重组荞麦胰蛋白酶抑制剂对人肝癌细胞的凋亡及半胱氨酸天冬酶活性的影响

先前的研究表明，基因重组荞麦胰蛋白酶抑制剂 (rBTI) 具有诱导不同肿瘤细胞凋亡的作用.为了揭示其诱导肿瘤细胞凋亡的可能机理，从基因水平上探讨与凋亡有关的分子事件，本研究用不同浓度的 rBTI 体外作用于人肝癌细胞 HepG2 后，采用 MTT 比色法检测抑制剂对epG2 细胞的抑制率，用 DNA 凝胶电泳和细胞核的形态学观察检测 HepG2 细胞的凋亡.结果表明，rBTI 在体外能够明显抑制 HepG2 细胞的增长，并诱导细胞凋亡.另外，细胞凋亡与Bcl-2/Bax mRNA 水平有关.通过 RT-PCR 检测发现，细胞经过rBTI处理后，抗凋亡基因Bcl-2 mRNA 水平下调，促凋亡基因 Bax mRNA 有所上调，而对照 GAPDH 无变化.对 HepG2细胞中 Fas/Fas 配体及半胱氨酸天冬酶（caspase）的研究证明，细胞经过 rBTI 处理后，对死亡受体 Fas mRNA没有影响; rBTI 可明显激活caspase-3 和 caspase-9 酶活性， 对caspase-8 活性几乎无影响.上述结果表明，rBTI 对HepG2 细胞具有明显的诱导凋亡作用，其诱导细胞凋亡的机制与 caspase-3 依赖性凋亡调节信号通路有关，未涉及 Fas/Fas 配体途径.

Effect of Recombinant Buckwheat Trypsin Inhibitor on Apoptosis and Caspases Activity in Human Hepatoma (HepG2) Cells

Recombinant buckwheat trypsin inhibitor (rBTI) has been reported to induce apoptosis in human cancer cells，such as IM-9，K562.To examine whether rBTI had an anticancer effect on HepG2 human hepatoma cells，and reveal the possible mechanisms，cells treated with rBTI were subjected to MTT，nuclear staining and DNA fragmentation assays for measuring growth inhibition and induction of apoptosis.In addition，as the Bcl-2 (an anti-apoptotic gene)/Bax (a pro-apoptotic gene) ratio is associated with the increase of apoptosis，the mRNA expression levels of both were determined.Following rBTI incubation，Bcl-2 was decreased，whereas Bax was increased.We also investigated the alteration of the Fas/FasL system after rBTI treatment in HepG2 cells. However，not significant changes were found.rBTI treatments markedly induced the activities of caspase-3 and caspase-9，while caspase-8 was responded slightly.These findings suggested that rBTI induced apoptosis was likely initiated at mitochondria and maybe related to the caspase-3 signaling pathway，but not via Fas/FasL.