抑制蛋白激酶Cα活性增加TNF-α对小鼠肝癌细胞毒性的实验研究  

Inhibition of PKC-α enhances the toxicity of TNF-α to H22 cell Lines

作　　者：侍作亮[1] 陈孝平[1] 张万广[1] 关剑[1] 靖凯[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肝脏外科中心,武汉430030

出　　处：《中华普通外科杂志》2006年第5期364-366,共3页Chinese Journal of General Surgery

基　　金：卫生部临床重点学科资助项目(2001-321)

摘　　要：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 effect of tumor necrosis factor alpha （TNF-α） in combination with G06976 on murine liver cancer cells （H22）. Method H22 cells were divided into two groups. Each group was further divided into four subgroups. Group 1 was treated with TNF-α , 20, 40, 60 ng/ml. Group 2 was treated with TNF-α 0, 20, 40, 60 ng/ml and Go6976 （4. 6 nmol/ml）. The apoptotic rate, protein kinase C alpha （PKC-α） expression and phosphorylation-PKC-α （p-PKC-α） were detected by flow cytometer and Western blotting respectively in 4 h, 8 h and 16 h. Result Treated with TNF-α （0, 20, 40, 60 ng/ml） for 4 h, the apoptotic rates of H22 cells were 2.44% s0. 31%, 1.80% ± 0. 32%, 2. 73% ±0. 14% and 3.05% ±0. 78% respectively, with no change on the expression of PKC-α and p-PKC-α; For 8 h, the expressions of PKC-α and p-PKC-α in H22 cells were up-regulated with increasing concentration of TNF-α; When PKC-a was inhibited with Go6976 at the same time, the apoptotic rates of cells increased significantly, being 2. 90% ± 0. 39%, 7. 76% ± 0. 35%, 11.43% ± 1.05% and 12. 96% ±2.44% , respectively. Moreover, PKC-α and p-PKC-α were down-regulated accordingly. When H22 cells were treated with TNF-α only or combined with G06976 for 16 h, the result was similar to that of 8 h; The apoptotic rate dropped in the group in which PKC-α was inhibited by G06976 with TNF-α at 60 ng/ml, but the proportion of necrotic cells increased. Conclusion TNF-α up-regulates the expression of PKC-α and p-PKC-α in H22 cells. Inhibiting the activity of PKC-α significantly enhances the toxicity of TNF-α to H22 cells.

关 键 词：肿瘤坏死因子d 蛋白激酶C 细胞凋亡 肝癌 细胞毒性 

分 类 号：R735.7[医药卫生—肿瘤]