苦参碱及联合丹酚酸B对肝细胞癌凋亡与自噬交互调节的影响及分子机制

Autophagy plays an important role in maintaining cellular homestasis. Recent researches found that apoptosis and autophagy could inhibit and transform mutually, and there were precise molecular regulations between them. Moreover, Chinese medicine active ingredients could regulate apoptosis and autophagy of tumor cells. Our previous studies showed that matrine could effectively inhibit proliferation, induce cell apoptosis, and dramatically increase the generation of autophagosomes in Hepatocellular Carcinoma (HCC) cell line HepG2. However, the effect and exact mechanism of generation of autophagosomes and the cross-regulation between autophagy and apoptosis in therapy of HCC by matrine and other Chinese medicine are still not clear. On the basis of our preliminary studies, we are to systemically investigate the roles and associated mechanisms of cross-regulation between autophagy and apoptosis by matrine or with salvianolic acid B in vitro, animal experiment and clinical specimens of HCC, using molecular biology and cytobiology techniques. In addition, we will screen the new autophagy-associated genes by gene-chip technique to explore their expression in biological behavior of HCC, such as differentiation, invasion and metastasis. The results may be supposed to provide sufficient experimental evidence for further researches on Chinese medicine monomers and combination therapy in HCC, and may be having a significant influence on modernization of Chinese medicine.

自噬在维持细胞自稳态中发挥重要作用，自噬和凋亡相互抑制并可相互转变，存在精确的分子调控机制。中药有效成分可调控肿瘤细胞凋亡和自噬，我们前期研究发现苦参碱作用于肝癌细胞株HepG2可有效抑制肝癌细胞增殖并诱导凋亡，同时发现自噬体显著增多。然而自噬与凋亡交互调节在苦参碱等治疗肝癌中的作用和相关机制尚不明确。在前期基础上，本研究应用分子生物学和细胞生物学的研究方法和手段，从体外培养、动物实验和临床标本检测三个方面，针对自噬、凋亡及交互调节在苦参碱、丹酚酸B及联合用药治疗肝癌中的作用及其相关机制，进行深入系统的研究；并应用基因芯片技术寻找新的自噬调控基因，探讨其表达对肝癌分化、侵袭、转移等生物学行为的影响及机制。课题的实施可为中药单体及联合用药治疗肝癌提供实验依据，对中药现代化具有重要意义。

细胞的自噬和凋亡存在精确的分子调控机制，然而二者交互调节在苦参碱治疗原发性肝癌中的相关机制尚不明确。本研究针对自噬、凋亡及二者交互会话在原发性肝癌治疗中的作用，进行深入系统的研究；并应用基因芯片技术寻找新的自噬调控基因，探讨其表达对肝癌分化、侵袭、转移等生物学行为的影响及机制。.本课题的研究分三部分进行。.首先，我们在肝癌细胞系HepG2和Bel7402中研究了苦参碱诱导的凋亡和自噬及其分子机制：发现苦参碱抑制肝癌细胞生长、增殖并诱导线粒体介导caspase依赖的细胞凋亡；且自噬水平增加，其机制可能与PI3K/ AKT/mTOR通路的抑制和自噬相关基因beclin-1上调有关；基因芯片检测得到lamp-1参与自噬溶酶体形成，可能为自噬相关基因；抑制自噬能够促进苦参碱诱导的凋亡增加。.其次，苦参碱作用于人肝癌细胞系HepG2和SMMC-7721，并应用基因芯片技术检测自噬相关基因，发现通过p53/AMPK信号通路诱导肝癌细胞的自噬，p53失活通过调节AMPK激酶，参与自噬体的调控，AMPK激酶的活性下降后导致自噬体水平下降，细胞凋亡增多；而p53的异构体参与自噬的产生。IFN相关基因，尤其是干扰素α诱导分子IFl27和跨膜蛋白l可能参与苦参碱诱导的自噬。.然后，我们发现苦参碱作用于肝癌MHCC97L和Huh-7细胞系，激活了caspase依赖的细胞凋亡，PARP被切割降解，且激活细胞自噬；苦参碱通过磷酸化JNK激活Bcl-2/Bcl-xL-Bax/Bak通路。SP600125抑制JNK活性后细胞活力增加，同时Bcl-2/Bcl-xL的磷酸化水平降低，细胞凋亡与自噬水平下降；苦参碱作用后，Bax由胞浆转移至线粒体并寡聚化，线粒体膜电位降低，细胞色素c由线粒体释放至胞浆；Bcl-2/Bcl-xL及其磷酸化后释放的Bax与Beclin 1相互作用，抑制肝癌细胞自噬。用小干扰RNA敲低Bax/Bak后，自噬水平升高，同时Bcl-2/Bcl-xL磷酸化水平也升高。Bax与Beclin 1相互作用可能是凋亡与自噬通路新的交互点；苦参碱能抑制裸鼠MHCC97L瘤块的生长。.综上，本研究阐述了苦参碱抑制肝癌细胞生长和增殖，诱导肝癌细胞凋亡和自噬的作用和相关分子机制，也发现了二者交互会话的可能的分子通路，为苦参碱用于肝癌的治疗提供了实验依据。