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家蚕(Bombyx mori)30K蛋白是家蚕五龄幼虫血淋巴中含量最高的一类蛋白质,具有多种生物学功能。30K蛋白作为一类昆虫载脂蛋白,推测其可能在哺乳动物细胞中也具有相似的功能,但迄今尚未见报道。为了研究其对哺乳动物细胞内脂类蓄积的影响,本研究利用含诱导液油酸(oleic acid,OA)和棕榈酸(palmic acid,PA)的培养基(OA∶PA=2∶1)诱导正常人(Homo sapiens)肝细胞系L-02 24 h,在体外建立了人脂肪肝细胞模型,并利用该模型分析了两种家蚕30K蛋白30Kc6和Lip-19G1对肝细胞内脂滴蓄积的影响。结果表明,当采用油红O染色法进行检测时,经30Kc6蛋白预处理后的正常肝细胞再经诱导液诱导后,细胞内脂滴的积分光密度(integral optical density,IOD)值/细胞总面积值与未经蛋白处理的对照组细胞相比明显减少(P<0.05),而Lip-19G1蛋白预处理组差异不显著;此外,经30Kc6或Lip-19G1蛋白处理后的脂肪肝细胞模型内脂滴IOD值/细胞总面积值与未经蛋白处理的对照组细胞相比也明显减少(P<0.01)。上述结果表明,家蚕30Kc6蛋白对肝细胞内脂滴蓄积具有一定的预防作用,同时家蚕30Kc6和Lip-19G1蛋白均可以有效减少脂肪肝细胞模型内脂滴的蓄积量。本研究将为家蚕30K蛋白载脂功能的深入研究提供基础信息,并为其作为一种新型的降脂药物开发提供理论依据。
Bombyx mori 30K protein is the most abundant type of protein in the hemolymph of the 5th instar larvae of silkworm and has a variety of biological functions. As a kind of insect apolipoprotein, 30K protein is presumed that it may have similar function in mammalian cells, but so far it has not been reported. In order to study its effect on lipid accumulation in mammalian cells, the present study was conducted using medium containing oleic acid (OA) and palmic acid (PA) (OA: PA = 2: 1) Homo sapiens hepatoma cell line L-02 for 24 h, a model of human fatty liver cell was established in vitro, and the effects of 30Kc6 and Lip-19G1 30Kc6 and Lip-19G1 on the lipid droplet accumulation in hepatocytes were analyzed using this model . The results showed that the integral optical density (IOD) value of intracellular lipid droplet / total cell count was significantly higher when normal red blood cells pretreated with 30Kc6 protein were induced by inducing fluid, (P <0.05), but there was no significant difference between the Lip-19G1 protein pretreatment group and control group (P <0.05). In addition, The lipid droplet IOD / total cell area was also significantly decreased (P <0.01) compared with the cells without protein treatment. The above results indicated that 30Kc6 protein of silkworm could prevent lipid droplets accumulation in hepatocytes, and both 30Kc6 and Lip-19G1 protein of silkworm could effectively reduce the accumulation of lipid droplets in fatty liver cell model. This study will provide the basic information for the in-depth study of 30K protein carrier function of silkworm, and provide a theoretical basis for its development as a novel lipid-lowering drug.