The role of hepatic endothelial c-Kit in regulating the homeostasis and pathogenesis of the liver

To investigate the role of hepatic EC-specific endothelial marker—c-Kit in the regulation of vascular homeostasis and liver pathogenesis. We firtly generated endothelial cell-specific conditional c-Kit knock out and their control mice. LSECs isolated from control and EC-specific c-Kit KO mice were subjected to RNA-seq. Second, c-Kit+ LSECs were purified using anti-CD117 magenetic beads and the eluted fraction was further incubated with anti-CD146 magnetic beads to obtain c-Kit- LSECs. c-Kit- LSECs and c-Kit+ LSECs were thereafter compared through transcriptome analysis as well. To further elucidate the function of c-Kit+ LSECs in modulating NASH pathogenesis of the liver challenged by MCD-diet, bulk CD146+ LSECs were purified from MCD mice (4w) subjected with c-Kit+ or c-Kit- LSECs infusion respectively at the last week, and compared by RNA-sequencing. Liver sinusoidal endothelial cells were isolated from indicated mice as described in the summary and were subjected to RNA sequencing, in triplicate or quadruplicate, using Illumina HiSeq 3000.

本研究旨在探究肝脏内皮细胞特异性内皮标志物——c-Kit在血管稳态调控与肝脏发病机制中的作用。首先，我们构建了内皮细胞特异性条件性敲除c-Kit的小鼠及其对照小鼠。从对照及内皮细胞特异性c-Kit敲除（KO, knockout）小鼠中分离的肝窦内皮细胞（liver sinusoidal endothelial cells, LSECs）将进行RNA测序（RNA-seq）。其次，我们使用抗CD117磁珠纯化得到c-Kit阳性LSECs，随后将洗脱组分与抗CD146磁珠进一步孵育，以获取c-Kit阴性LSECs。随后，我们同样通过转录组分析对c-Kit阳性与阴性LSECs进行比较。为进一步阐明c-Kit阳性LSECs在调控甲硫氨酸-胆碱缺乏（MCD, methionine-choline deficient）饮食诱导的肝脏非酒精性脂肪性肝炎（NASH, non-alcoholic steatohepatitis）发病机制中的功能，我们从喂食MCD饮食4周的小鼠中纯化得到批量CD146阳性LSECs，并在实验最后一周分别输注c-Kit阳性或阴性LSECs，随后通过RNA测序对其进行比较分析。按照前述方法从指定小鼠中分离肝窦内皮细胞，设置3次或4次生物学重复，采用Illumina HiSeq 3000平台进行RNA测序。