Gene Therapy For Hepatoma Cells Using Diphtheria Toxin A Under The Control Of Promoter Of The Human α-Fetoprotein Gene

Backgroud and objective Diphtheria toxin is poisonous to human and many animals, even one or two molecules can kill a cell. Diphtheria toxin A chain is known to kill cells by ribosylating the EF2 translation-initiation factor and inhibiting protein synthesis. Genetic recombination immunotoxin conjugated by the DTA and different vectors can kill the cells, which express special protein. Primary liver carcinoma is one of the most common malignant tumors in China, which episode is very hidden, many patients have lost the opportunity of operation when they are diagnosed, and the recurrence rate is very high after resection, with low effect under the radiotherapy, chemotherapy, immunotherapy and even interventional therapy. Gene therapy is a hot spot for the primary liver carcinoma, which can express the definite gene in the target cell by the specific initiators of the cell and the tissues, and to decrease the toxin on the normal tissues as more as possible. So far expression of the target gene by AFP transcription modulator is the main method for dealing with the liver carcinoma. In this study DTA expression vector with AFP promoter was constructed. After transfection into the liver carcinoma cell in vitro, the kill effect of the cell by DTA was confirmed, which can present a new method for specific gene therapy for the liver carcinoma.Methods 1. New vector was constructed by orientation clone and verified. AFP promoter was cloned into the site of KpnI and HindIII, to take the place of SV40 promoter of pS2-DTA . EGFP fragment was cloned into the site of NcoI and XbaI, to take the place of DTA of pAF-DTA. 2. By intervention of liposomes, pAF- EGFP was transfected, and fluorescence was detected, to evaluate the efficienfy of AFP promoter. 3. After the transfection of pAF-DTA, expression of DTA was detedcted by PCR and immunohistochmistry, cell apoptosis was detected by TUNEL, and cell survival rate was detected by trypan blue resist-dye, and the kill effect was observed by cell morpholoty andbiological changes.Results 1. By the enzyme cut evaluation and sequencing, AFP promoter of pAF-DTA was found in the upper stream of A chain of diphtherotoxin. 2. By fluorescence detecting, after pAF-EGFP transfected, the expression of green fluorescence protein was trace in cell with AFP negative, however was high in cell with AFP positive. 3. After pAF-DTA transfected, the biological changes of the cell and the kill effect on the cell hints: (1) In AFP positive carcinoma cells, cell division phas was seldom, with low survival rate and slow growth. However the control cells proceed into the logarithm growth phase quickly. (2) In AFP positive carcinoma cells, the mRNA and protein came from DTA increased significantly, and the TUNEL was increased significantly contrast against the contol cells (PO.01).Conclusions 1. AFP promoter was cloned successed. 2.By orientation clone, a new eukaryon vector with AFP promoter and chain A diphtherotoxin was constructed. 3. The AFP promoter has high activity of transcription, which can accelerate the expression of green fluorescence protein. 4. pAF-DTA can highly express in the cells with AFP positive, and can promote selective killing, inhibiting its growth.