The Tansduction And Evaluation Of MDHFR Drug Resistant Gene In SCID-hu Mice

Chemotherapy is one of the curative treatment modalities for several types of tumors, which has resulted in substantial improvement in the outcome of some cancer patients. However, systemic chemotherapy of malignant disease is usually limited by myelosuppression because of the nonselective action on both tumor cells and normal cells. Gene therapy by transferring specific drug-resistance genes into hematopioetic progenitors may be a useful approach in ameliorating chemotherapy toxicity on bone marrow, which will allow higher than conventional doses of chemotherapeutic agents to be administered safely. In addition, It is also possible that patients are rescued from potentially lethal side effects of high doses of drug and cured completely.Thus far, one of the major troublesome problems in human drug-resistance gene therapy are the lack of animal model to assess the safety and efficiency of drug-resistance gene therapy involving in human hematopoietic stem cell in vivo. To circumvent the drawback, the research work is mainly focusing on the parts as follows.In order to improve transduction efficiency by retroviral vector, we firstly conducted research on purity and expansion of human hematopoietic stem cells in vitro. The CD34+ cells from mobilized peripheral blood and umbilical cord blood separated by anti-CD34+ miniMACS system have been shown that the purity of the CD34+ cells could reach 82.9%-97.0%(average: 91.8%) and yield rate is 65.7% ,while the purity of CD34+cells selected by Panning only 21.7%. The data demonstrated that the miniMACS system is better than the latter.To elucidate the role of recombinant human hematopoietic growth factors (HGF_S) in expansion of umbilical cord blood(UCB) CD34+cells, the effects of different combinations of GF_s(including SCF, 1L-3,IL-6,G-CSF, EPO)on the proliferation and differentiation of those cells were studied in liquid culture system .All kinds of combinations of GF_S could increase expansion of total hematopoietic cells, but, a combination of five GF_S( including SCF+IL-3 + IL-6+G-CSF+EPO) was identified as the most potent combination tested. However, in those combination adding specific-lineage growth factor, expansion of progenitor colony forming decrease dramatically in two weeks, regimens without specific-lineage growth factor, such as SCF/IL-3/IL-6, could maintain expansion of hematopoietic progenitor longer. The expansion reach 25 folds in the third week in SCF/IL-3/IL-6 group. Similar results were obtained from expansion of mobilized peripheral blood (PB) CD34+ cells. However, Comparative study showed that the expansion of total cells and progenitors from UCB CD34+ cells is 1.7-2.7 and 1.4-2.5 times higher than that one from PB CD34+ cells in one week to three weeks. And this result imply that UCB stem cells have the higher proliferation potential...