SDR9C7基因调控肝脏脂质代谢的研究

The liver plays a central role in lipids metabolism, and the lipophagy-lysosome pathway has been proved to play a pivotal role in cellular homeostasis and lipids metabolism. Human SDR9C7 and mouse SDR9C7 evolutionally conserved with 83% identity in amino acids. Our studies verified that mSDR9C7 mRNA specifically highly expressed in liver in the adult mice, and hSDR9C7 protein granularly distributed in cytoplasm. However the role and exact subcellular localization of SDR9C7 is still unknown. In addition to those, our results showed that partial hSDR9C7 protein granularly colocalized with lysosome-associated membrane protein 1, and was related with lipophagy in hepatocytes. Based on previous studies, this study objects to further investigate the subcellular localization of hSDR9C7 protein by using LC3Ⅱ, Perilipin A and pDs-Red Golgi as markers for autophagosome, lipid droplets and Golgi separately. And the relationship between SDR9C7 and lipid metabolism will be explored while inhibition or activation of autophagy in L02 cell, HepG2 cell and primary mouse hepatocytes. The wild type and mSDR9C7 gene knockout mice fed with chow diet or high fat diet will be used to explore the relationship between mSDR9C7 and lipid metabolism, and the further molecular mechanism in vivo. Our aim is to evaluate the role of SDR9C7 in regulating liver lipids metabolism，invetigate the molecular mechanism to lay a theoretical foundation for the possible application.

肝脏是动物体脂类代谢的中心，脂自噬溶酶体途径是脂类代谢的重要途径，在调节细胞能量平衡方面起重要作用。人hSDR9C7与鼠mSDR9C7蛋白氨基酸序列相似性高达83%。我们证实hSDR9C7在HepG2细胞中成颗粒状聚集，成年鼠体内mSDR9C7基因仅在肝脏组织中特异性高表达，但SDR9C7生物学功能和亚细胞定位仍未知。我们新发现部分hSDR9C7与溶酶体膜相关蛋白1（LAMP1）共定位，与肝细胞脂自噬相关。为此，我们将以LC3Ⅱ、Perilipin A、pDs-Red Golgi为标志蛋白进一步对SDR9C7的亚细胞定位进行研究；利用L02、HepG2或鼠原代肝脏细胞为模型，在自噬抑制或激活条件下体外检测SDR9C7基因表达对脂质含量的影响；利用野生型鼠和mSDR9C7基因敲除鼠研究在普通饲料和高脂饲料条件下SDR9C7基因表达对脂质代谢的影响；并阐明其分子机理，为应用奠定基础。

（1）SDR9C7蛋白亚细胞定位的实验结果表明，SDR9C7蛋白为全细胞分散分布，没有呈颗粒状与脂滴、自噬体、高尔基体的共定位分布。首次阐明SDR9C7蛋白呈非颗粒状分布。.（2）在小鼠各组织中的表达情况的检测结果表明，Sdr9c7基因在肝脏、血液、骨髓、皮肤、肺为高丰度表达，首次检测了Sdr9c7基因的各组织表达情况。.（3）L02细胞脂肪变性后hSDR9C7基因的表达没有显著变化、杂合子实验结果表明Sdr9c7+/-小鼠，Sdr9c7+/-小鼠的生长发育正常，及高脂饲喂条件下血液生理生化、肝脏系数和肝脏石蜡组织切片中细胞空泡面积无明显差异，推测Sdr9c7基因对肝脏脂肪变性没有影响。.（5）mSdr9c7-/-基因型小鼠出生后5-12小时内死亡。.（6）mSdr9c7-/-基因型小鼠出生后皮肤异常，角质化不全，屏障功能缺失；Sdr9c7-/-新生鼠皮肤中的TARC以及p-STAT3的表达增高。预计对鱼鳞病等皮肤疾病的机理和临床治疗具有提示意义。.（7）Sdr9c7-/-新生鼠出现弥漫性凝血，血液TARC含量增高。预计对血凝、血栓等疾病发生的机理和临床治疗具有提示意义。.（8）mSdr9c7-/-鼠胚胎期肺发育正常，但出生后表现出肺张不全，肺泡间隔增厚，SPC和T1α表达增加。预计对新生儿（尤其是早产儿）肺张不全、呼吸衰竭的发生机理和临床治疗具有提示意义。.(9)制备了Sdr9c7基因条件敲除LoxP鼠、CRISPR-Cas9法制备Sdr9c7基因全敲除鼠，目前CRISPR-Cas9法制备Sdr9c7基因杂合子敲除鼠已成功。