| Objective: Nowadays, malignant tumors are the common serious diseases which are threatening the health and life of human being. The combined therapy of malignant tumors includes operation, chemotherapy, radiotherapy, biotherapy, immunotherapy and so on. Among them, chemotherapy has an irreplaceable function on the prevention of turmor recur and metastasis. But it also has two disadvantages which influence the therapeutic efficacy. In clinical chemotherapy, the multidrug resistance of tumor cells and depression of bone marrow 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, therefore, they are particularly susceptible to be killed by MDR-sensitive drugs, which cause the serious bone marrow toxicity. In our study, we transferred the mdr1 gene into bone marrow mononuclear cells of male Balb/c mice by a retrovirus-mediated vector that contains a full-length cDNA of human mdr1 gene in vitro, then detected the expression of transferred mdr1 gene in bone marrow mononuclear cells. After that, we transplanted the hematopoietic cell with mdr1 gene into the female Balb/c mice with H22 liver cancer, to observe the influence to the hematopoietic system, the protection to bone marrow after overdosage chemotherapy and the curative effects, to investigate the expression, distribution and safety of the foreign mdr1 gene in vivo in gene therapy, and to study the enriching effect in the bone marrow and peripheral blood mononuclear cells.Methods: The retroviral packaging cell line PA317-HaMDR1/A containing human mdr1 gene was screened by colchicines; Viral supernatants were harvested from confluent layers of the cells and concentrated by ultracentrifugation and then the foreign mdr1 gene was transferred into the bone marrow mononuclear cells of male Balb/c mouse by co-culture with concentrated viral supernatant and cytokines. Then the transferred cells were transplanted to the female Balb/c with H22 liver cancer which had been pretreated by 60Co-γray, through the vena caudalis. After that, we cured the female Balb/c with H22 liver cancer with adriamycin, and detected the protection to bone marrow of mdr1 gene and the curative effects. At the same time, the effect of enrichment and distribution of foreign mdr1 in the bone marrow mononuclear cells, peripheral blood mononuclear cells, tumour and chief organs were detected by immunohistochemistry(IC), reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization(FISH) at the level of molecule and protein level; the homing rate of the bone marrow monitored by FISH and the planting rate of mdr1 gene in bone marrow tested by IC; the safety was detected by hematogenetic function and hematoxylin and eosin stain and electron microscope to inspect of chief organs.Results: (1) The efficient integration and expression of mdr1 gene in genome of bone marrow mononuclear cells can be tested by RT-PCR array; (2)The transduction efficiency of bone marrow expressing P-glycoprotein (P-gp) were 31.24% tested by IC array; (3)The percentage of P-glucoprotein expression of the bone marrow mononuclear cells and peripheral blood mononuclear cells were(9.36±1.84)% and(8.52±1.26)% respectively before chemotherapy, and it was confirmed that 6 week after chemotherapy mdr1 gene was expressing stably and effectively in bone marrow and peripheral blood mononuclear cells, As the dose of chemotherapeutant increased, the expression of mdr1 gene increased too; (4)There was no expression of the foreign mdr1 gene in chief organs and tumor tested by RT-PCR array and FISH; (5)In chemotherapy experiments, the mouse in mdr1-transferring group could be survival after treated with 3-fold dosage chemotherapy, meanwhile, the tumor were treated effectively, and the survival rate in this group was higher than the control group (P>0.05). The mouse in control groups were died of the severe bone marrow depression caused by over dosage chemotherapy; (6) White cell count in peripheral blood of the mouse in mdr1-transferring group is more than the control group, There was statistically significant between the two groups 2 weeks later after chemotherapy (P<0.01); (7)The homing rate of the bone marrow and the planting rate of mdr1 gene in bone marrow were respectively 61.6% and(9.73±1.64)% on first week after chemotherapy; (8)Hematoxylin and eosin stain and electron microscope manifested that except inflammatory exudation and edema in liver, lung and intestina parva in earlier period, no other changes were observed.Conclusions: By retrovirus vector the foreign mdr1 gene could be efficiently transferred into bone marrow mononuclear cells of mouse in vitro, and it was confirmed that mdr1 gene can be integrated into the genome of bone marrow mononuclear cells and expressed stably and effectively; There was stably and effectively expression of the foreign mdr1 gene in bone marrow mononuclear cells and peripheral blood mononuclear cells, but no expression in chief organs and tumor; The transferring mdr1 gene was no effect on hematogenesis function and chief organs; The enrichment of P-gp in bone marrow mononuclear cells and peripheral blood increased gradually along with intensified chemotherapy; The homing rate of mdr1 gene was high in the transplanted female Balb/c pretreated by 60Co-γray; The protection of bone marrow induced by transferred mdr1 gene was especially obvious in overdosage chemotherapy with asriamycin. |
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