| [ Background ] Tumor necrosis factor a (TNF- a ) is a multipolar cytokine eliciting a wide range of biological responses, such as inflammation, cell proliferation, differentiation, and apoptosis. It was approved that tumor necrosis factor a has great role on immune response and antitumor of the body. But many tumor cells resisted to TNF-induced cytotoxicity. Therefore probing mechanism of resistance to TNF- a can help find a new strategy for treatment of cancer. TNF- a -induced cytotoxicity to tumor cells was affected by the cellular intrinsic and acquiring factors. Some research showed that the cellular apoptosis induced by TNF- a was greatly enhanced by inhibiting cells to synthesize protein., which suggested that the sensitivity of tumor cells to TNF- a has relationship with the synthesized protein . However, the principle that regulates synthesizing protein in tumor cells is still unknown.[Objective] This study attempt to observe the effect of NF-KB pathway activation to the cytotoxic reaction and the cell cycle of TNF-induced using cultural SMMC-7721 cell line. And the paper also discuss that activation of nuclear factor- K B pathway induced TNF- a effects on IL-6 expression. It is hoped to elucidate that role of IL-6 in the tumor cell resistance to treatment andeffect of nuclear factor- K B pathway on resistance to TNF- a in the SMMC-7721 cell line.[Methods] (1) The expression of IL-6 mRNA and secretion of IL-6 were measured when SMMC-7721 was treated by TNF- a in different times and different concentrations using RT-PCR and RIA .(2) The NF- K B pathway activation was measured respectively when SMMC-7721 was activated by TNF- a in different times and different concentrations using the different methods of immunocytochemistry and ELISA. The apoptosis rate and cell cycle of SMMC-7721 induced TNF-a was measured using flow cytometry at the same time.(3)Above indexes were observed further when activation of the NF- K B pathway was blocked by TPCK .[ Results ] (l)Resting SMMC-7721 cell line only secreted a few IL-6. But IL-6 expressions increased obviously after treatment with TNF-a. And it increased continuously with the time and dosage increasing of treatment with TNF-a in SMMC-7721 cell line. Results showed that the secretion of IL-6 arrived at the peak in 8 hours and slightly decreased from 12 hours to 24 hours in condition of 10ng/ml TNF- a . Then the secretion of IL-6 kept steady level in 48 hours. Similarly, Resting SMMC-7721 cell line only expressed a few IL-6 mRNA. The IL-6 mRNA expression increased with the time and dosage increasing of TNF- a excitement in SMMC-7721 cell line. The expression of IL-6 mRNA arrived at the peak in 6 hours and slightly decreased after 8 hours in the condition of lOng/ml TNF-a. Then the expression of IL-6 mRNA has been keeping steady level after 8 hours.(2) Cells proliferation was observed in SMMC-7721 cell line after treatment with IL-6. But survival rate of SMMC-7721 obviously decreased after treatmentwith Anti-IL-6 or Anti-IL-6R. The sensitivity to TNF- a increased in SMMC-7721. However the results showed that IL-6, Anti-IL-6 and Anti-IL-6R had no obvious affection to apoptosis of SMMC-7721 induced by TNF- a .(3) NF- K B already had the significant activation and nuclear translocation when NF- K B pathway was stimulated by TNF- a after 30 minutes, which reached the peak at 2 hours, and then it gradually declined. Besides, the NF- K B activation increased with the increasing of TNF- a dosage in 2 hour. ICC showed the same results.(4) The activation of NF- K B pathway can be blocked by TPCK. Blockage of the activation can course decreasing of living rate and secretion of IL-6 , increasing of apoptosis induced by TNF- a in SMMC-7721.(5) Apoptosis was not obvious after treatment by TNF- a in SMMC-7721. TPCK had no obvious affection to apoptosis of SMMC-7721,too. However, TNF- a treatment increased the apoptosis rate of the cells with TPCK precondition .(6) SMMC-7721 cell lines were synchronized in G0 /G1 by serum starvation...
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