The Safety Of Bone Marrow Transplantation With Mdr1 Gene Transfection In Mice

Objective: Malignant tumors have been becoming one disease which threatening human death seariously. Though there has been making great progresses in tumor therapy,combined therapy consummating,new therapy emerging continuously,the percentage of long-term surviving in children who have got malignant tumor is only 60~70%.Chemotherapy plays a great role that can't be replaced in preventing tumor recurrence and metabasis.There are two obstacles severely influencing chemotherapy's curative effect:(1)tumor cells generates resistance to the drug,leading to tumor cells insensitive and resulting in chemotherapy failure;(2)while chemotherapeutics killing tumor cells it also causes grave bone marrow depression,and owing to patient not enduring chemotherapy schedule can't be enforced and eventually the therapy fails.It is already certified that there are intimate relevance between multidrug resistance gene 1 and tumor drug resistance. Highly expressing mdr1 gene,tumor cells is insensitive to chemotherapeutics and can't be killed efficaciously;in contrast marrow bone cells expressing mdr1 gene lowly are sensitive and vulnerable to chemotherapy drug.In our study, the mdr1 gene was transferred into bone marrow mononuclear cells of mouse by a retrovirus-mediated vector that contains a full-length cDNA of human mdr1 gene in vitro,after the transfection we assess the functional expression of transferred mdr1 gene in bone marrow mononuclear cells,and we set up a model in mouse to survey influence of hematopoietic cell transfected mdr1 gene impacting on the receptor's hematopoietic recovery and the changes in hematopoietic cell itself,so as to monitor the safety of influences on mouse hematological system by hematopoietic cell which was transfected by mdr1 gene mediated with retrovirus when the cells transplanted back into the body of mouse.Methods: The retroviral packaging cell line PA317-HaMDR1/A containing human mdr1 gene was screened by colchicines (120ng/ml); viral supernatants were harvested from confluent layers of the cells and concentrated by ultracentrifugation and the foreign mdr1 gene was transferred into the bone marrow mononuclear cells of mouse by co-culture with concentrated viral supernatant and cytokines;adopting RT-PCR assay, immunohistochemistry(IC) assay to assess the expression level in molecule and protein level;in marrow pretreatment procedure we apply 1.5 Gy,3.0 Gy and 6.0 Gy three different dose of 60Co-γin irradiation and then survey variation of the hematopoiesis recovery in every group;observe the hematopoiesis in Balb/c mice which were transplanted with haemopoietic cell transfected by mdr1 gene,and adopt immunohistochemistry assay and RT-PCR assay to monitor the expression of mdr1 gene,and at the same time we detect expression of some important multiplication and apoptosis factors in bone marrow MNC,which include bcl-2,Bax,Ki-67,p53 and PCNA.Results: (1) After screened by colchicines, the viral titre of supernatant was raised for 1.49-fold(1.71×106( CFU/ml ) /1.15×106(CFU/ml)),and raised for 2.12-fold(3.63×106(CFU/ml)/1.71×106( CFU/ml ) )by centrifugalization concentration; (2)The efficient integration and expression of mdr1 gene in genome of bone marrow mononuclear cells can be tested by RT-PCR array;(3) The transduction efficiency of bone marrow expressing P-glycoprotein (P-gp) were 22.40% tested by IC array;(4)after irradiation all of the 6.0Gy group mice were dead in 9 days and at the third day the minimum percentage of bone marrow cell in 1.5Gy group and 3.0 Gy group were 42.1% and 26.7% to the blank group,but at the ninth day the quantitative of BMC doubled 3.9 times and 2.5 times respectively to the minimum of BMC in 1.5Gy group and 3.0 Gy group in nine days;(5)in one month after transplantation there was no significant difference between the group transplanted with hematopoietic cell without transfction and the group transplanted with hematopoietic cell which was transfeced with mdr1 gene(P>0.05),both recoverying to nomal level of mouse peripheral blood leucocyte(PBL),but there appeared difference between these two group 2 months after transplantion,while the former PBL was 7.42×109/L and the latter 9.06×109/L ,there exsisted apparently difference in statistics analysis(P<0.01);(6)in body of the receptor mice which had been transplanted with mdr1 gene transfected MNC ,their bone marrow MNC's P-gp expression percentage were respectively 7.93% and 6.54% at 1 month and 2 month after transplantation;(7)there was no obviously differences in bone marrow MNC's multiplicaiton factor(MF) between the mice which transplanted with MNC transfected by mdr1 gene and the blank mice's,and so does the apoptosis factor P53,but the expression of Bax obviously declined in the experimental group,leading to the ratio Bcl-2 to bax much higher than the blank group.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 was expressing stably and effectively in cells; in short terms there is no significant influence on the bone marrow MNC by transfection of mdr1 gene,but in the long run(in our study we had surveyed 2 months),there is a decline in the expression of apoptosis factor Bax in the MNC which transfected by mdr1 gene,in favour of the recovery of the receptor mouse;on the other hand it also poses a blance problem between multiplication and apoptosis in the process of receptor hematopoiesis after transplanting HSC which was transfected with mdr1 gene.