帕米磷酸二钠对骨癌性疼痛模型小鼠的镇痛作用及相关分子机制研究
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摘要
目的探讨帕米磷酸二钠对骨癌性疼痛模型小鼠镇痛作用及肿瘤坏死因子(TNF-α)、细胞凋亡相关蛋白Bax、Bcl2及Caspase-3表达的影响。方法选取小鼠45只随机分为对照组、模型组、观察组,每组15只。将前列腺癌细胞RM-1注射入股骨远端的骨髓腔,制造小鼠骨骼痛模型。对照组将前列腺癌细胞RM-1灭活后注入股骨远端骨髓腔;观察组给予帕米磷酸二钠治疗,模型组与对照组均使用等量生理氯化钠溶液代替。观察各组小鼠热辐射刺激及机械刺激的疼痛变化,疼痛行为检测完毕后,立即处死小鼠,取小鼠右侧股骨骨髓标本在液氨中保存备用。Western blot、实时PCR法对TNF-α、Bax、Bcl2及Caspase-3蛋白及mRNA表达进行检测。结果术后14d,与对照组比较,观察组和模型组各项疼痛指标差异有统计学意义(P<0.01);给药后6、13d时,与模型组比较,观察组各项疼痛指标差异有统计学意义(P<0.05)。观察组TNF-α、Bax、Caspase-3蛋白及其mRNA表达明显高于模型组,低于对照组((P<0.05,P<0.01);观察组Bcl2蛋白及Bcl2mRNA表达明显高于模型组(P<0.05)。结论帕米磷酸二钠能有效缓解骨癌性疼痛模型小鼠疼痛,其作用机制与上调骨癌性疼痛模型小鼠TNF-α表达和改善Bax、Caspase-3、Bcl2细胞凋亡相关蛋白表达有关。
Objective To investigate the analgesic effect of pamidronate disodium on two mouse models of bone cancer pain and on tumor necrosis factor(TNF-α),apoptosis-related proteins Bax,Bcl2 and caspase-3.Methods Fortyfive mice were randomly divided into observation group,model group and control group(n=15).Prostate cancer cell RM-1 was injected into the bone marrow cavity of the distal femur,so that a model of skeletal pain in mice was established.In the control group,prostate cancer cell RM-1 was inactivated and injected into the bone marrow cavity of the distal femur.The observation group received pamidronate disodium.The model group and control group were given physiological saline instead.The pain changes of thermal stimulation and mechanical stimulation were observed in each group.After the pain behavior was detected,the mice were killed immediately and the right femur bone marrow specimens were collected and stored in liquid nitrogen.Western blot and real-time PCR were used to detect TNF-α,Bax,Bcl2,Caspase-3 protein and mRNA expression in order to explore the mechanism of action.Results The pain indexes of patients in the observation group and model group 14 dafter operation were significantly different from those of the control group(P <0.01).After administration of 6 and 13 d,the difference of pain indexes was statistically significant between the model group and the observation group(P <0.05).The expressions of TNF-α,Bax,Caspase-3 and mRNA in the observation group were significantly higher than those in the model group,but lower than those of the control group(P <0.01,P <0.05).The expressions of Bcl2 and Bcl2 mRNA in the observation group were significantly lower than those in the model group,but higher than those of the model group(P <0.05).Conclusion Pamidronate disodium can effectively relieve pain in the mouse model of bone cancer pain.The mechanism may be related to the up regulation of TNF-αexpression in the mouse model and to the improvement of apoptosis-related protein expressions of Bax,Caspase-3 and Bcl2 in cells.
Objective To investigate the analgesic effect of pamidronate disodium on two mouse models of bone cancer pain and on tumor necrosis factor(TNF-α),apoptosis-related proteins Bax,Bcl2 and caspase-3.Methods Fortyfive mice were randomly divided into observation group,model group and control group(n=15).Prostate cancer cell RM-1 was injected into the bone marrow cavity of the distal femur,so that a model of skeletal pain in mice was established.In the control group,prostate cancer cell RM-1 was inactivated and injected into the bone marrow cavity of the distal femur.The observation group received pamidronate disodium.The model group and control group were given physiological saline instead.The pain changes of thermal stimulation and mechanical stimulation were observed in each group.After the pain behavior was detected,the mice were killed immediately and the right femur bone marrow specimens were collected and stored in liquid nitrogen.Western blot and real-time PCR were used to detect TNF-α,Bax,Bcl2,Caspase-3 protein and mRNA expression in order to explore the mechanism of action.Results The pain indexes of patients in the observation group and model group 14 dafter operation were significantly different from those of the control group(P <0.01).After administration of 6 and 13 d,the difference of pain indexes was statistically significant between the model group and the observation group(P <0.05).The expressions of TNF-α,Bax,Caspase-3 and mRNA in the observation group were significantly higher than those in the model group,but lower than those of the control group(P <0.01,P <0.05).The expressions of Bcl2 and Bcl2 mRNA in the observation group were significantly lower than those in the model group,but higher than those of the model group(P <0.05).Conclusion Pamidronate disodium can effectively relieve pain in the mouse model of bone cancer pain.The mechanism may be related to the up regulation of TNF-αexpression in the mouse model and to the improvement of apoptosis-related protein expressions of Bax,Caspase-3 and Bcl2 in cells.
引文
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[2]徐洁.趋化因子CXCL1在骨癌性疼痛中的作用及其机制研究[D].南通大学,2013:4
[3]黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2014,9(9):1069-1072
[4]马正良,夏小萍,朱巍,等.小鼠骨癌痛模型的建立及痛行为学和骨损害的观察[J].中国疼痛学杂志,2007,13(5):288-292
[5]Zhang ZJ,Cao DL,Zhang X,et al.Hemokinecontribution to neuropathic pain:respective induction of CXCL1and CXCR2in spinal cord astrocytes and neurons[J].Pain,2013,154(10):2185
[6]梁瑞娜,马珊珊,田毅,等.人脐带沃顿胶肝细胞对急性大鼠脊髓损伤的作用及脊髓组织中TNF-α和BDNF表达的影响[J].郑州大学学报医学版,2014,49(6):765-767
[7]史婷婷,白建平,梁月琴,等.芹菜素对大鼠缺血/再灌注心肌细胞凋亡及相关蛋白Bax、Bcl2、Caspase-3表达[J].中国药理通报,2011,27(5):666-671
[8]李宾,张卉,张辉.二磷酸盐对骨癌痛模型小鼠趋化因子CXCL1及受体CXCR2表达影响的研究[J].中国生化药物杂志,2015,35(2):31-35
[9]陈素艳,张振香,高峰,等.常压氧疗对大鼠脑缺血再灌注后脑组织Caspase-9及Caspase-3mRNA表达的影响[J].郑州大学学报:医学版,2014,49(2):203-205
[10]Li Q,Guo Y L,Li X,et al.The interference of picrosideⅡon the expressions of Caspase-3and PARP follwing cerebral ischemia reperfusion injury in rats[J].Chin Pharmacol Bull,2010,26(3):342-345