【作者】 李响；

【导师】 郭曲练；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 目的：通过生物信息技术对数据库芯片结果进行筛选,以发现可能与慢性疼痛(神经病理性疼痛)发病相关的新致病基因,并对所过滤的差异基因表达谱进行分析。同时构建大鼠脊神经结扎(SNL)模型,观察大鼠疼痛行为学变化,并检测所获致病基因在损伤及非损伤背根神经节(DRG)的表达情况及在不同神经元中的分布,以验证生物信息学筛选基因的可行性,并探讨所获致病基因对神经病理性疼痛发病的可能影响机制。方法：1、从GEO数据库中搜索并下载与神经病理性疼痛相关的芯片数据,应用BRB ArrayTools软件对数据进行标准化、过滤及差异基因比较,通过基因功能挖掘平台挖掘文献,并与已有Meta分析比对筛选出未在SNL模型损伤及非损伤侧DRG中发现的致病基因,同时,应用DAVID6.7数据库对非损伤DRG中的差异基因进行分析,探讨非损伤DRG中可能发生的病理生理变化。2、将65只大鼠随机分为正常组(n=15)、Sham组(n=25)及SNL组(n=25),SNL组建立大鼠左侧SNL模型,Sham组仅暴露左侧L5脊神经而不结扎,正常组未作处理。术前1天及术后1、4、7、10、13、16、19、22、25、28天测定各组大鼠左后足机械痛阈变化情况,术后3、7、14、21、28天分批处死,取术侧及健侧L4、L5DRG,采用RT-PCR及Western-Blot技术分别对生物信息学方法所获致病基因的mRNA及蛋白表达变化水平进行检测。3、将36只大鼠随机分为正常组(n=9)、Sham组(n=9)及SNL组(n=18),SNL组建立大鼠左侧SNL模型,Sham组仅暴露左侧L5脊神经而不结扎,正常组未作处理。术前1天及术后1、3、5、7、9、11、14天测定各组大鼠左后足机械痛阈变化情况,术后3、7、14天分批灌注处死,取术侧L4、L5DRG,采用免疫荧光技术检测生物信息学方法所获致病基因在NF200、IB4和CGRP免疫阳性DRG神经元中的表达变化情况。结果：1、采用生物信息技术共获得123条同时在损伤及非损伤DRG中差异表达的基因,经过进一步文献挖掘发现34条与神经病理性疼痛发病相关,其中20条基因未见报道NSF在已有Meta分析中被认定为与疼痛发生具有最明显联系,此外,在非损伤DRG中基因差异表达主要导致了离子通道及离子运输、神经元损伤、免疫应答、囊泡转运等方面的变化。2、术前三组大鼠机械痛阈无显著差异(P>0.05),术后SNL组与空白组及Sham组相比,机械痛阈显著下降(P<0.05),同时术侧后肢抬起、足趾并拢、足底轻度外翻,而健侧及其它两组大鼠无明显变化。3、术后各时间点RT-PCR及Western-Blot结果均显示SNL组大鼠术侧L4、L5DRG内NSF的mRNA及蛋白水平较健侧、正常组及Sham组L4、L5DRG显著下降(P<0.05)。4、免疫荧光结果显示NSF在正常大鼠三种亚型DGR中分布广泛,NSF(?)日性神经元主要分布于中小神经元。NSF在SNL大鼠IB4及CGRP免疫阳性神经元中表达下降,同时NSF的表达逐渐由中小神经元转向大中神经元。结论：1、利用生物信息技术能够寻找到与神经病理性疼痛发病相关的致病基因,并能通过分子生物学和组织学试验得到验证,提示其可以作为寻找疾病致病基因的一种有效方法。2、大鼠脊神经结扎后NSF的表达下调可能与神经病理性疼痛的发生发展有关。3、生理状态下NSF可能参与DRG各亚型神经元中无害及有害感觉信息的传递,而脊神经结扎后IB4及CGRP阳性神经元中的NSF表达变化可能在神经病理性疼痛的发生发展中起着更为重要的作用。更多还原

【Abstract】 Objective:Using bioinformatics technology to mine the new pathogenic genes and analyze the expression profile of differential expressed genes. Besides, to observe the time-dependent variation tendency of pain behavior and investigate the mRNA and protein expression level as well as the distribution in different DRG neuron subtypes, and to verify the possibility of the gene mining method based on bioinformatics technology and the potential effect of mined new pathogenic gene in the neuropathic pain.Methods:1. The microarray data was downloaded from online database GEO. The data were normalized, filtered and compared by BRB ArrayTools. In order to make sure that the expression of obtained new pathogenic genes was not reported in the L4and L5DRG of SNL model, the differential genes were detected in data mining platforms and compared with the existing Meta analysis research. Meanwhile, DAVID6.7database was applied for the analysis of differential expressed genes and related physiopathologic change in uninjured DRG.2.65adult male SD rats were randomly divided into3groups: Normal group (n=15), Sham group (n=25) and SNL group (n=25). For the rats of SNL group, left L5spinal nerve was ligated, while L5spinal nerve was only exposed without ligation for the rats of sham group, and the normal group was not treated by any surgery. The mechanical threshold values were measured before the surgery and1,4,7,10,13,16,19,22,25and28days after SNL surgery. The rats were scarified respectively at3,7,14,21and28days after SNL surgery, and the L4and