Effects Of The Retrovirus Vector Containing Retro-hTR Gene On Growth Of SGC7901 Cell Line

Objective: To study the effects of a retroviral vector containing retro-hTR gene on apoptosis of gastric carcinoma cell line of SGC7901.Methods: We got the recombinant retroviral vector plasmid pLXSN-hTR, which carried the retro-hTR gene. It was expected that the transformed gastric 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, the supernatant was collected and the viral titer was determined with NIH3T3 cell line, we transfected SGC7901 cell with the newly collected and concentrated retroviral supernatant. The transformed gastric 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 gastric cell line and slows down the growth of the transformed cells of the malignant tumor. The existence and expression of the aim gene in SGC7901 cell was confirmed through PCR, and observe the apoptosis of the SGC7901 cell through inversion microscope. MTT was used to determine the cell growth condition.Results: The retrovirus vector containing retro-hTR gene was successfullyconstructed, and then PT67 cell was transfected successfully through electroporation. Flow cytometry analysis showed that 27.2% of the treated PT67 cells were positive of GFP gene expression. It was 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 anti-sense virus could reach 2.6 X 105cfu/ml and 3.8 X 105cfu/ml. Then the SGC7901 cell was transfected with the recombinant retrovirus supernatant. PCR approved that the retro-hTR gene had been integrated into the target cell gene successfully. The inserted retro-hTR gene was able to express itself and the productions could decrease the telomerase activity in the target cell. Therefore, when the cell infected proliferated, 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 got shorter and shorter. When the times of the cell division increased to some extent, the apoptosis program would be triggered up, which would lead to the death of the infected cells. Cells growth curve showed that retro-hTR gene lowered the growth of SGC7901 cell line significantly and the inhibition rate was found higher than that of control group (p<0.05 ) . Significant inhibiting effect of the infection on SGC7901 cells in the control group was not found.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 this study, retrovirus vector containing target hTR gene not only inhibited cell proliferation and lowered telomerase activity, but also induced cell apoptosis. Retro-telomerase can inhibit the growth of SGC7901 cell by gene therapy. This study provides a new gene therapy method and foundations for the clinical applicability of retro-telomerase in gastric carcinoma gene therapy.