The Effects On Protecting Hematopoietic Cells From Anticancer Agents In Vivo By MDR1-Transferred Into Cord Blood Mononuclear Cells

Objective: Leukemia is a disseminated non-solid malignancy of hematological system. The therapy of leukemia is different from that of solid malignancies. Leukemia cannot be excised by operation. Obviously chemotherapy is especially important to it. Chemotherapy is a typical, traditional, preferred and important treatment for leukemia but there are two barriers in it :one is the tumor cells resistance to anticancer agents, the other is the toxicity of chemotherapeutic agents to hematopoiesis. Some studies confirmed that the both barriers related to the expression of multidrug resistant gene (MDR1) and it's production P-glycoprotein (P-gp). The over expression of P-gp in tumor cells directly led to drug resistance of the cells and chemotherapy failure by refluxed anticancer agents from the cells; while it's low-level expression in hematopoietic stem cells indirectly led to chemotherapy failure by over accumulated drug in the cells and damaged hematopioetic cells and limited drug doseand course of treatment. We performed to transfer MDR1 gene into hematopoietic cells of human cord blood mononuclear cells (CBMNC) by a retrovirus-mediated vector that contains a full-length cDNA of human MDR1 gene and explored a secure, stable and efficient method that can transduce MDR1 gene into the cord blood hematopoietic cells. We assessed the security, feasibility and function expression of transduction MDR1 gene into cord blood mononuclear cells in vitro and provided conditions for protecting marrow hematopoietic cells from damage of the drug in vivo undergoing high-dose anticancer agent. Methods: MDR1 gene was transferred into mononuclear cells of human cord blood with plasmid pHaMDR1/A by the retrovirus- mediated. The expression of MDR1 gene in the cord blood mononuclear cells in vitro; the transferring positive rate on different transferring term and the P-gp function were tested in vitro by PCR, immunohistochemistry (IC) method and flow cytometry (DNR extrusion test) in the different level of gene, protein and molecule. The cell cycle of the CBMNC after transduction were tested by flow cytometry.Results: (1) A secure, stable and efficient method of transferring MDR1 gene into human CBMNC has been established successfully. (2)The effective integration of MDR1 gene in human CBMNC can be tested by PCR method. (3) 2 days,4days and 6days after transduction the transferring positive rates of CBMNC were 18%,29.83%,34.67%respectively by determining of IC method. (4) It was confirmed by DNR extrusion test that the product (P-gp) of transferred MDR1 gene maintained its biological function. (5) The cell cycle of transferred CBMNC conserved normal biological characteristics.Conclusion: It is secure and available that MDR1 gene was transferred into human CBMNC. The stable and efficient expression of MDR1 gene can be tested by in vitro. These data provide a foundation and evidence for the experiment on prevention marrow from toxicity of anticancer drug in vivo.