核蛋白GADD45α促进部分肝切除后大鼠肝细胞增殖的作用机制研究

GADD45α is a cell growth arrest and DNA damage-regulated protein implicated in a variety of cellular stress responses, plays important roles in the regulation of cellular damage response and suppression of tumor development. Upregulation of GADD45α expression has been shown to link to the G2/M checkpoint of the cell cycle by inhibiting CDC2 kinase activity. In addition, GADD45α also functions in modulating genomic DNA demethylation and DNA damage repair responses, and therefore contributing to the genome stability. Our laboratory found that GADD45α was involved in rat liver regeneration after partial hepatectomy. GADD45α protein have a rapid accumulation and sustained expression throughout the liver regeneration process. Therefore, it is worthy of further study that GADD45α how to control the survival and proliferation of liver cells after damage by partial hepatectomy and how to regulate the process of rat liver regeneration. To this end , the project intends to start further research to reveal the detail process of GADD45α regulation. We plan to execute GADD45α overexpression, RNA interference, suppression methods in liver body and cultured primary hepatocytes, and then use the real-time PCR, Western Blot and other methods to detect these materials to find the response variation of GADD45α and its up and downstream signaling molecules. then, the impact of cell proliferation changes caused by GADD45α will be detected with methods of cell growth curve and flow cytometry. and then analyzes the role of GADD45α in hepatocyte proliferation control after partial hepatectomy and conform its role in regulation of rat liver regeneration. This project attempts to clarify the mechanism of hepatocyte proliferation controled by GADD45α, and to provide new targets for the clinical treatment of liver diseases.

GADD45α是一个细胞生长阻滞和DNA损伤调节蛋白，在细胞对外界损伤反应调控网络中扮演举足轻重的角色，在维持基因组稳定性和抑制肿瘤发生发展的过程中发挥重要的作用。本实验室在前期研究中发现GADD45α参与肝再生调控，部分肝切除后GADD45α蛋白迅速大量积累，并在整个肝再生进程持续表达。因此，GADD45α如何调控肝细胞存活和增殖进而调控肝再生值得深入研究。本项目拟进一步用GADD45α超表达、基因干涉等方法处理培养的原代肝细胞和大鼠部分肝切除模型，然后用real-time PCR、Western Blot等方法检测上述材料中GADD45α及其上下游信号通路分子的表达变化，用生长曲线、免疫组织/细胞化学、流式细胞术、肝功分析等检测GADD45α对细胞增殖的影响，进而分析其参与再生肝细胞增殖调控的作用。本研究试图阐明GADD45α对再生肝中肝细胞增殖的调控机理，为肝病临床治疗提供新的靶标。

为了解GADD45α对大鼠肝再生的作用及调控机理，本项目在基于本实验室对再生肝的基因表达谱分析的基础上，用qRT-PCR验证了芯片检测结果的可靠性。用IPA、GenMAPP、KEGG、BIOCARTA、QIAGEN和Biocompare等几个数据库以及查阅相关文献构建了GADD45α的信号通路图并分析其可能的作用机理，然后利用过表达和基因干涉的方法获得了GADD45α过表达或者下调表达的BRL-3A细胞，对调节GADD45α正常BRL-3A细胞以及FZD/UVC诱导的BRL-3A细胞的的增殖、凋亡、周期以及DNA损伤修复进行了初步研究。MTT结果显示过表达GADD45α明显抑制了BRL-3A细胞的细胞活力，增强了FZD/UVC诱导的BRL-3A细胞的活力，而干涉GADD45α的表达增强了BRL-3A细胞的细胞活力，抑制了FZD/UVC诱导的BRL-3A细胞的活力；流式细胞术检测细胞周期结果发现GADD45α过表达导致正常BRL-3A细胞G1和S期细胞数量减少，G2/M期阻滞，同时增加了FZD/UVC诱导的S期细胞周期停滞，而干涉GADD45α的表达导致正常BRL-3A细胞G1细胞数量减少，G2/M和S期期细胞数量增多，同时减少了FZD/UVC诱导的S期细胞周期停滞；采用qRT-PCR和Western Blot等方法检测GADD45α表达变化对正常BRL-3A细胞GADD45α的信号通路相关基因/蛋白的表达变化的影响，结果显示，P38MAPK、JNK、CDC2/CCNB1、AKT和MTOR等信号通路相关基因/蛋白的表达发生显著的变化。结果表明GADD45α通过P38MAPK、JNK、CDC2/ CCNB 1、AKT和MTOR等信号通路调控BRL-3A细胞的增殖，通过Myc、Bcl-2、Ccnd1、Ccnb1、Caspase8、Caspase9和Bax等基因调控BRL-3A细胞DNA损伤修复。