L5DRG at both sides were obtained to verify the expression level of new pathogenic genes by RT-PCR and Western Blot experiments.3.36adult male SD rats were randomly divided into3groups: Normal group (n=9), Sham group (n=9) and SNL group (n=18). For the rats of SNL group, the left L5spinal nerve was ligated, while L5spinal nerve was only exposed without ligation for the rats of sham group and the normal group was not treated by any surgeries. The mechanical threshold values were measured1day before the surgery and1,3,5,7,9,11and14days after SNL surgery. The rats were then perfused and scarified respectively at3,7and14days after SNL surgery, and the L4and L5DRG at the operative side were obtained to verify the distribution of new pathogenic gene in different DRG neuron subtypes by immunofluorescence technology.Results:1.123genes which expressed differentially in L4and L5DRG were obtained by bioinformatics technology. After further literature mining,34genes were related with the neuropathic pain, and expressions of20genes in them were not reported. NSF has the strong and correlational evidence with neuropathic pain based on the exsited Meta analysis. Besides, the differentially expressed genes induced the change of ion channel, ion transportation, neuron damage, immune response and vesicle transportation in the uninjured L4DRG.2. The50%mechanical paw withdrawal threshold (50%MWT) values were not significantly different among3groups before SNL surgery (P>0.05). After the SNL surgery,50%MWT values of SNL group rats decreased significantly compared with Normal and Sham group (P<0.05). Meanwhile, some behavior changes such as everted foot and closed toes were observed on the rats of SNL group.3. Through the analysis of RT-PCR and Western Blot, the mRNA and protein expression level of NSF decreased significantly in ipsilateral L4and L5DRG of SNL rats while compared with the DRGs in contralateral sides, Normal and Sham group.4. In the normal rats, the NSF was distributed widely in NF200-, IB4-and CGRP-positive DRG neurons. And most of the NSF positive neurons were small and medium size neurons. In the SNL rats, the NSF positive rates in IB4and CGRP positive neurons decreased significantly (P<0.05), and the area distribution of NSF positive neurons moved to the large and medium size. Conclusions:1. The bioimformatics technology can effectively mine the potential pathogenic genes of neuropathic pain, which can be verified by further molecular biology and histology experiments.2. The significant decreases of NSF mRNA and protein levels in L4and L5DRG of SNL rats as well as the persistent pain-related behaviors were observed, which can indicate that the expression change of NSF plays a role in the neuropathic pain.3. The wide distribution of NSF in normal DRG neurons implies that NSF can regulate the transmission of both innocuous and noxious sensory information. After SNL surgery, the area of NSF positive neurons were changed, and the positive rates in IB4and CGRP positive neurons were deceased, which implies a more important role of the NSF expression in IB4and CGRP positive neurons.更多还原