磷脂酶C-γ2（PLCG2）调节大鼠再生肝的肝细胞凋亡机理研究

Liver is one of the few organs which have the capacity to regenerate rapidly after injury. Around one week after 70% partial hepatectomy, the liver recovers to its original mass and volume. Liver regeneration (LR) is a complex process under precise regulation from numerous proliferative and inhibitory factors. At present, many studies mainly focus on the triggering and proliferative mechanism involved in LR. And many factors (hormones, growth factors, cytokines) playing important roles in initiation and progression of LR have been extensively studied. Inhibitory regulation mechanism, such as LR termination, apoptosis of excessive hepatocytes and maintenance of normal liver mass, remain to be studied. In our previous work on proteomics of rat liver regeneration, we found that phospholipase C-γ2 (PLCG2) has a key protein for liver regeneration; bioinformatics analysis showed that PLCG2 possibly induces cell apoptosis during liver regeneration through PKCD-dependent p38 and JNK cascades activation. Taking this scientific hypothesis as the main line, we firstly control the expression change of PLCG2 in the isolated primary hepatocytes from rat regenerating liver through gene addition and gene interference, and then detect the influence of PLCG2 on cell apoptosis and proliferation using various approaches including Hoechst/PI double-staining, flow cytometry and so on. Meanwhile, the expression changes of PLCG2 and PKCD-activating signaling-related components will be detected to analyze its possible its possible pathway by qRT-PCR, Western Blot, administration of signal pathway inhibitors and so on. This study will be helpful in unraveling the role and action mechanism of PLCG2 in liver regeneration, which prepares good conditions for the continued in vivo study and its clinical application.

肝再生是机体正反两方面因素调控下的复杂过程。目前，对肝再生研究主要集中在正调控机制上，而对肝再生负调控机制的研究相对较少，肝再生终止以及再生肝量精细调节等问题至今仍不清楚。我们在进行大鼠肝再生研究时发现，PLCG2与肝再生关系密切；根据以往研究并结合我们的生物信息学分析，认为PLCG2在肝再生中可能依赖PKCD激活p38和/或JNK途径促进细胞凋亡。本项目以此科学假设为主线，以腺病毒感染体外培养的大鼠再生肝肝细胞为切入点，通过基因添加和干涉等方法研究肝细胞中PLCG2的表达，用Hoechst/PI双染、流式细胞术等方法检测PLCG2对细胞凋亡和增殖的影响、用信号通路抑制剂、qRT-PCR、Western Blot 等方法检测上述材料中PLCG2及下游信号通路相关基因和蛋白的变化，以及信号通路改变与细胞凋亡之间的关系，从而解析其作用及作用途径，以期为后续体内研究和临床应用积累资料。

我们早期进行大鼠肝再生基因组学研究时发现，PLCγ2与肝再生关系密切。通过生物信息学分析并结合他人研究，认为PLCγ2依赖PKCD激活P38或JNK信号途径调节肝再生的细胞凋亡。为证明此假设，本课题拟以大鼠肝细胞为检测对象，通过基因添加和基因干涉研究PLCγ2对肝细胞凋亡的影响及作用机理。为此，本文用PLCγ2过表达和PLCγ2 shRNA干扰重组腺病毒分别感染大鼠肝细胞，并依次检测了PLCγ2蛋白及mRNA含量、PLCγ2亚细胞定位；肝细胞增殖活性、细胞周期和凋亡率、PLCγ2下游信号通路活化情况，并通过信号通路抑制实验进一步检测PLCγ2发挥功能的细胞信号途径。结果显示：（1）PLCγ2过表达和PLCγ2 shRNA干扰重组腺病毒成功感染大鼠肝细胞，肝细胞PLCγ2 mRNA和蛋白水平发生显著变化。（2）过表达PLCγ2后大鼠肝细胞增殖呈时间依赖性减弱。感染24h后，G1期细胞比例显著增加，S期比例显著减少，凋亡率明显增加；干涉PLCγ2后细胞增殖和凋亡情况刚好相反；（3）过表达PLCγ2后大鼠肝细胞内Caspases活性均明显增加；干涉其表达则引起Caspases活性减弱；（4）PLCγ2过表达引起肝细胞PKCD、JNK1/2和P38磷酸化水平显著增加，PLCγ2干涉后三者的磷酸化水平显著降低；（5）PKC抑制剂处理肝细胞48h后，细胞活性较过表达组显著增加，细胞增殖数量明显增加，凋亡细胞数减少，PKCD、JNK1/2和P38磷酸化水平显著降低。结果表明，PLCγ2通过PKCD介导的JNK1/2和P38信号通路促进大鼠肝细胞凋亡。该研究结果不仅有助于我们了解PLCγ2对肝细胞凋亡的调控作用及作用途径，并为后续体内研究和肝衰竭等肝病发生机制的研究提供资料。