摘要
目前,口腔颌面部急慢性疼痛的机制不明,而神经系统致敏的问题正是神经科学领域研究的难点和热点。在疼痛研究中,各种牙髓损伤动物模型具有很多优点,而急性牙髓炎是较为理性的病理性疼痛动物模型,对于研究牙髓和口颌面疼痛的机制具有重要价值。
目的:本研究结合临床实际,采用开髓封LPS的方法,建立大鼠急性牙髓炎的病理性疼痛动物模型;研究不同牙髓炎症阶段脑干中相关核团c-fos和GFAP的变化时程,探讨中枢致敏机制;进而应用蛋白质组学新技术,筛选与鉴定急性牙髓炎致敏时三叉神经节异常表达蛋白,寻找痛觉相关蛋白。
材料与方法:雄性SD大鼠,设正常对照组和实验组。实验组动物左上第一磨牙开髓,封LPS,于6小时、12小时、24小时、48小时取牙齿、三叉神经节和脑干组织,用HE染色方法观察牙髓炎症的发展变化,用免疫组化、western-blot的方法分析脑干三叉神经脊束核尾侧亚核(Vc)、三叉神经脊束核极间亚核(ⅵ)、三叉神经孤束核(NTS)核团内c-fos和GFAP的变化时程,用蛋白质组学方法分析三叉神经节功能表达差异蛋白。
结果:
1.HE染色结果:随着LPS封药时间的延长,穿髓点下方的炎症范围逐渐扩展,程度加重。12-24小时出现典型的急性牙髓炎的病理学变化,穿髓点下方大量中性粒细胞浸润伴有局限性小脓肿形成,牙髓组织充血、水肿渗出明显,成牙本质细胞排列紊乱,部分牙髓变性坏死。48小时部分动物病变扩展至根尖周组织。
2.脑干c-fos蛋白变化时程:在LPS致急性牙髓致敏后,与对照组相比,实验组Vc核c-fos阳性神经元表达数目明显增加,并呈时间依赖性,24小时达到最高;对侧Vc核及V_i核的c-fos表达数目少于实验侧,但变化趋势与实验侧相近;NTS的c-fos阳性神经元数目显著增加,6h最高,随时间逐渐降低,48小时仍高于对照组。
3.急性牙髓炎脑干中星形胶质细胞GFAP的变化时程:在LPS致急性牙髓炎致敏后,Vc变化时程慢于c-fos阳性神经元,24 h至48h数量增多,此时星形胶质细胞胞体变大,突起增粗,染色加深,与c-fos阳性神经元分布区域相近。对侧Vc未见显著变化。而三叉神经节的卫星胶质细胞48小时才出现较弱的阳性表达。
4.蛋白质组分析鉴定出48个差异点蛋白质斑点,其中21个表达量上调,27个下调,经质谱检测出10个点,分别为甲状腺素运载蛋白;内质网蛋白ERp29;低分子量神经丝蛋白;盘膜边缘蛋白;血清白蛋白前体;外周髓磷脂P0蛋白;组织蛋白酶B;丙酮酸脱氢酶E1;复制蛋白A和细胞骨架肌动蛋白。
结论:
1.开髓封LPS可以有效的造成急性牙髓炎动物模型,是较好的痛觉致敏模型;
2.在急性牙髓炎致敏过程中,随着炎症的进展,脑干相关核团的神经元和胶质细胞先后被激活,相互作用,共同造成神经系统的致敏;
3.在鉴定的10种蛋白质中,包括了细胞结构蛋白、分子伴侣蛋白、生物合成相关蛋白、以及在应激反应和能量代谢等重要生命活动中起关键作用的酶,细胞骨架破坏、能量代谢改变、信号转导异常等共同参与了LPS急性牙髓致敏的形成;
4.内质网蛋白ER29、神经丝蛋白及髓磷脂P0蛋白可能成为评价牙髓急性痛敏时程、疼痛程度及预测预后的生物指标,为进一步深入研究急性牙髓炎性致敏的分子机制提供重要的线索,对研究三叉神经节功能及口颌面部疼痛的诊断和治疗具有参考价值。
At present,the mechanism of orofacial pain still remain unclear,the central sensitization is becoming one hot and difficult area of neuroscience research.In the pain research,many dental pulp injury models with different advantages have been employed,of which the acute pulpitis model is an ideal pathological pain model,which is of great value for the research of dental pulp and orofacial pain.
Object:Based on the dinical characteristics of acute pulpitis,a rat acute pulpitis model was induced by Lipopolysaccaride(LPS),the expression of c-fos and GFAP in related nudeus of brain stem was observed to explore the mechanism of central sensitization,after that,the abnormally expressed proteins in the trigeminal ganglion were screened with a proteomics method to find pain-related proteins.
Materials and methods:male rats were divided into experiment and control group.The access to dental pulp of the left upper first molar was prepared and LPS was placed into cavity,at 6h,12h,24h,48h after operation,the teeth were extracted,immunohistochemistry and western blot method were used to analyze the expression of c-fos and GFAP in brain stem,and proteomics method was carried out to detect the differential expression of function proteins in trigeminal ganglion.
Results:
1.HE staining:showed that with the increased stimulating duration of LPS,the range of inflammation extended gradually,and the inflammation became more severe.From 12 to 24h,the typical pathological change of acute pulpitis appeared,the pulp tissue became congested along with apparent exudate, great amounts of neutrophilic granulocyte infiltrated to the area below the perforation of pulp chamber,local abscess formed,necrosis appeared in partial dental pulp tissue,and odontoblast became malalinemented.After 48h, this pathological change extended to periapical tissue in partial animals.
2.After central sensitization induced by LPS,the c-fos positive neuron number in Vc increased significantly compared with control group,which is also time-dependent and reach the peak at 24h after operation.Though the c-fos positive neuron number in contralateral side of Vc was lower than that in ipsilateral side,the development trend was similar with ipsilateral side.The number of c-fos positive neuron in NTs also increased significantly till 6h after operation,and then decreased gradually,but was sill higher than control group.
3.The change of GFAP expression in Vc appeared later than c-fos,from 24 to 48h the number of positive neuron increased,the astrocyte became larger and the process became thicker,the staining also became stronger,the distribution of GFAP positive neuron was similar with c-fos.No apparent change was found in the contralateral side.The astrocyte in trigeminal ganglion only showed weak staining at 48h after operation.
4.Total 48 protein spots were founded,among which 21 were upregulated,27 were downregulated.Ten proteins were identified as thyroxine binding globulin,ERp29,neurofilament protein,peripherin,serum albumin precursor, peripheral myelin P0 protein,Cathepsin B,pyruvate dehydrogenase E1, replication protein A and actin.
Conclusion:
1.Acute pulpititiscould be induced by LPS plcaed in exposed dental pulp in rat, which is a suitable model for hyperalgesia research;
2.In this acute pulpitis,model,with the development the inflammation,neuron and astrocyte in related nucleus were activated in succession during the process of central sensitization,both the neuron and astrocyte acted reciprocally leading to central sensitization.
3.Ten proteins identified in this research included structural protein,chaperone proteins,biosynthesis related protein,and some enzymeswhich play a key role in the stress and energy metabolism,based on this diversity,we conclude that the destruction of cytoskeleton,the change of energy metabolism and abnormal signal transduction all together take part in formation of central sensitization induced by acute pul pitis;
4.ERp29,neurofilament protein and peripheral myelin P0 protein might be the biological indicators for the duration,degree and prognosis of acute pulpitis and the important dues for mechanism of orofacial pain,which might also provide the references for the diagnosis and treatment of orofacial pain.
引文
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