进一步证明NR2B在骨癌痛发生过程中起重要作用；
5.MAPK信号转导通路(ERK通路和p38通路)在骨癌痛小鼠模型中被激活；NR2B参与了MAPK信号通路介导的骨癌痛的中枢敏化；
6.NR2B参与介导的骨癌痛可能与GFAP的过表达相关,其机制有待进一步研究。
Objective:In order to investigate the mechanisms of bone cancer pain, a mouse bone cancer pain model is established to study the role of NR2B in the initiation and maintenance of bone cancer pain by observing the expression and distribution of NR2B in the anterior cingulate cortex and L4-6 spinal cord of the animal.
1. The establishment and evaluation of the new model of mouse cancer pain:thirty adult male C57BL/6 mice were randomly assigned to four groups:the test group (n=15):10μl PBS containing 2×106 Lewis lung cancer cells were inoculated into the marrow cavity of distal femur of the animal; the heat-inactivated group (n=5):the same number of Lewis lung cancer cells that had been heat-inactivated were inoculated; PBS group (n=5):the same volume of PBS solution was inoculated; normal control group (n=5):no treatment. We observed the spontaneous lifting time and mechanical allodynia of mice on alternate days from the 7th day after inoculation. The structural damage of the operated femur induced by tumor was monitored by radiological analysis by comparing with the other femur bone of the animal and observed respectively on the 7th,15th,23rd day. In the test group, five femurs from operated animals that were randomly selected were stained with hematoxylin and eosin (HE) after radiography; bone destruction was observed on 7th,15th,23rd day, respectively. Other groups were observed only on the 23rd day.
2. The expression and distribution of NR2B in the anterior cingulate cortex and spinal cord in the test group:eighty-one adult male C57BL/6 mice were randomly assigned to three groups:cancer pain group (N=27); sham-operated group (N=27); normal control group (N=27).10μl of D-Hanks solution containing 2×106 Lewis lung cancer cells were inoculated in the cancer pain group; the same volume of solution without cancer cells was used in the sham-operated group; the normal control group:no treatment. Spontaneous lifting time and mechanical allodynia of mouse hind paw were measured on the 6th,10th,14th,18th,22nd day after inoculation. Three mice were taken to observe the expression of NR2B mRNA by using the qRT-PCR detection at the same time. According to the results of the PCR, three mice from each group were decapitated to extract the L4-6 spinal cord and anterior cingulate cortex to do fluorescence quantitative RT-PCR and immunohistochemical staining to observe the expression and distribution of NR2B, the remaining three mice were used to detect NR2B protein by Western Blot on the 14th and 18th day after inoculation.
3. The effects of NR2B gene silencing in bone cancer pain mouse: 72 adult male C57BL/6 mice were randomly divided into three groups: cancer pain group (N=21):Lewis lung cancer cells 2×106/10ul were inoculated as stated above; interference group (N=21):PEI/NR2B siRNA complex was intrathecally injected 12 days later after cancer cell inoculation; control group of the interference (N=21):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 and Western Blot to detect the expression levels of NR2B in each group.
4. The effects of NR2B were mediated by MAPK signaling pathway in the mouse bone cancer pain:forty-eight adult male C57BL/6 mice were randomly divided into four groups:normal group, cancer pain group, interference group and control of interference group. Spontaneous lifting time and mechanical allodynia in each group were measured pre-inoculation and on the 10th,12th,14th,16th and 18th day after inoculation. L4-6 spinal cord of the animals from each group was used to detect the expression levels of NR2B, SP, c-fos and GFAP gene by real-time quantitative PCR, and the expression levels of NR2B, p-ERK, p-p38 and GFAP protein were detected by Western Blot on the 14th and 18th day.
1. Evaluation of bone cancer pain model:compared with the controls, the test group displayed a gradual development of spontaneous pain showing longer flinch time from the 11th day on; there was obvious mechanical allodynia from the 13th day on after inoculation as demonstrated by a 50% reduction of von Frey threshold. On the 23rd day after operation, X-ray showed that medullary cavity of lower end of femur on ipsilateral side had disappeared and some cortical bone had been lost. Pathological examination showed that tumor cells penetrated the medullary canal and invaded peripheral tissues. Therefore, the model was validated by data from the above three aspects.
