Mdr1 Gene Protection And Treatment Of The Role Of Bone Marrow In The Rabbit Vx2 Carcinoma Chemotherapy

Objective: Nowadays, malignant tumors,the common serious diseases,threaten the health and life of human being, and they have been the second reason to cause death. The therapeutic alliance for the tumors including operation, chemotherapy, radiotherapy, biotherapy, and immunotherapy is necessary. Chemotherapeutic medicines are injected into the vessels and produce a marked effect, and the small metastatic sites all over the body as well as the original sites of the tumors are eliminated. So metastasis and relapse of the malignant tumors are prevented. In clinical chemotherapy, the multidrug resistance of tumor cells and bone marrow depression caused by chemotherapeutic medicines are the main obstacles to healing of tumors, and both aspects are related to mdr1 gene. It has been proven that the over expression of mdr1 gene in tumor cells is the main reason to cause the multidrug resistance of the malignant tumor cells, so they could not be killed effectively. However, normal bone marrow cells have little or no expression of the mdr1 gene and, therefore, are particularly susceptible to killing by MDR-sensitive drugs, which cause the serious bone marrow toxicity. In the study, the mdr1 gene was transferred into bone marrow mononuclear cells of New Zealand rabbits by a retrovirus-mediated vector that contains a full-length cDNA of human mdr1 gene in vitro, and to explore a perfect, stable and efficient method for mdr1 transduction. The functional expression of transferred mdr1 gene in bone marrow mononuclear cells was assessed. The results supply basis for further study in vivo of protecting bone marrow from damage of high-dose anticancer agents. Methods: The retroviral packaging cell line PA317-HaMDR1/A containing human mdr1 gene was screened by colchicines (90ng/ml), and viral supernatants were harvested from confluent layers of the cells and concentrated by ultracentrifugation. The foreign mdr1 gene was transferred into the bone marrow mononuclear cells of rabbits by co-culture with concentrated viral supernatant and cytokines. The chimerism and functional expression of mdr1 gene in bone marrow mononuclear cells were detected by polymerase chain reaction (PCR), Streptavidin-Peroxidase immunohistochemistry (IC) staining and daunorubicin (DNR) efflux assay in the different aspects. The relationships among transferring time, transduction efficiency and functional expression were researched .Results: (1) After screened by colchicines, the viral titre of supernatant was raised for 6.67-fold; (2) A stable and efficient method of transferring mdr1 gene into bone marrow mononuclear cells of rabbit has been established successfully; (3) The efficient integration of mdr1 gene in genome of bone marrow mononuclear cells can be tested by PCR method; (4) The transduction efficiency of 2 days, 4 days and 6 days were 22%, 35% and 37% respectively tested by IC method; (5) It was confirmed by DNR efflux assay that the product (P-glycoprotein) of transferred mdr1 gene maintained its biological function.Conclusions: The foreign mdr1 gene could be transferred into bone marrow mononuclear cells of rabbit efficiently by retrovirus vector in vitro, and the stable and efficient expression of mdr1 gene in cells could be tested. These data provide a foundation and evidence for the further research on protecting bone marrow from toxicity of anticancer drugs in vivo .