TGF-β1及其诱导的自噬激活在老年肝切除术后肝脏再生过程中的作用及机制

As the life extension and precise surgical technique, aging patient who underwent hepatectomy due to liver tumor are gradually increasing in China. Reduction of aging liver regeneration of the remnant liver is an important factor that prevent recovery and prognosis. We found TGF-β1 and TβR expression was increased in the early stage of hepatectomy, accompanied with high expression of ATG5 mRNA and LC3-II protein. TGF-β1, the main negative regulator of liver regeneration, could inhibit cell proliferation and promote cell apoptosis. TGF-β1 could also induce autophagy, which will promote cell apoptosis. We hypothesize that TGF-β1 could induce autophagy through Smad pathway and/or p38 MAPK signaling pathway, which will promote cell apoptosis and inhibit liver regeneration. We will compare the autophagy level by LC3-II/I, autophagy flux and autophagosome, and evaluated cell apoptosis with cleaved caspase3 in aging mice after hepatectomy or in aging hepatocyte after treatment with TGF-β1. Then we will compare the difference with the treatment of TGF-β1 inhibitor or autophagy inhibitor. We will focus on investigating the roll of TGF-β1 induced autophagy, trying to find some way to control the cell apoptosis. Finally, we hope to find some methods promoting liver regeneration and improving prognosis in aging patients.

随着寿命延长和外科技术提高，我国因肝脏肿瘤行肝切除的老年患者显著增多，而老年人术后肝再生能力及速度下降严重影响康复和长期存活。前期研究发现老年小鼠肝切除术后TGF-β1和TβR表达升高，并伴随着自噬激活和细胞凋亡增多。TGF-β1是肝再生过程中重要的负调节因子，能阻滞细胞周期，也能激活自噬和诱导细胞凋亡，且自噬与凋亡密切相关。推测老年肝再生过程中TGF-β1可能通过Smad及TAK1-p38 MAPK通路激活肝细胞自噬，促进凋亡，进而抑制肝再生。拟构建老年小鼠肝切除模型、分离培养原代肝细胞，检测LC3-II/I表达、自噬流、自噬小体验证自噬激活；检测活化Caspase3表达比较细胞凋亡差异；利用siRNA及抑制剂干预，探明TGF-β1与自噬及自噬与凋亡的关系。重点研究TGF-β1诱导自噬激活在老年肝再生中的作用，探索调控肝细胞凋亡的靶点，为促进老年肝再生提供可行方案，具有重要的临床意义。

随着寿命延长和外科技术提高，我国因肝脏肿瘤行肝切除的老年患者显著增多，而老年人术后肝再生能力及速度下降严重影响康复和长期存活。前期研究发现老年小鼠肝切除术后TGF-β1表达升高，并伴随着自噬激活和细胞凋亡增多。我们构建了小鼠70%肝切除模型，年轻组(6周)，老年组（64周），分别留取0h、术后4h、8h、12h、24h、48h、72h肝脏组织进行研究。电镜显示年轻小鼠0h肝细胞内自噬小体较多，肝切除术后再生启动阶段（4h）老年小鼠自噬激活，可见大量自噬小体。随后自噬小体数量均下降至较低水平，两组无明显差异。Western blot检测LC3I/II与p62蛋白等自噬相关蛋白，显示老年小鼠自噬水平较年轻小鼠高。Capase3，Caspase7蛋白表达差异及Tunel染色证实肝切除术后老年肝细胞凋亡增加，TGF-β1过量表达可能是诱导肝细胞凋亡的因素。为明确可能调节自噬激活的通路，通过Western blot方法检测TGF-β1下游信号通路Smad蛋白以及P38Mapk，p-P38Mapk的表达。发现p-P38Mapk及p-P44在老年小鼠和年轻小鼠表达均升高，而在老年小鼠p-P38Mapk及p-P44表达水平较年轻小鼠表达水平低，而Smad信号通路相关蛋白差异不明显。老年老鼠在肝切除术后，mTor磷酸化水平明显下降。为进一步筛选参与自噬激活的相关LncRNA，进行测序及生信分析，筛选出一条差异表达LncRNA（NONMMUT025897.2），相应人同源lncRNA为MAP3K13variant4。TGF-β1处理小鼠AML-12及NCTC1469细胞24h，明确TGF-β1调控NONMMUT025897.2的表达。ShRNA敲降NONMMUT025897.2在AML12细胞表达后，发现细胞凋亡增加。而ShRNA敲降人HepG2细胞中MAP3K13表达后，CCK8及Brdu染色提示细胞增殖下降，提示该同源LncRNA与肝细胞增殖及凋亡相关。综上，我们发现了肝切除术后TGF-β1可能通过p38Mapk及mTor通路激活肝细胞自噬及促进凋亡，LncRNANONMMUT025897.2为调控的机制之一。我们的研究发现了影响老年肝再生下降的可能机制，为促进老年肝切除术后肝再生的研究提供依据。