溶质转运载体SLC7A7复杂杂合突变导致赖氨酸尿性蛋白不耐症并发肺泡蛋白沉着症的作用机制研究

SLC7A7 mutations have been suggested to be involved in Lysinuric protein intolerance, y+LAT-1 which encoded by SLC7A7 heteromerizes with SLC3A2-encoded 4F2hc to form an exchanger for cationic and neutral amino acids(CAA), thereby providing the main export route for CAAs from both epithelial and non-polar cells . The mechanism of SLC7A7 mutations affecting cationic amino acid transport and the same mutations lead to different clinical phenotypes has not been fully elucidated. New trials of this project is trying to use peripheral blood of the pedigree with SLC7A7 heterozygous mutation, the original generation of cultivating immortalized lymphocyte and the small intestine epithelial cells model of mutation to analysis the mechanisms complex heterozygous mutation affects expression and the interaction/positioning with 4F2hc protein using Co-IP, Western Blot, and immunofluorescence. To research the mechanisms of the mutations concurrent pulmonary alveolar proteinosis specifically, the methods of ELISA and RT-PCR have been used to test the activity of NO pathway and the related gene expression. To verify the mechanisms of different clinical phenotypes lead by the same mutation, activator and inhibitor are used to change the gene expression in cells. Then the relations between SLC7A7 and related genes and signaling pathways are researched in SLC7A7 knockout mouse model. In summary, a scientific mechanism behind how SLC7A7 mutations can lead to Lysinuric protein intolerance and complicated with different clinical phenotypes will be studied and powerful experimental proof will be elucidated with the completion of this project.

SLC7A7突变与赖氨酸尿性蛋白不耐症的发生相关，其编码的y+LAT-1与4F2hc蛋白形成二聚体，参与阳离子氨基酸的转运。SLC7A7突变影响阳离子氨基酸转运及相同突变导致不同临床表型的机制尚未完全阐明。本研究拟采用携带SLC7A7复杂杂合突变的家系外周血标本，结合原代培养的永生化淋巴细胞和构建的突变小肠上皮细胞模型，运用Co-IP、免疫荧光等方法，研究复杂杂合突变影响表达及与4F2hc蛋白相互作用/定位的机制；运用ELISA和qPCR等方法测定NO信号通路活性，分析复杂杂合突变并发肺泡蛋白沉着症的机制；运用激活剂和抑制剂改变细胞中相关基因的表达，验证赖氨酸尿性蛋白不耐症中相同突变导致不同临床表型的可能机制，进而在SLC7A7敲除的小鼠模型中分析并验证SLC7A7与相关基因及信号通路的关系。本课题为阐明SLC7A7突变导致赖氨酸尿性蛋白不耐症的发生以及并发不同临床表型的机制提供实验依据。

SLC7A7突变与赖氨酸尿性蛋白不耐症的发生相关，其编码的y+LAT-1与4F2hc蛋白形成二聚体，参与阳离子氨基酸的转运。SLC7A7突变影响阳离子氨基酸转运及相同突变导致不同临床表型的机制尚未完全阐明。本研究拟采用携带SLC7A7复杂杂合突变的家系外周血标本，结合原代培养的永生化淋巴细胞和构建的突变人肺上皮细胞模型，运用免疫荧光等方法，研究复杂杂合突变影响slc3a2c蛋白表达定位的情况;运用ELISA等方法测定NO含量，分析复杂杂合突变并发肺泡蛋白沉着症的机制。研究证实突变可能通过影响细胞增殖、凋亡以及与SLC3A2相互作用导致疾病的产生。