高糖状态下整合素αVβ3介导的肝窦毛细血管化分子机制的研究

Integrinαvβ3 is one of the cell adhesion molecules, which plays an important role in the cell inflammation, adhesion, activation, proliferation, and so on. Accumulating evidences have been proved that structural and functional change of liver sinusoidal endothelial cells was the foundation of sinusoidal capillarization. Besides, the expression of integrinαvβ3 has a positive correlation with sinusoid capillarization, which may associated with the development of diabetic fatty liver disease.In this study, we considered the relationship between integrinαvβ3 and sinusoidal capillarization as pointcut. We detect the level of intracellular ROS, expression of integrinαvβ3 and inflammatory cytokines from perspective of cell level and animal modle, and further discuss the likely molecular mechanism for the development of diabetic fatty liver disease. Besides, the technology of siRNA gene silencing will be used to discuss whether integrinαvβ3 gene can regulate the expression of intracellular ROS, inflammatory cytokines, basement membrance proteins after the gene of integrinαvβ3 knockdown. The present study will not only contribute to clarify the molecular mechanism of the capillarity hepatic sinusoid mediated by integrinαvβ3, but also giving a well understanding in the pharmacologic of diabetic fatty liver disease , thereby may provide part of the pharmacologic basis for the treatment of diabetic fatty liver disease.

整合素αvβ3是一种细胞黏附分子，参与细胞的炎症反应、粘附、活化、增殖等。肝窦内皮细胞(LSEC)结构和功能的改变是肝窦毛细血管化的基础。肝脏整合素αvβ3的变化与肝窦毛细血管化密切相关，它很可能是2型糖尿病非酒精性脂肪肝形成中的重要因子。本研究以整合素αvβ3与肝窦毛细血管化之间的关系为切入点，从细胞水平和动物模型研究高糖状态下LSEC整合素αvβ3、细胞内活性氧（ROS）及炎性细胞因子的表达水平；进一步探讨整合素αvβ3介导的基底膜蛋白表达和LSEC窗孔变化的关系及分子机制。利用siRNA技术探讨整合素αvβ3基因沉默对调控大鼠LSEC ROS水平、基底膜蛋白及炎性细胞因子表达效应的影响。通过本研究将有助于阐明高糖状态下整合素αvβ3介导肝窦发生毛细血管化的分子机制，为寻找治疗糖尿病合并脂肪肝的靶点药物提供一定的理论基础。

目的：探究高糖状态下层粘连蛋白（Laminin，LN）在人肝窦内皮细胞(Human liver sinusoidal endothelial cells, HLSECs)中的表达及活性氧(reactive oxygen species ,ROS)、整合素αvβ3（integrin αvβ3）在其中的作用，并探讨高浓度棕榈酸（Palmitate）对HLSECs integrinαvβ3表达的影响。方法：培养人肝窦内皮细胞，MTT检测细胞活力，DCFH-DA探针和荧光酶标仪检测对照组、高糖组（25mmol/L）、甘露醇组（20mmol/L）的HLSECs在6h、24h、48h时ROS的表达水平；实时荧光定量PCR(Real time quantity, RT-PCR)、Western-blotting技术分别检测对照组、高糖组、甘露醇组、高糖+抗氧化剂组的HLSECs 6h、24h、48h整合素αvβ3在核酸及蛋白水平的表达变化；细胞免疫荧光技术检测对照组、高糖组、甘露醇组、高糖+整合素αvβ3抑制剂LM609组HLSECs 6h、24h、48h LN的表达变化；用Western blotting检测BSA对照组，棕榈酸干预（50，100,200,400,600,800µmol/L）组整合素αvβ3表达。采用SPSS19.0进行统计学分析。结果：与对照组相比，高糖组ROS、整合素αvβ3、LN表达明显增加（P<0.05），而甘露醇组ROS、整合素αvβ3、LN表达的变化不明显（P>0.05）；与高糖组比较，高糖+抗氧化剂组的整合素αvβ3表达水平显著下降（P<0.05），高糖+整合素αvβ3抑制剂组的LN表达显著下降（P<0.05）。HLSECs在棕榈酸浓度大于200μmol/L时活力明显下降；棕榈酸诱导HLSECs整合素αvβ3表达上调且呈浓度时间依赖性，整合素αvβ3在100μmol/L,培养48h后表达量最高，加入NAC后整合素αvβ3表达下调；棕榈酸诱导HLSECs生成ROS具有浓度依赖性； LM609对 ROS生成无显著影响；100μmol/L棕榈酸干预组较BSA对照组荧光信号明显增强，抑制整合素αvβ3和ROS后LN荧光信号强度表达明显减少。结论：高糖能上调人肝窦内皮细胞ROS、整合素αvβ3、LN的表达水平，这与葡萄糖自身代谢有关，与渗透压的改变无关；高糖