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目的探讨肿瘤坏死因子α(tumor necrosis factor α,TNF-α)及联合蛋白激酶Cα (protein kinase C alpha,PKC-α)抑制剂Go6976对小鼠肝癌(H22)细胞凋亡的影响。方法将处于对数生长期的H22细胞分成两组,每组分四部分,一组仅以不同浓度的TNF-α(0,20、40、60 ng／ml)处理,另一组TNF-α处理同时以Go6976(4．6 nmol／ml)抑制PKC-α活性,分别于4 h,8 h,16 h采用流式细胞仪检测小鼠肝癌细胞的凋亡率,Western blotting方法检测PKC—α和磷酸化PKC-α蛋白的表达情况。结果 TNF-α(0、20、40、60 ng／ml)处理H22细胞4 h,细胞凋亡率分别为2．44％±0．31％、 1．80％±0．32％、2．73％±0．14％、3．05％±0．78％,PKC-α和磷酸化PKC-α表达无显著改变;同时用 TNF-α和Go6976处理H22细胞,凋亡率、PKC-α和磷酸化PKC-α的表达与单独使用TNF-α的结果相似。TNF-α处理8 h,细胞凋亡率分别为2．11％±0．43％、1．83％±0．31％、3．40％±0．47％、6．05％± 0．78％,PKC-α及磷酸化PKC-α的表达随TNF—α浓度增加而上调;TNF—α处理同时抑制PKC-α活性,细胞凋亡率显著升高,分别为2．90％±0．39％、7．76％±0．35％、11．43％±1．05％、12．96％± 2．44％,PKC-α和磷酸化PKC-α表达相应下调。仅以TNF-α处理或TNF—α处理同时抑制PKC-α活性16 h,其结果与8 h的结果基本一致;于Go6976抑制细胞PKC-α活性组,TNF-α60 ng／ml时细胞凋亡率较TNF-α40 ng／ml时有所下降,但坏死细胞的比例却明显升高。结论 TNF-α上调PKC—α和磷酸化PKC-α表达;抑制PKC-α活性,显著增加TNF-α对H22细胞的细胞毒性。
Objective To investigate the effects of tumor necrosis factor α (TNF-α) and apoptosis-associated protein kinase C (A) inhibitor Go6976 on hepatocellular carcinoma (H22) cell apoptosis in mice. Methods H22 cells in logarithmic growth phase were divided into two groups with four in each group. One group was treated with different concentrations of TNF-α (0, 20, 40, 60 ng / ml) At the same time, the activity of PKC-α was inhibited by Go6976 (4.6 nmol / ml). The apoptosis rate of mouse hepatoma cells was detected by flow cytometry at 4 h, 8 h and 16 h, respectively. The protein expression of PKC-α And phosphorylated PKC-α protein expression. Results The apoptosis rates of H22 cells treated with TNF-α (0, 20, 40 and 60 ng / ml) for 4 h were 2.44% ± 0.31% and 1.80% ± 0.32%, respectively. 73% ± 0.14% and 3.05% ± 0.78%, respectively. No significant changes were found in the expressions of PKC-α and phosphorylated PKC-α. The apoptosis rate, PKC-α And phosphorylated PKC-α were similar to those of TNF-α alone. The apoptotic rates of TNF-α treated for 8 h were 2.11% ± 0.43%, 1.83% ± 0.31%, 3.40% ± 0.47%, 6.05% ± 0, respectively. The expression of PKC-α and phosphorylated PKC-α were up-regulated with the increase of TNF-α concentration. 78% of cells treated with TNF-α also inhibited the activity of PKC-α, the cell apoptosis rate was significantly increased by 2.90% ± 0 .39%, 7.76% ± 0.35%, 11.43% ± 1.05%, 12.96% ± 2.44%. The expressions of PKC-α and phosphorylated PKC-α were down-regulated. The results showed that the activity of PKC-α was similar to that of the control group at 8 h after treatment with TNF-α alone or TNF-α for 16 h. Rate of TNF-α40 ng / ml decreased, but the proportion of necrotic cells was significantly increased. Conclusions TNF-α up-regulates the expression of PKC-α and phosphorylated PKC-α, inhibits the activity of PKC-α and increases the cytotoxicity of TNF-α to H22 cells.