Studies On Transformation Of Bel7402 Cell Line Mediated By A Retroviral Vector Containing Retro-hTR Gene

Objective: To study the effection on apoptosis of hepatic carcinoma cell line of Bel7402 mediated by a retroviral vector containing retro-hTR gene.Methods: We got the recombinant retroviral vector plasmid pLXSN-hTR, which carry the retro-hTR gene. We expect that the transformed hepatic cancer cell mediated by the retrovirus vector could transit anti-sense telomerase RNA, which could combine with the normal telomerase RNA, then stop the expression of telomerase and induce the malignant tumor cell into apoptosis. When pLXSN-hTR was transferred into packaging cell line PT67 by electroporation, and selected with G418, we got a stable cell line, which could produce retrovirus. After the recombinant retrovirus supernatant was collected and the viral titer was determined with NIH3T3 cell line, we transfected Bel7402 cell with the newly collected and concentrated retroviral supernatant. The transformed hepatic cancer cell conformed by the retrovirus vector containing pLXSN-hTR could produce anti-sense telomerase RNA. The anti-sense RNA would attach to the normal telomerase RNA and bring down the activity of telomerase, which leads to the apoptosis of the hepatic cell line and slows down the growth of the transformed cells of the malignant tumor. We could confirm the existence and expression of the aim gene in Bel7402 cell through PCR, and observe the apoptosis of the Bel7402 cell through inversion microscope. MTT was used to determine the cell growth condition.Results: We had successfully constructed the retrovirus vector containing retro-hTR gene, and then PA317 and PT67 cell was transfected successfully through electroporation. Flow cytometry analysis shows that 27.2% and 33.6% of the treated PA317 and PT67 cells are positive of GFP gene expression. It is found that the positive percentage rose when the cells were screened with G418.We selected positive cell clone that could produce retrovirus continuously, and harvested the recombinant retrovirus supernatant. Through centrifuge and concentration, the titer of sense and antisense virus could reach 2.7×105cfu/ml and 3.9 × 105cfu/ml. Then the Bel7402 cell was transfected with the recombinant retrovirus supernatant. PCR approved that the retro-hTR gene has been integrated into the target cell gene successfully. The inserted retro-hTR gene is able to express itself and the productions could decrease the telomerase activity in the target cell. Therefore, when the cell infected proliferates, it's telomere would get short and could not been prolonged to its former length due to the insufficient activity of telomerase. With the continuous dividing of the infected cell, its telomere gets shorter and shorter. When the times of the cell division increase to some extent, the apoptosis program will be triggered up, which will lead to the death of the infected cells. We detect the cell growth with the method of MTT, and draw the cells growth curve to show the results apparently. We find that retro-hTR gene lowered the growth of Bel7402 cell line significantly and the inhibition rate was found higher than that of control group (p<0.01) . We did not find significant inhibiting effect of the infection on Bel7402 cells in the Control group.Conclusions: Retroviral vector is a good gene therapy tool. It can transfect cancer cells only, and have no effects on most normal cells. This means that the method has a target-oriented effect. What's more, it can express the target gene continuously and need not to be used repeatly. All these characters make retrovirus vector used popularly in modern gene therapy research. In my study, retrovirus vector containing target hTR gene not only inhibited cell proliferation and lowered telomerase activity, but also induced cell apoptosis. We can make out clearly that the growth of Bel7402 cell treated with gene therapy was inhibited greatly. The difference between the experiment group and the control group is significant (p<0.01) .The result of our study provides a new gene therapymethod and foundations for the clinical applicability of retro-telomerase in hepatic carcinoma gene therapy.