肝星状细胞逆境适应与活化致高脂膳食性NAFL非炎性纤维化机制

Fibrosis is an important stage in the development of non-alcoholic fatty liver (NAFL), the activation of hepatic stellate cell (HSC) is its basic cellular basis. Trigger Kupffer cells and inflammatory reaction by necrosis of liver cells is the main activation factor. However in previous studies we found that fibrosis in the high fat diet feeding NAFL significantly earlier in the liver cell necrosis and inflammation, its scope is significantly greater than the damage and inflammatory lesions but associated with lipid deposition severity. We speculated that there may be a direct activation mechanism on HSC from high lipid microenvironment and hepatocyte lipid deposition. The lipid activated fibrosis that do not depend on inflammation is rarely reported. This study builds on the previous high fat diet NAFL rat model and the high lipid portal vein serum in vitro microenvironment model, by hyperlipidemic serum culture, Transwell co-culture with liver cells, mixed culture, conditioned medium co-culture to study the adaptation and survival features of HSC in NAFL high lipid Disse microenvironment. By detecting the expression of some key molecules, such as TGF beta, HGF, etc., study the activating mechanism of HSC in high fat microenvironment, and clarify the new non-inflammatory fibrosis mechanism of high fat diet NAFL resulted from the adversity adaptation and activation of HSCs.

纤维化是NAFL进展的重要阶段，HSC的活化是其基本细胞学基础。肝细胞坏死所触发的kupffer细胞及炎性反应被认为是其主要激活因子。然而在前期研究中我们发现高脂膳食性NAFL的纤维化过程明显早于肝细胞坏死和炎症，其范围也显著大于损伤和炎症病灶，而与脂质沉积程度相关。我们猜测其中可能存在高脂微环境和肝细胞脂质沉积对HSC的直接激活机制。目前对不依赖炎性激活的脂性纤维化还鲜见报道。本研究在前期建立的高脂膳食性NAFL大鼠模型基础上，利用门静脉高脂血清构建体外高脂微环境模型，通过高脂血清培养、与肝细胞Transwell小室共培养、混合培养、条件培养基培养，研究NAFL时HSC在肝细胞、高血脂构成的Disse间隙高脂微环境中的适应和生存特征，通过对TGF-β、HGF等关键分子表达水平的检测，研究高脂微环境下HSC的活化机制，最终阐明肝星状细胞逆境适应与活化导致高脂膳食性NAFL非炎性纤维化机制。

纤维化是NAFL进展的重要阶段，HSC的活化是其基本细胞学基础。在前期研究中我们发现高脂膳食性NAFL的纤维化过程明显早于肝细胞坏死和炎症，其范围也显著大于损伤和炎症病灶，而与脂质沉积程度相关。我们猜测其中可能存在高脂微环境对HSC的直接激活机制。本研究探索NAFL时HSC在肝细胞、高血脂构成的Disse间隙高脂微环境中的适应和生存特征，研究高脂微环境下HSC的活化机制，阐明肝星状细胞逆境适应与活化导致高脂膳食性NAFL非炎性纤维化机制。.本项目通过建立高脂膳食性NAFL大鼠模型，从组织学水平证实高脂膳食性NAFL的非炎症性纤维化以及HSCs的活化。通过肝细胞与星状细胞的混合培养，建立Disse间隙微环境的细胞学模型，发现混合培养以及肝细胞脂肪变性都可能参与纤维化的过程，其中肝细胞可能发挥着主导作用，星状细胞可能发挥着抑制作用。通过real time-PCR、western blot的方法检测细胞内α-SMA的表达水平，确定棕榈酸及高脂门静脉血清对LX-2的活化及功能无显著影响。通过建立压力培养系统，明确机械压力显著降低整合素分子αvβ3的表达（4 mmHg、8 mmHg，48 h），并影响LX-2的细胞形态和细胞骨架（F-actin）的正常分布，促进I型胶原的表达。.本项目从组织学、细胞学和炎症分子等多水平证实NAFL阶段存在非炎症性纤维化机制，取得了重要的科学发现——肝细胞可能是间质纤维化的来源之一，对非酒精性脂肪肝的非炎症性纤维化机制是突破性的发现。本项目建立了机械压力、高脂微环境细胞学模型，在肿瘤侵袭和转移机制的研究中具有非常重要的应用价值。此外，本项目还对肠-肝轴的菌群机制进行了初步探索，并取得了很有价值的科学发现。