簇集蛋白（Clusterin）在六价铬[Cr(Ⅵ)]诱导的肝细胞早熟性衰老中的作用研究

Recently, the events of water pollution caused by chromium occurred frequently. It has been reported that oral ingestion of Cr(Ⅵ) through drinking water led to elevated primary liver cancer mortality, which highlighted that we should pay much attention to the study on Cr(Ⅵ)-induced liver damage. Our group found that long-term and low-dosage exposure of Cr(Ⅵ) in hepatocytes resulted in premature senescence, which accompanied with the increased expression of Clusterin (CLU), but the functional role of which in premature senescence has never been explored. It is well known that CLU plays an important role in tumorigenesis and premature senescence is a roadblock on the way to tumorigenesis, thus we inferred that CLU could promote malignant transformation by inhibiting premature senescence. By over-expressing and knocking out CLU in L-02 hepatocytes, we will explore the occurrence of premature senescence after Cr(Ⅵ) exposure to demonstrate the functional role of CLU in premature senescence. We will also conduct the mechanism study to see if TGF-β pathway and PI3K/Akt pathway are involved in the inhibitory effect of CLU. Cr(Ⅵ) has become serious threats to public health. The present study will demonstrate the functional role of CLU played in premature senescence and the related mechanism, which may provide experimental evidence for the mechanism study on Cr(Ⅵ)-induced liver cancer especially for the study on how do the cells that have the potential to malignantly transform escape from premature senescence.

近年来水体"铬污染"事件频发。研究表明饮用水被六价铬[Cr(Ⅵ)]污染后导致居民原发性肝癌发生率明显增高，故Cr(Ⅵ)的肝毒性研究需引起重视。本课题组发现Cr(Ⅵ)长期低剂量暴露后诱导肝细胞发生早熟性细胞衰老（简称早衰），并伴有簇集蛋白（Clusterin，CLU）表达水平明显增高，但CLU在早衰中的作用及其分子机制尚不明确。已知CLU能促进恶性肿瘤的发生发展，而早衰被认为是细胞癌变过程中的屏障，故假设CLU可抑制早衰而促进细胞恶性转化。研究内容包括在L-02肝细胞中过表达或沉默CLU基因，经Cr(Ⅵ)作用后判断早衰的发生情况以证实CLU的作用，并探讨其机制是否与影响TGF-β、PI3K/Akt通路有关。Cr(Ⅵ)已对人类健康造成严重威胁，通过阐明CLU在Cr(Ⅵ)诱导的早衰中的作用及其相关机制，将为Cr(Ⅵ)致肝癌作用的研究特别是有恶性转化倾向的细胞逃避衰老的机制的阐明提供实验依据。

作为最常见的重金属污染物，Cr(VI)及其化合物已对人类健康和生态环境造成严重威胁。自1990年铬被IARC列为确认人类致癌物以来，Cr(VI)主要被认为是由吸入途径致癌的环境污染物而引发呼吸系统癌症。而近年发生的云南曲靖的水体“铬污染”以及药用“铬胶囊”事件，加上已有研究证实饮用水被Cr(VI)污染而导致居民原发性肝癌的发生率明显增高的报道，Cr(VI)经口污染导致的肝脏毒性甚至癌变逐渐引发毒理学研究者的重视。本课题组发现Cr(VI)长期低剂量的暴露诱导L-02肝细胞发生早衰，并伴有CLU表达的明显增高。但CLU在肝细胞早衰中的作用及其分子机制尚不明确。本研究构建L-02-CLU（OE）及L-02-CLU（shRNA）细胞模型，Cr(VI)染毒后对各组细胞进行早衰特征性指标检测（β-Gal染色、细胞周期阻滞、衰老标志物CTGF、SMP30）以及早衰通路分析。发现Cr(VI)作用后L-02-CLU（OE）细胞早衰发生抑制而L-02-CLU（shRNA）细胞早衰发生加重，明确CLU对早衰的抑制作用；通过检测TGF-β通路核心蛋白，包括TGF-β、p-Smad2/3、Smad2/3、Erk1/2以及PI3K/Akt通路相关蛋白PI3K、p-Akt、Akt、Cyclin D1，发现CLU对Cr(VI)诱导的肝细胞早衰的抑制与TGF-β通路的抑制与PI3K/Akt通路的激活有关。以往关于Cr(VI)的细胞毒性研究集中在凋亡，对早衰的研究甚少。已证实正常的细胞经较大的刺激后发生凋亡，经轻中度刺激后发生早衰，环境中人群对Cr(VI)及其化合物的暴露多为长期低剂量的暴露，故研究早衰更有实际意义。早衰是细胞恶性转化的屏障，早衰逃逸的细胞具有恶性转化倾向，克服衰老是肿瘤细胞不断增殖永生化的重要步骤。Cr(VI)诱导早衰细胞中CLU表达增高，CLU与肝癌的发生发展以及恶性增殖密切相关。如果CLU在Cr(VI)诱导的细胞早衰中发挥抑制作用，则Cr(VI)暴露后CLU对TGF-β通路的抑制以及对PI3K/Akt通路的活化将在细胞恶性转化中发挥积极作用。鉴于Cr(VI)暴露后肝癌的发生是多基因、多因素、多步骤的过程，对于CLU在早衰中作用机制的探讨将对Cr(VI)致肝癌作用的研究特别是肝癌细胞逃避衰老机制的阐明提供一定的实验依据，并为铬及其他环境毒物所致肝癌的靶向治疗提供新的思路和策略。