【作者】 孙宏丹；

【导师】 仲来福；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 成人肝脏被手术部分切除（partial hepatectomy）70%以后,仍然具有可重新再生的能力。没有被切除的肝组织可快速再生,并在术后一至两周内完全恢复至初始重量。值得注意的是,率先对再生刺激做出反应并进入细胞周期循环的是肝细胞。过去的几十年里人们虽然对这一现象进行了大量、深入的研究,但对触发成人肝细胞进入细胞周期循环的关键性体液因子却不十分清楚。 C-Jun氨基末端激酶（C-Jun N-Terminal Kinase,JNK）,也被称为应激活化蛋白激酶,是丝氨酸-苏氨酸蛋白激酶家族的一员。在多种外源刺激下,JNK将信号由细胞膜传导至细胞核内。这些信号可对细胞进行一系列的功能调节,包括细胞的增殖、分化、转化及凋亡等。研究表明,JNK信号传导系统在肝细胞的增殖和肝脏再生方面起重要的作用,但对触发该信号早期激活的上游作用因子尚不明了。 近年来,细胞外ATP已成为重要的信息分子,在细胞应激条件下,ATP可从细胞内分泌释放出来,它通过激活细胞表面受体对多种组织和器官的多种功能进行调节,也包括对再生的调节作用。尽管人们已经知道细胞外ATP可对肝脏的许多功能进行调节,但它在肝细胞增殖和再生中所起的作用迄今尚不清楚。 本课题研究的目的是探讨细胞表面的嘌呤信号是否能通过激活JNK来促进肝细胞的增殖及肝脏的再生,及P2嘌呤受体所介导的Ca²⁺和蛋白激酶C（PKC）信号系统在JNK信号的诱导方面的作用。 实验结果表明,应用ATPγS（非水解性ATP的类似物）处理原代肝细胞时再现了与肝脏再生有关的一系列早期信号,如快速而短暂的JNK信号系统的激活,立即早期基因（immediate early genes）—c-jun和C-fos的mRNA的表达增高及活化蛋白-1（AP-1）DNA结合活性的增强等。激活剂选择性试验的结果显示UTP>ATP>ATPγS。这表明细胞外ATP是通过激活P2Y2受体而起作用。体外试验结果也表明,当ATPγS单独作用或与表皮生长因子（EGF）协同作用可促进与细胞周期进展有关更多还原

【Abstract】 Adult liver has a remarkable ability to regenerate after the loss of hepatic tissue. After surgical removal of 70% of the mass of liver （partial hepatectomy）, the remaining tissue undergoes rapid regeneration and the original mass is restored in its entirely, typically within a week or two after surgery. Interestingly, hepatocytes are the first to enter the cell cycle in a concerted manner in response to regenerative stimulus. Despite intense investigations for the past several decades, the identity of the key humoral factors which trigger cell cycle progression in adult hepatocytes remains elusive.C-Jun N-Termianl Kinase （JNK） is a family of serine/threonine kinase that transducer signals from the cell membrane to the nucleus in response to a wide range of stimuli which control a spectrum of cellular processes, including cell growth, differentiation, transformation and apoptosis. C-Jun N-Teminal Kinase signaling cascade plays a key role in hepatocyte proliferation and liver regeneration. However, the factors responsible for the activation of JNK early on during regeneration remain unknown.In recent years, extracellular ATP which is discretely released from cells under stress, has proven to be a bona-fide signaling molecule influencing a variety of cellular functions via activation of cell surface receptors, but its role in hepatocyte growth and regeneration is unknown, In this study, we sought to determine if purinergic signaling can lead to the activation of c-Jun N-terminal kinase. （JNK）, a known central player in hepatocyte proliferation and liver regeneration. Moreover, if P2 purinergic receptor mediated activation of Ca²⁺ and protein kinase C signaling networks play a key role in the induction of JNK signaling in hepatocyte also remain unclear. Hepatocyte treatment with ATPγS, a non-hydrolyzable ATP analog, recapitulated early signaling events associated with liver regeneration, i.e. rapid and transient activation of JNK signaling, induction of immediate early genes c-fos, and c-jun, and Activator Protein-1 （AP-1） DNA-binding activity. The rank order of agonist preference, UTP>ATP>ATPγS, suggests that the effects ofextracellular ATP is mediated via the activation of P2Y2 receptors in hepatocytes. ATPyStreatment alone, and in combination with epidermal growth factor （EGF）, substantiallyincreased cyclin Dl, and PCNA protein expression and hepatocyte proliferation in vitro.Extracellular ATP as low as 10 nM was sufficient to potentiate EGF-induced cyclin Dlexpression. Partial hepatectomy can induce a rapid and robust activation of JNK signalingand potent proliferation response in the remnant liver, based on the immunoblotting ofphosphor-JNK, phosphor-c-jun, Cyclin Dl and PCNA expression andimmunohistochemical analysis for Ki67 and PCNA. Infusion of ATP via the portal veindirectly activated hepatic JNK signaling, while infusion of a P2 purinergic receptorantagonist prior to partial hepatectomy inhibited JNK activation and cell cycle proliferationand liver regeneration in vivo. In order to test the role of PKC as a potential upstreammediator of JNK activation, hepatocytes were treated with a battery of small molecule PKCinhibitors, which exhibit differential specificity towards multiple PKC isoforms—prior tothe treatment with ATPyS. Our results suggests that extracellular ATP via the activation ofP2 purinergic receptors activates multiple Ca-dependent, Ca-independent and atypical PKCisoforms which includes PKCu. Furthermore, extracellular ATP can rapidly induce PKCactivation and translocation to membranes. Based on the inhibition of JNK by the PKCinhibitior G66976 and the apparent lack of inhibition by PKC inhibitors G66983 andG66850, we believe PKCu is a potential key upsteam mediator of ATP-mediated JNKsignaling in hepatocytes. We also find immunohistochemical evidence that extracellularATP leads to the activation of PKCu in hepatocytes. Within 10 min of treatment of ATPyS,PKCu is translocated into the nucleus from the cytoplasma. We also find that ATPySinduced JNK activation in not dependent on Ca2+ or the activation of PLC. In conclusion,extracellular ATP is a hepatic mitogen that can activate JNK signaling and hepatocyteproliferation in vitro and initiate JNK signaling in regenerating liver in vivo. ExtracellularATP also activates multiple PKC isoforms as determined by the translocation andphosphorylation of PKC in hepatocytes. Duo to JNK and PKC activation are early eventsin hepatocyte proliferation and liver regeneration, these findings implicate that extracellularATP as a potential humoral mediator responsible for the induction of a cascade of signalingevents which collectively induce hepatocyte proliferation.更多还原