脑梗死患者GMFB蛋白通过血脑屏障释放至血液中的分子机理及其临床诊断价值探究

Cerebral infarction is the second largest causes leading to mortality in the world, and its incidence has increased year by year. Unlike the injury of the cardiac and liver tissues, there is no effective biochemical or immunological diagnostic indicators for cerebral infarction. Glia maturation factor beta, GMFB, is a small protein molecules specific expressed in the central nervous system of vertebrate and plays an important role in the regulation of neuron and glial cell growth and differentiation. Our early study showed that, as a kind of neurogenic protein, GMFB has the prerequisites to become an excellent chemical marker to diagnosis for cerebral infarction. As a novel biochemical indicator, GMFB protein has the following characteristics: the smaller molecular weight, stronger specificity, higher sensitivity and more easily detected, etc. However, it is still largely unknown whether GMFB expression can penetrate the blood brain barrier into the blood during brain infarction, and whether GMFB can be served as a novel indicator for the diagnosis of diseases such as brain injury. As a cerebral infarction indicator, the specificity and sensitivity of GMFB are still not clear and need to be solved, as well as its specific molecular mechanism of the change. In this study, we will explore its specific molecular mechanism of the change and assess the clinical diagnosis value of GMFB by immunofluorescence staining, immunohistochemistry staining, live/dead staining, ELISA analysis, TTC staining, micro-PET, nerve cell hypoxic-ischemic model and animal cerebral infarction model in the level of cell, animal and clinical patient.

脑梗死，继心血管疾病后的第二大人类杀手，目前仍无有效的生化指标用于脑梗死的临床诊断，我们早期研究发现神经源性蛋白---胶质成熟因子-β（Glia maturation factor beta，GMFB）具备成为一项优异的化学标志物为脑梗死等疾病进行诊断的先决条件，作为一项新的生化检测指标，GMFB蛋白具有分子量小、特异性强、敏感性高及检测方法简便等优点。然而，在大脑梗死过程中GMFB蛋白能否通过血脑屏障释放至血液，GMFB蛋白作为脑梗死诊断指标的特异性和敏感性如何，以及其变化的具体分子机理如何？以上问题仍不明确，亟需解决。本研究采用免疫荧光染色、免疫组织化学染色、Live/Dead染色、ELISA检测、TTC染色、Micro-PET、体外缺血缺氧模型及大鼠脑梗死模型等新技术，在临床、动物及细胞三个水平上探究血清中游离GMFB蛋白升高的分子机理及其在脑梗死等疾病诊断中临床价值。

【背景】胶质成熟因子-β（GMFB），由142个氨基酸残基组成的分子量为17kDa的蛋白质小分子，主要表达于脊椎动物的大脑中，目前对其功能及表达特点所知甚少，特别是神经损伤后，其表达变化，国内外未见报道。.【主要研究内容】本研究采用免疫荧光染色、免疫组织化学染色、Live/Dead染色、ELISA检测、TTC染色、Micro-PET、体外缺血缺氧模型及大鼠脑梗死模型等新技术在临床、动物及细胞三个水平上探究血清中GMFB蛋白升高的分子机理及其在脑梗死等疾病诊断中临床价值。.【重要结果及关键数据】免疫组化染色显示，与大脑皮质、髓质及大脑基底核相比，GMFB在大脑的海马锥体细胞中表达最为明显；通过免疫荧光染色发现，GMFB主要表达于星形胶质细胞的细胞核中，而在神经元中，除细胞核外，在其轴突和树突中也有明显表达。利用星形胶质细胞缺血缺氧模型，我们发现随着缺血缺氧处理时间的增加，星形胶质细胞死亡比例逐渐增加，同时，培养基中游离GMFB的含量也逐渐升高。本研究通过大鼠脑梗死模型证实，脑梗死后血清中游离的GMFB也可显著升高，且与其梗死程度呈正相关 (R2=0.96)。进一步地临床标本研究证实，脑梗死病人血清中GMFB也可显著升高，且呈一定的时间依赖性，在脑梗死发病的早期（24小时内）血清中GMFB就可升高，且持续时间达一周以上。.【科学意义】 本研究首次明确了GMFB在脑内分布的特点及在胶质细胞与神经元中表达分布的差别，也首次在细胞、整体动物及临床标本水平上证明GMFB可作为神经缺血缺氧损伤的新指标，有望作为一项新的颅脑损伤检测指标，对脑梗死等疾病进行诊断或辅助诊断。