摘要
目的婴儿痉挛症(Infantile spasms,IS)是一种特发于婴儿期的年龄依赖性癫痫脑病，临床上以反复痉挛发作、脑电图高度失律、明显的精神运动发育落后为特征。70%～90%的患儿可伴有重度认知功能障碍，智力倒退等严重脑损伤，病因可多达200余种，临床流行病学提示，母孕期精神应激对子代健康的具有影响，婴儿痉挛症的发病与母亲产前不良应激明显相关。婴儿痉挛症属于难治性癫痫，几乎所有的传统抗癫痫药物均药效欠佳，但促肾上腺皮质激素释放激素（ACTH）对其有独特疗效。因此，本研究通过建立婴儿痉挛症动物模型，利用差异蛋白质组技术，旨在研究探讨婴儿痉挛症的发病机制、孕期应激因素的影响及ACTH对婴儿痉挛的治疗机制。
方法利用未成熟脑对N-甲基-D-天门冬氨酸(NMDA)高度敏感，制备乳鼠痉挛发作模型，差异蛋白质组技术分析以下三类动物模型间的差异：（1）痉挛模型组与正常对照组比较，探讨发病机制；（2）孕期应激痉挛模型组与痉挛模型组比较，分析孕期应激作用机制；（3）ACTH治疗组与非治疗组比较，研究ACTH的治疗机制。将动物模型的脑组织样本进行双向电泳分离，获得双向胶图，依据实验分组，Imager marster软件分析各组间蛋白表达差异，选取差异蛋白进行胶内酶切，质谱鉴定，生物信息学方法分析并构建差异蛋白相互作用网络，探讨生物功能的调节网络。对部分差异表达蛋白进行western-blot验证。并收集了5例婴儿痉挛症患儿的手术切除的颅内致痫灶样本，通过免疫组织化学的方法初步验证差异蛋白的表达，探讨其生物学功能。
结果（1）孕期应激痉挛动物模型组较痉挛动物模型组，痉挛发作的潜伏期短（t=3.96，P＜0.001），发作次数多（t=8.65，P＜0.001）；孕期应激痉挛模型ACTH治疗组较孕期应激痉挛模型组，痉挛发作潜伏期长（t=6.9，P＜0.001），发作次数少（t=5.75，P＜0.001）。以上均有统计学差异。（2）痉挛模型组与正常对照组差异蛋白质组分析，发现痉挛模型组中Pkm2、ATP6V1B2、Prps2、ARHGDIA、CFL1、DPYSL3等蛋白表达下调。（3）孕期应激痉挛模型与痉挛模型差异蛋白质组分析，发现孕期应激YWHAZ下调。(4)ACTH治疗组与非治疗组差异蛋白质组分析发现ACTH治疗后Tuba1a、CFL1、SNAP-25、ARHGDIA、MDH、DDAH1和ANNEX3等表达改变。（5）ARHGDIA在5例婴儿痉挛症患儿的致痫灶中的表达存在差异，在经ACTH治疗过的患儿中表达更明显（由于样本少，未行统计学分析）。
结论（1）母鼠孕期应激，NMDA可诱发其乳鼠更为严重的痉挛发作，而给予ACTH治疗可减轻痉挛发作程度。这更好的反应了动物模型对ACTH治疗敏感的特点，更符合婴儿痉挛症这一疾病的特点。（2）痉挛模型组与正常对照组比较，提示婴儿痉挛脑损伤机制可能涉及能量障碍、脑发育异常及神经重塑等。（3）孕期应激痉挛模型组与痉挛模型组比较，YWHAZ蛋白的下调，可能为导致痉挛加重的重要因素之一。（4）ACTH治疗组与非治疗组比较，差异蛋白涉及细胞骨架、神经突触、能量代谢、血管调节、信号传导等方面，治疗机制可能与神经传导，突触重建、乙酰化密切相关。（5）通过差异蛋白ARHGDIA在5例临床样本中表达水平的检测，初步发现ACTH治疗与ARHGDIA表达可能相关，有进一步的开展基础与临床研究的价值。
Objective Infantile spasms (IS) is s an age-specific epileptic encephalopathycharacterized by flexor, extensor, mixed flexor-extensor spasms, hypsarrhythmia andmental retardation. About70%to90%of patients associated with severe cognitiveimpairment. The causes of disease may be over200. From clinical epidemiologyinvestigation, we found that the onset risk of infantile spasms correspondinglyincreased with the degree of maternal prenatal stress. Infantile spasms is a refractoryepilepsy, poor response to nearly all traditional anti-epileptic drugs exceptadrenocorticotropic hormone (ACTH). The study aimed to investigate thepathogenesis of infantile spasms, effect of prenatal stress, and protective mechanismof ACTH treatment.
Methods An infantile spasm rat model induced by N-methyl-D-aspartate(NMDA) in neonate rats was used. A proteomics-based approach was used to detectthe differential expression proteins as following:(1) Spasm Model group vs NormalControl group to study pathogenesis of infantile spasms;(2)Prenatal Stress-SpasmsModel group vs Spasms Model group to study effect of prenatal stress;(3) PrenatalStress-Spasms Model-ACTH treatment group vs Prenatal Stress-Spasms Modelgroup to reseach protective mechanism of ACTH. Gel image analysis was followedby mass spectrometric protein identification, bioinformatics analysis.Draw differential expression proteins interaction networks to explore the biologicalfunctions. Some differential expression proteins were verified by western-blotanalysis. Five paraffin-embedded surgery resected epileptogenic foci tissues ofinfantile spasms patients were obtained. And the very important differential expression protein was verified by immunohistochemistry.
Results (1) Compared with Spasms Model group, the Prenatal Stress-SpasmsModel group exhibited a shorter latency period (t=3.96, P <0.001), and an increasedspasm frequency (t=8.65, P <0.001); compared with Prenatal Stress-Spasms Modelgroup，the Prenatal Stress-Spasms Model-ACTH treatment group exhibited a longerlatency period (t=6.9, P <0.001), and an decreased spasm frequency (t=5.75, P<0.001).(2) Compared with Normal Control, Pkm2, ATP6V1B2, Prps2, ARHGDIA,CFL1, DPYSL3were down regulation in Spasm Model.(3) The YWHAZ proteindown regulated in Prenatal Stress-Spasms Model compared with Spasms Model.(4)Proteomic analysis for ACTH treatment group and non-treatment group, we founddifferential expression proteins including Tuba1a, CFL1, SNAP-25, ARHGDIA,MDH, DDAH1and ANNEX3.(5) ARHGDIA expressions were more obvious (nostatistical analysis was performed due to the small sample size) in epileptogenic focitissues of ACTH treated patients.
Conclusion (1) Prenatal stress exposure resulted in more severe seizures andbetter response to ACTH treatment, which is correlative with the infantile spasms.(2)Pkm2, ATP6V1B2, Prps2, ARHGDIA, CFL1, DPYSL3were down regulation inspasm model, it suggests that the energy metabolism dysfunction,encephalodysplasia and synaptic reorganization maybe the damage factors of neuron.(3) The down regulation of YWHAZ maybe one of important causes for aggravatingspasm in prenatal stress group.(4) Compared ACTH treatment group withnon-treatment group, the differential proteins involved in the cytoskeleton, synapses,energy metabolism, vascular regulation, and implied that protective mechanismmaybe related to signal transduction, nerve conduction, synaptic reorganization andacetylation.(5) Differential expression of ARHGDIA in5cases of IS patientssurgical specimens, which maybe related to ACTH treatment and worth furtherstudying.
引文
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