T-细胞激活蛋白家族LAT2基因在严重的先天性中性粒细胞减少症中的作用和机制研究

The linker for activator of T-cells family member 2 (LAT2) gene is mainly expressed in the hematopoietic cell, which has been investigated in depth with regard to its functional importance in several immune responses and participates in leukemia due to the deficit differentiation of myeloid. Although the deficit differentiation of myeloid also leads to severe congenital neutropenia (SCN), there is no study on the relationship between LAT2 gene and SCN. Interestingly, we detected a homozygous mutation of the LAT2 gene in a patient with SCN. Genetic analysis revealed that the mutation phenotype was co-segregation and highly conserved in variant species, sequence analysis and function prediction show this mutation is deleterious. Both LAT2 and G-CSFR are combined with GRB2, increase expression of LAT2 or enhance phosphate level of LAT2 may inhibit differentiation of myeloid cell following G-CSF treatment. We proposed to separate the human bone marrow cells(CD34+), and LAT2 overexpression and LAT2 knock down CD34+ myeloid cells were produced by the retroviral vector pMYSiGIRES-GFP. To explain the role of a different expression level of LAT2 during myeloid differentiation, differentiation of normal, LAT2 overexpression, LAT2-/- myeloid cells toward to granulocyte by G-CSF was observed. The function and mechanism of LAT2 involved in SCN was explored, all three type of myeloid cells were left treated with several doses of G-CSF, or the normal myeloid cells were treated with the combination of several doses of G-CSF and LAT2, and the LAT2 mRNA; LAT2, GRB2 and Lyn protein; phosphorylation of LAT2 and Lyn; and intracellular localization was analysis by RT-PCR, western blot, and immunofluorescent staining. This study may lay a foundation for gene therapy about SCN.

LAT2基因主要表达于血液细胞中，负调控骨髓细胞的分化，参与“成熟障碍”导致的白血病。严重的先天性中性粒细胞减少症(SCN)由骨髓细胞“成熟障碍”所致，尚无与LAT2基因的相关性研究。本研究组前期发现，SCN患者携带LAT2纯合突变，功能预测突变位点高度保守且有害。由于LAT2和G-CSFR均与GRB2结合，推测LAT2通过表达上调或磷酸化水平增加竞争性抑制G-CSF刺激的骨髓细胞向中性粒细胞分化。为验证这一推测，我们拟采用人骨髓细胞构建LAT2高表达和基因敲除的细胞模型，给与G-CSF刺激后，观察骨髓细胞的分化情况。同时将不同浓度G-CSF和不同浓度的G-CSF与LAT2重组蛋白的组合作用于骨髓细胞，通过比较骨髓的分化程度以及标志物的变化，包括LAT2 mRNA、蛋白和磷酸化蛋白的表达、细胞内定位、GRB2蛋白和Lyn去磷酸化，阐明LAT2在SCN中作用和机制，为基因治疗奠定基础。

LAT2参与“成熟障碍”导致的白血病，而严重的先天性中性粒细胞减少症(SCN)也是由骨髓细胞“成熟障碍”所致。本课题通过临床实验，分子生物学，细胞生物学，动物模型等技术，对LAT2在SCN中的分子机制进行初步研究。通过临床血液样本，进行基因突变检测，分析在中性粒细胞减少组和对照组中是否存在致病或易感突变位点，同时进行基因表达分析，明确在不同组内的是否存在表达差异。结果未发现致病或易感突变位点和表达差异，分析其原因可能与病例的收集有关，SCN患者罕见，我们收集的3岁以内的单次中性粒细胞计数降低患儿并不能代表SCN病例组。随后我们使用CRISPR-Case9方法制备了点突变细胞模型和小鼠模型，在点突变小鼠模型中观察到尾部异常，包括断尾，无尾和尾部褪色趋透明化等表型，所有具有异常尾部特征的小鼠均为纯合型。同时，我们使用细胞爬片和荧光免疫杂交方法，检测活化后淋巴细胞LAT2蛋白表达和磷酸化水平，结果提示，在活化的淋巴细胞中，检测到LAT2蛋白表达，以及轻度磷酸化。通过动物模型表型和细胞实验，LAT2具有必要的生物学功能，但具体作用通路还需要进一步研究。.此外，在项目的资助下，我们还鉴定了一个凝血因子VII缺乏家系的致病基因突变和一例新发的地中海贫血基因突变，为患者治疗以及后续遗传咨询提供必要的资料。同时在出生缺陷相关的研究中，还检测到一例外周血，皮肤，生殖器嵌合病例。上述病例已有一篇发表，另外两篇待发表。.项目资助发表中文论文1篇，英文论文2篇，待发表英文论文2篇。指导一名本科生毕业论文。项目直接经费21.0万元，配套经费31.5万元，合计使用26.4951万元，剩余经费26.0049万元，后续经费将用于进一步功能研究。