Studies for the induction of apoptosis in human junctional and gingival epithelial cells and for protective effect of HGF against apoptosis of these cells

肝细胞生长因子(HGF)，又称散射因子和肿瘤细胞毒因子，是许多上皮细胞最强大的有丝分裂原，被认为是支持正常发育和组织再生的过程。在本研究中，我们首先检测了血清剥夺诱导人交界处和牙龈上皮细胞的凋亡，并探讨了HGF对这些细胞凋亡的保护作用。目前，我们获得了以下结果：1.在无血清培养24小时后，细胞发生了凋亡，以DNA寡核体的产生和DNA片段的原位末端标记法进行检测。HGF(10 ng/mlHGF)在无血清条件下可明显抑制细胞DNA断裂。在无血清培养24小时后，我们检测了这些细胞提取物中的白介素1β转换酶(ICE，新近命名为caspase-1)样蛋白的活性，发现caspase-3的活性，而不是caspase-1的活性，与在10%血清中孵育的对照细胞相比，增加了约2倍。Caspase-3的特异性抑制剂乙酰天冬氨酸-谷氨酸-天冬氨酸醛不仅能抑制caspase-3的激活，还能抑制血清剥夺诱导的DNA片段化，提示caspase-3的激活参与了血清剥夺诱导的细胞凋亡。我们用Western blotting方法检测了HGF对凋亡抑制蛋白Bcl2表达的影响，发现HGF促进了Bcl2的表达。提示血清剥夺通过增加caspase-3的活性来诱导人牙龈上皮细胞的凋亡，HGF通过增加Bcl2的表达进而抑制caspase-3的激活来抑制细胞的凋亡。

Hepatocyte growth factor (HGF), also known as the scatter factor and tumor cytotoxic factor, is a most potent mitogen for many epithelial cells and is suggested to support the process of normal development and tissue regeneration.In this study, we first examined the induction of apoptosis in human junctional and gingival epithelial cells induced by serum deprivation, and investgated the protective effect of HGF against apoptosis of these cells. At present, we obtained following results :1. When these cells were cultured in serum-free medium for 24h, the cells underwent apoptosis, judged by generation of oligonucleosomal fragmentation of nuclear DNA and by in situ detection of fragmented DNA by TUNEL method. However, DNA fragmentation was markedly inhibited when the cells were incubated with HGF (10 ng/ml) in serum-free medium.2. We measured interleukin-1beta converting enzyme (ICE, named recently as caspase-1)-like protease activity in these cell extracts after incubation for 24h in serum-free medium and found that caspase-3 activity, but not caspase-1 activity, was increased about 2 times as compared with those of control cells incubated in the presence of 10% serum.3. Specific inhibitor for caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde inhibited not only the activation of caspase-3 but also the DNA fragmentation induced by serum deprivation, suggesting that activation of caspase-3 is involved in induction of apoptosis in these cells by serum deprivation.4. We examined the effect of HGF on the expression of Bcl-2, which is one of apoptosis suppressor proteins, by Western blotting analysis, and found that HGF increased the expression of Bcl-2.These results suggest that serum deprivation induces apoptosis in human junctional and gingival epithelial cells by increasing caspase-3 activity and that HGF suppresses the apoptosis by increasing the expression of Bcl-2, followed by inhibiting caspase-3 activation.