少突胶质细胞分化障碍在低剂量铅暴露致脱髓鞘相关疾病中的作用研究

Environmental low-dose lead exposure is still a major public health problem. Many population studies have shown that low-dose lead exposure is closely related to demyelination-related diseases of the central nervous system, such as multiple sclerosis, and is a risk factor for the occurrence and development of the disease. However, the specific mechanism is still unclear. At present, it is believed that the key to the occurrence of oligodendrocyte demyelination-related diseases is the high expression of histone methylation modifying enzyme EZH2, which plays an important role in oligodendrocyte differentiation. Previous studies have shown that lead exposure can lead to the decrease of EZH2 expression in cerebral cortex and neural progenitor cells. On the basis of previous studies, this project intends to establish a low-dose lead exposure model during pregnancy. The relationship among lead exposure, oligodendrocyte differentiation disorder, EZH2 and demyelination of cerebral cortex is clarified by detecting lead content, PET imaging and differentiation markers in cerebral cortex of offspring at different stages. On this basis, oligodendrocyte precursor cells are isolated and cultured to establish differentiation. The role of EZH2 in oligodendrocyte differentiation disorder induced by lead exposure and its possible molecular mechanism were explored by RNA interference and high expression, and immunoprecipitation of chromatin. This study provided theoretical basis for demyelination-related diseases caused by lead exposure and new targets for prevention and treatment.

环境低剂量铅暴露仍是目前重大公共卫生问题。多项人群研究表明低剂量铅暴露与中枢神经系统脱髓鞘相关疾病如多发性硬化症密切相关，是疾病发生发展的风险因素，然而其具体机制尚不清楚。目前认为少突胶质细胞分化异常脱髓鞘相关疾病发生的关键，组蛋白甲基化修饰酶EZH2高表达在少突胶质细胞分化中起重要作用，前期研究结果显示铅暴露可导致大脑皮质和神经祖细胞中EZH2表达降低。本项目拟在既往研究的基础上，首先建立孕期低剂量铅暴露模型，通过对不同时期子代大脑皮质进行铅含量、PET影像学、分化标志等检测，明确铅暴露、少突胶质细胞分化障碍、EZH2以及大脑皮质脱髓鞘之间的关联；在此基础上，分离培养少突胶质前体细胞建立分化模型，通过RNA干扰和高表达、染色质免疫共沉淀等方法，探讨EZH2在铅暴露致少突胶质细胞分化障碍中的作用及其可能分子机制，为铅暴露致脱髓鞘相关疾病提供理论依据以及为预防和治疗提供新靶点。

环境低剂量铅暴露是造成的人类尤其是婴幼儿神经系统损伤重要因素之一。多项人群研究表明低剂量铅暴露与中枢神经系统脱髓鞘相关疾病如多发性硬化症密切相关，但其具体机制尚不清楚。目前认为少突胶质细胞分化异常脱髓鞘相关疾病发生的关键，而组蛋白甲基化修饰酶EZH2高表达在少突胶质细胞分化中起重要作用。本项目首先建立孕期低剂量铅暴露模型明确铅暴露、少突胶质细胞分化障碍、EZH2以及大脑皮质脱髓鞘之间的关联；同时分离培养少突胶质前体细胞建立分化模型揭示EZH2在铅暴露致少突胶质细胞分化障碍中的作用及其分子机制。结果显示，①母鼠在孕产期暴露于对照组、低中高铅染毒组（1 mg/100g BW、3 mg/100g BW、9 mg/100g BW）后，铅暴露组仔鼠血液和脑组织中均发现铅且成剂量反应关系，并且随着时间的推移而降低并在仔鼠出生后第42天后无法检测出；出生后第1天仔鼠大脑皮质中EZH2、髓鞘碱性蛋白（MBP）和髓鞘少突胶质细胞糖蛋白（MOG）表达呈现出随母体暴露剂量升高而降低，而A2B5、O4表达则相反，随着时间的推移中高剂量组中EZH2、MBP、MOG的表达水平均降低；②对分离培养的大鼠少突胶质细胞进行铅染毒试验，浓度为25μmol/L时细胞活力出现明显下降并呈现剂量反应关系，浓度为100μmol/L时Caspase3活性明显升高并呈现剂量反应关系；10μmol/L醋酸铅染毒少突胶质细胞前体细胞，结果显示与对照相比在培养的第14天铅染毒组中A2B5和O4表达增高，而EZH2和MBP表达降低；同样在EZH的RNA干扰株中，与对照细胞株相比，培养的第14天铅染毒组中A2B5和O4表达增高，MBP表达降低，而在EZH高表达的情况下这一现象可以得到逆转；③通过免疫共沉淀试验发现Olig1、Olig2、Hes5、NCX3、CNPase和YY1基因均可获得扩增，进一步试验发现铅染毒少突胶质细胞前体细胞分化过程中Hes5、CNPase和YY1基因表达出现与EZH2表达呈现一致下降关系。这些结果提示EZH2可通过调控Hes5、CNPase和YY1基因表达阻碍少突胶质细胞分化过程，从而引起相关疾病，这一结果可能分子机制，为铅暴露致脱髓鞘相关疾病提供理论依据以及为预防和治疗提供新靶点。