Transfection And Expression Of RI Gene On Human Umbilical Blood Stem Cells

Tumor is one of the most harmful diseases, but those anti-tumor methods are not very satisfactory. In this case, a special kind of anti-tumor method with high efficiency and low side-effect is heated expected. Many experiments show that angiogenesis plays an indispensable role in the growth of solid tumors, new blood vessels supply the tumor with oxygen, nutrition and deplete metabolite. If the tumor is 1-2mm3 without angiogenesis, the tumor tissue will degenerate. So anti-angiogenesis will be an efficient method in controlling the growth and transfer of solid tumors. Since Ang (Angiogenin) is an important angiogenic factor and RI (Ribonuclease Inhibitor) is a high efficient inhibitor of Ang, it can be said that RI may be a latent anti-angiogenic drug. Umbilical blood stem cells have been well known as a kind of good carrier in gene transfection, in respect that they have many virtues, such as high success rate of transplantation, powerful proliferation potence, low incidence of GVHD, immuno-killing tumor cells as well as abundant source and convenient collection. Since the first case of umbilical blood transplantation was successfully practised to cure Fanconi anaemia in 1989, thegene transfer of umbilical blood stem cells has been noticed gradually. Now with their uncomparative virtues, umbilical blood stem cells are widely applied in the gene therapy of many diseases, such as tumor and hereditary diseases. In this experiment, RI gene was transfected by retrovirus carrier and highly expressed in purified human umbilical blood stem cells, in order to make the base of the anti-tumor method of stem transplantation (after selection, cultivation and proliferation, those stem cells with RI gene will be given back to tumor patients to control the growth and transfer of solid tumors by restraining the angiogenesis of tumor tissues.) This new kind of anti-tumor method has not been reported in the world.Objective: To study the transfection and expression of RI gene on human umbilical blood stem cells.Methods: (1)Extracting pLNCX-ri with the method of alkali separation; (2)Transfecting RI gene into package cells PA317 and measuring the positive cell clones with G418; (3)Measuringvirus titer with the target cells NIH3T3 and selecting the cells of high titer; (4)Separating human umbilical blood stem cells, enriching CD34+ cells with MACS and measuring the content of CD34+ cells with FCM; (5)Transfecting pLNCX-ri into CD34+ cells with retrovirus carrier, measuring the efficiency of transfection and selecting positive cell clones with G418; (6)Measuring the pLNCX-ri on chromosomes with PCR; (7)Measuring the expression quantity of RI gene with Western-blot after transfection.Results: (1)The purity of extracted pLNCX-ri was high, which was measured by agarose gel electrophoresis; (2)With 500μg/ml of G418,the positive cell clones were fuond;(3)The virus titer measured with NIH3T3 cells was 2.5×105CFU/ml;(4)The content of CD34+ cells measured with FCM was 96.15%; (5)With 500μg/ml of G418, the transfection efficiency of RI gene into CD34+ cells was 17.8%, and positive cell clones were found; (6)The pLNCX-ri on chromosomes was found by PCR; (7)The positive expression of RI gene was measured by Western-blot after transfection.Conclusion: The RI gene could be transfected into human umbilical blood CD34+ cells and the expression quantity was high.