| Background:During this decay, gene therapy is a new way of cancer therapy. Though it is still in the time of laboratory, some results from the experiments are inspiring. Since 1990s the NF- K B gene (nuclear factor kappa B gene ) and its inhibitor I K B a gene have become the hotspot in cancer gene therapy. NF- K B gene exists in almost all mammalian cells. It is a nuclear transcriptional factor which maintain the cell vitality and protect the cell from apoptosis. In the cancer cells the activation of NF- K B gene can regulate the expression of a numerous oncogenes, which take part in the proliferation ,invasion and metastasis of the cancer cells. NF- K B is activated of some degree in these cells. Moreover it is controlled by its inhibitor I K B a .When the cell are not stimulated, I K Bα combines NF- K B of a inactivated state anchoring in the cytoplasm. Whereas the stimulator such as carcinogen, radiation, cytokine and chemicals, can lead I K B a to degrade. In this moment NF- K B become active and translocate into the nucleus to function as a transcription factor.Active NF- K- B regulates the transcription of genes in the target cells. It can promote the proliferation of the cancer cells. Mutant I K B a (ml K B a ) is the mutant of I K B a .It is more stable than I K B a .When it associates with NF- K B, the trimmers forms stably, which can keep NF- K. B from being activated. Objective:To observe whether ml K B a can inhibit the growth of Hep G2,the hepatocellular carcinoma cells, and enhance the radiosensitivity . The molecular mechanisms of its effect are also discussed. Methods and Results:Part 1. The gifted mutant iKBa(mlKBa) plasmid was identified by restriction enzyme analysis and PCR sequence test. The adenoviral vector which contained ml K B a (Adv- m I K B a ) was constructed by bacterial homogenous recombination using AdEasy system. Packaged in the 293 cells, the adenovirus containing m I K B a was harvested. Hep G2 cells were infected by this harvested adenovirus. When the green fluorescence protein appeared in the Hep G2 cells, successful adenovirus-mediated m I K B a gene transfer was conformed. The extracted cytoplasm of these cells was analyzed with Western blot, In which exogenous ml K B a protein expression was detected positively.Part 2. To observe the growth of the Hep G2 cells containing exogenous m I K B a , plating efficiency analysis, clone formation ability in soft agar, growth curve were done. The results suggested that the growth of the m I K B a transfected Hep G2 cells was inhibited. In situ cell apoptosis detection with TUNNEL assay for visualization under light microscopy, the ml K B a transfected Hep G2 cells showed strongly positive.Part 3. Cells were divided into three groups: 1. Ad-mi K B a group: Hep G2 cells transfected with adenovirus containing ml K B a vector; 2. Adv group: Hep G2 cells containing Ad vector; 3. Hep G2 group: HepG2 cells untreated.Cells were treated with different dosage of high-energy X-ray irradiation.The clone formation rates were compared among the three groups with statistical "L-Q model" and the cell survival curve was obtained. The results showed a 3.2 radiation sensitivity ratio.Cells were irradiated with 6Gy high-energy X-ray. After that in situ cell apoptosis detection with TUNNEL assay was done. In the Ad- m I K B a group, the apoptosis rates were 18.2% and 68.3% before and after irradiation, between which there was a remarkable difference (P<0.01). In the Adv group, the apoptosis rates were 1.4% and 11.7% before and after irradiation, between which there was a remarkable difference (P<0.01). In Hep G2 group, the apoptosis rates were 1.6% and 8.9% before and after irradiation, between which there was a remarkable difference(P<0.01). Also there was a remarkable difference (P.01) among the three groups after irradiation. The cell apoptosis rate of Ad- m I K B a group (68.3%) were remarkably different from those of the Ad- group(8.9%) and Hep G2 group(l 1.7%). But there was no difference of the cell apoptosis rates...
|
PDF Full Text Request