2. The expression and distribution of NR2B in the anterior cingulate cortex and spinal cord of mouse with bone cancer pain:
Real-time quantitative florescence PCR detection in the anterior cingulate cortex:there was no significant difference between the cancer pain group and the sham operated group on the 6th and 10th day after inoculation, but the NR2B expression in both groups was higher than that in the control group; on the 14th and 18th day, the expression of NR2B in the cancer pain group increased significantly as compared to the groups of normal control and the sham operated (p<0.05), while there was no significant difference between the sham operated group and the normal control group. On the 22nd day, the expression of NR2B in the cancer pain group was reduced, but there was no statistical significance as compared to the other two groups. Real-time quantitative florescence PCR detection of NR2B expression in L4-6 spinal cord:on the 6th day after inoculation, there was no significant difference between the cancer pain group and the sham operated group, but the expression of NR2B in these two groups was increased significantly as compared to the normal control group. On the 10th,14th,18th and 22nd day, the expression of NR2B in the cancer pain group was increased significantly as compared to the groups of normal control and the sham operated (p<0.05), while there was no significant difference between the sham operated and the normal control group.
(2) The results of immunohistochemical examination in the anterior cingulate cortex and L4-6 spinal cord:the expression of NR2B in the cancer pain group was mainly located in the superficial dorsal horn, central aqueduct, anterior horn of the spinal cord and the anterior cingulate cortex, the expression level of NR2B was highest in the anterior horn, followed bysuperficial dorsal horn and lowest levels were observed in the central aqueduct. The optical density (OD) values of NR2B-positive cells in L4-6 spinal cord and area of contralateral anterior cingulate cortex in the cancer pain group were significantly different as compared to the sham-operated and the normal controls on the 14th and 18th day after inoculation (P<0.05), whereas there was no significant difference between the later two groups (P>0.05).
(3) The results of the Western Blot in L4-6 spinal cord:the expression of NR2B in the spinal cord of cancer pain group was significantly increased as compared to the groups of the normal control and the sham operated on day 14 and 18 after inoculation (p<0.05), while there was no significant difference between the later two groups (P>0.05).
3. The effects of NR2B gene silencing in cancer bone pain mouse:
(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.
4. The effects of NR2B were mediated by MAPK signaling pathway in bone cancer pain in mouse:
(1) Real-time quantitative PCR detection:the expression levels of NR2B, SP, c-fos and GFAP genes in the L4-6 spinal cord of cancer pain group was significantly increased as compared to the normal control group on the 14th and 18th day after inoculation (P<0.05); the expression of these genes in the L4-6 spinal cord in the interference group was significantly lower than that of the cancer pain group; while there was no significant difference between the cancer pain group and the control of interference group(p>0.05).
(2) Western Blot detection results:the expression of NR2B, p-ERK, p-p38 and GFAP protein in the spinal cord of cancer pain group was significantly increased as compared to that in the normal group on the 14th and 18th day after inoculation (P<0.05); the expression of these proteins in the L4-6 spinal cord of the interference group was significantly lower than that in the cancer pain group (P<0.05); while there was no significant difference between the cancer pain group and the control group of interference group(p>0.05).
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.
2. The observation that NR2B was expressed in large quantity in both the L4-6 spinal cord and anterior cingulate cortex suggests that the generation and maintenance of bone cancer pain not only can be activated in the spinal cord, but also is related to the anterior cingulate cortex.
3. In this study we found that the expression of NR2B was strongly positive in the anterior horn cells of spinal cord of the bone cancer pain model, which provided some new insights about the mechanism of cancer pain and might be useful in later research.
4. 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.
5. The MAPK signaling pathway was activated in the bone cancer pain model, suggesting that MAPK signal transduction pathway (ERK pathway and p38 pathway) is involved in the generation and maintenance of bone cancer pain.
6. Bone cancer pain is mediated by NR2B receptor system, and may be related to the over-expression of GFAP, yet the mechanisms involved need to be further explored.
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