大鼠骨癌痛模型的建立及其脊髓GFAP表达水平的变化
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摘要
目的
     采用Walker 256大鼠乳腺癌细胞建立大鼠胫骨癌痛模型,并验证模型建立的成功,建立大鼠慢性炎症及神经病理性疼痛模型并与骨癌痛模型进行疼痛行为学比较。用免疫组织化学实验(IHC)观察三种疼痛模型大鼠脊髓神经胶质纤维酸性蛋白(GFAP)表达水平的变化。
     方法
     将36只SD雌性大鼠,随机分为4组:①肿瘤组(Sarcoma n=8):接种肿瘤细胞;②假手术1(Shaml n=10):接种不含肿瘤细胞的大鼠腹水;⑧假手术2(Sham2 n=10):接种生理盐水;④正常对照组(Naive n=8)。参照Medhurst等报道的方法将含1×10~5Walker 256大鼠乳腺癌细胞悬液注入大鼠胫骨上段制备骨癌痛模型。Sham 1和Sham 2分别注入等体积的不含肿瘤细胞的大鼠腹水和生理盐水,观察疼痛行为学(一般情况、体重、机械缩足反射阈值MWT和热缩足潜伏期PWTL)、骨放射学和组织学。同样规格大鼠52只,随机分为6组:①慢性炎症疼痛模型(CFA,n=10),注射CFA;②假手术组(NS,n=8):注射NS;③坐骨神经部分结扎模型(PNL,n=9);④假手术组(Sham,n=9);⑤⑥正常对照组(Naive n=8);观察大鼠疼痛行为学。三种疼痛模型及其对照组分别在疼痛高峰期间处死取脊髓标本,冰冻切片后行免疫组化,观察三种模型大鼠脊髓GFAP表达水平的变化。
     结果
     从疼痛行为学、影像学、组织学多方面观察的结果证实,Walker 256大鼠乳腺癌细胞能成功制备大鼠胫骨癌痛模型。Sarcoma组造模后第7天开始体重不增或降低、MWT显著降低(P<0.01),第11天PWTL显著降低(P<0.01)。疼痛高峰期间,三种疼痛模型中CFA大鼠MWT最低(P<0.01),Sarcoma组和PNL组无明显差异(P>0.05),三种疼痛模型PWTL无明显差异(P>0.05)。Sarcoma和PNL大鼠脊髓GFAP的上调水平高于CFA、Sham及Naive组(P<0.01);Sarcoma组脊髓GFAP的上调水平高于PNL组;且统计学上有显著差异(P<0.05)。CFA大鼠脊髓GFAP表达水平与NS组相比无明显差异(P>0.05)。
Objective
     The aim of this investigation was to set up tibial cancer pain model with Walker 256 mammary gland carcinoma cells in rats,and compare differences of painful behaviors with inflammatory & neuropathic pain model.GFAP that has been implicated in the generation and maintenance of chronic pain was then examined in spinal cord by IHC method and evaluated whether its expression was different in three chronic pain models.
     Methods
     36 female SD rats were randomly divided into 4 groups:①Sarcoma(n=8,tumor cells were injected);②Sham1(n=10,rat ascetic fluid without tumor cells was injected);③Sham 2(n=10,NS was injected);④Na(i|¨)ve(n=8).According to the method described by Medhurst et al,10~5 syngenenic Walker 256 mammary gland carcinoma cells,ascetic fluid without tumor cells and NS were respectively injected into the tibia medullary cavity via intercondylar eminence.Series of tests were carried out including general condition,weight bearing ability,ambulatory pain,MWT,PWTL together with bone radiology and bone histology.Another 52 female SD rats were randomly divided into 6 groups:①CFA(n=10);②NS(n=8);③PNL(n=9);④Sham(n=9);⑤⑥Na(i|¨)ve(n=8), and underwent as above.All rats were sacrificed at the period of remarkable pain.GFAP was then examined in spinal cord by IHC method and evaluated whether its expression was different in three chronic pain models.
     Results
     Rats that developed of hyperalgesia and allodynia after intra-tibial injection of Walker 256 cells were proved successful.MWT and PWTL were measured during the experimental days,and the findings revealed Sarcoma rats started to exhibit mechanical allodynia on the 7~(th) day and thermal hyperalgesia on the 11~(th) day after the operation(P<0.01).At the period of remarkable pain,CFA rats exhibited mechanical allodynia stronger than Sarcoma and PNL rats(P<0.01),but no difference was observed between Sarcoma and PNL rats(P>0.05).The difference of thermal hyperalgesia between three chronic pain models was not remarkable(P>0.05).The up-regulation of GFAP expression in spinal cord of Sarcoma rats was significantly greater than that induced by PNL(P<0.05) and CFA(P<0.01) model.The up-regulation of GFAP in PNL model was significantly greater than that induced by CFA model(P<0.01).Compared to Sham or Na(i|¨)ve group, no change of GFAP was observed in the spinal cord of CFA.The data suggests that the mechanisms of bone cancer pain are complicated and different from inflammatory and neuropathic pain.
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