Transfection And Expression Of HRI Gene On Human Umbilical Blood Stem Cells And Gene Therapy For Mouse Melanoma

ObjectiveRibonuclease inhibitor (RI) is an acidic cytoplasmic glycoprotein , and it can inhibit the activity of ribonuclease A(RNase A). RI also can inhibit Angiogenin (Ang) strongly. Ang is produced and secreted by tumor cells and normal cells, and it has a robust capacity to induce blood vessel formation in vivo and in vitro. Because RI can form the tight combination with Ang, so it can inhibit the activity of Ang.Many studies demonstrated that angiogensis, the growth of new vessculature, is an essential condition for the development of tumors and their metastatic dissemination. And exogenous restrictions on angiogenesis severely impair tumor growth and sometimes lead to a complete regression. Nowadays, many angiogenetic inhibitors havebeen found to inhibit angiogenesis in vivo and in vitro experiments. The findings of these specific endothelial inhibitors provide an important therapy method for tumors and other angiogenesis-depend diseases. But one shortage of these inhibitors is that only relatively high concentration can inhibit tumor growth effectively. But the complicated purification process limits their clinical application. Gene therapy can resolve this problem in certain degree. Hematopoietic cells are favorable target cells for gene therapy. Applying hematopoietic cells as carriers of the angiogenetic inhibitor can bring new hope for tumors and other angiogenesis-depend diseases. Our research group have explored many studies about the anti-tumor activity of RI, and have conformed its partial inhibition to tumor growth.The purpose of this study is to introduce human ribonuclease inhibitor gene into human CD34+cells using retrovirus vector system and induce its high expression in cells. At the same time, this study also explored the infection efficiency of human CD34+ cells and demonstrated the reliability of this infection system. Further investigation is to transplant the infected human CD34+ cells into Y-ray radiated mice and hope ri gene can be expressed for a long time in the body of mice and the expressed RI can inhibit the growthof melanoma implanted in the mice by inhibiting angiogenesis.Materials and MethodsThe study is composed of three parts:1. Establish retrovirus packaging cells and help virus analysis. The recombinant vector carrying human ribonuclease inhibitor gene, pLNCX-ri was transfected in retrovirus packaging cell~PA317 using the lipofect AMINE-mediated method. After selection in the presence of G418 in culture medium, cell clones were obtained. The titers of virus were measured using mice fibroblast NIH3T3. And conduct help virus analysis of the transfection system. No replication-competent virus could be tested in the supernatant of the transfected NIH3T3 cells.2. The retrovirus mediated hRI genes transfected CD34+ cells in vitro and analysised the infection efficiency. Separating human umbilical blood stem cells, enriching CD34+ cells with MACS and measuring the content of CD34+ cells with flow cytometry. Virus fluid infected CD34+ cells which were dealed with different cytokine groups. Calculating the cell cloning formations and PCR analysis. Comparing CD34* cells transfection efficiency and expending efficiency in vitroin different cytokine groups: IL-3 and IL-6, IL~3 and SCF, IL-6 and SCF, IL-3^ IL-6 and SCF and comparison group.3. Analysis the expression of retrovirus mediated hRI gene in vivo and its effect on the growth of B16 melanoma cells in C57BL mice. CD Implanting CD34" cells transfected with RI gene into radiated mice, and analysis the expression of RI gene in mice blood by ELISA assay. The expression of RI in bone marrow, liver, spleen, thymus and lymph nodes was identified by PCR, RT-PCR, Western-blot and immunofluorescence assay respectively. (2) To demonstrate the anti-tumor activity of RI, melanoma cells were implanted subcutaneously into four groups of radiated mice: radiated without bone marrow remove group, remove with uninfected human CD34+cells group, remove with blank vector infected human 0034*06118 group and remove with RI infected human CD34" cells group. Comparing mice mortality, tumor formation rate, tumor formation time and the weight of tumors. The density of blood vessel was compared by immuno-histochemistry and the tumor cells apoptosis was compared with flow cytometry.Results1. The recommbinant vector carrying human ribonuclease inhibitor gene, pLNCX-ri was transfected in retrovirus packaging cell-PA317. After selection in the presence of G418 in culture medium, cell clones were obtained. The titers of virus were measured using mice fibroblast NIH3T3. The highest titer of virus carrying RI gene and blank vector is 1. 5X 10sCFU/ml and 0. 6X 105CFU/ml respectively. Help virus analysis of the transfection system demonstrated that no replication-competent virus could be tested. RI gene had been integrated on genome of NIH3T3 cells and had stable expression, which is identified by mRNA expression analysis, electrophoresis and RT-PCR on RI gene in virus infected NIH3T3 cells.2. Detect the transfection efficiency of RI gene in CD34' cells by colony assay. The transfection efficiency in IL-3 and IL-6 group, IL-3 and SCF group, IL-6 and SCF group and no cytokine group is 19. 6%, 25.5%, 17.8%, 1% respectively. And the transfection efficiency in IL-3, IL-6 and SCF group is 35%. Using PCR the transfection efficiency in IL-6 and SCF group and IL-3, IL-6 and SCF group is 18. 2% and 40%. In IL~3, IL-6 and SCF group, the transfection efficiency and amplification efficiency is obviously higher than other four groups.3. After implanting CD34+cells transfected with RI gene 4 days, the expression could be detected. But then the content began todecrease. On the 19th day, the lowest content is 0. 169 u g/ml. Then the content of RI began to increase and reached 0. 249n g/ml on the 32nd day. After that the content began to decrease again and dropped to 0. 157 u g/ml on the 59lh day.4. After implanting CD34+cells transfected with RI gene 30 days, RI expression was detected in all murine bone marrow by RT-PCR. The expression efficiency in peripheral blood , spleen, lymph nodes and thymus is 50%, 40%, 43% and 38% respectively. But in liver, the expression efficiency is the lowest, which is about 20%.5. Results of PCR indicated that RI could be expressed in all murine bone marrow. The expression efficiency in peripheral blood, spleen, lymph nodes, thymus and liver is 33%, 32%, 36%, 28% and 15% respectively.6. Immunofluorescence showed the expression of RI in bone marrow and peripheral blood. But it is more obvious in bone marrow.7. Western-blot can detect the expression in bone marrow, peripheral blood, spleen and liver.8. The mortality of the radiated mice is lower in implantation group than in nonimplantation group. The mortality of mice is the lowest in implantion CD34+cells which were transfected with RI gene.9. Mice in transfected group developed smaller tumors than other three groups. In that group, the growth of tumor was slower and the tumor formation efficiency was also lower.10. The results of immunohistochemistry assay showed that the number of blood vessel in nonimplantation group, nontransfection group and transfected with blank vector group was 92 ±8. 94, 89± 6. 49 and 92±8. 93 respectively. But in the transfected with RI group the number of blood vessel was 31 + 5.44.11. Detecting the apoptosis of tumor cells by flow cytometry, the results showed that the apoptosis increased in transfected with RI group.Conclusions1. The recombinant vector carrying human ribonuclease inhibitor gene, pLNCX~ri can be transfected in retrovirus packaging cell~PA317, and the highest titer of virus earring RI gene is 1. 5X KTCFU/ml. The transfection system is safe and is free of help virus.2. Transfecting CD34+ cells with different cytokine combination, the transfection efficiency and amplification efficiency are different. In IL~3, IL-6 and SCF group the transfection efficiency and amplification efficiency have been elevated.