Mdr1 In The Efficacy Of Cancer Chemotherapy And Bone Marrow Protection Of Experimental Research

Objective: Cloning the target gene MDR1 into the plasmid vecter of adenovirus,and transfecting the plasmid vecter of adenovirus into the package cell HEK293, finally,generate the recombinant adenovirus expressing the target gene.Methods: Cloning the target gene in vitro by PCR and intergrating it into the shuttle vecter of pAdTrack-CMV by restrict clone , and recombinating it with adenovirus skeleton vecter Adeasy-1 by homologous recombination. Finally,the product were transfected into package cell HEK293 by lipofectamine,the recombinant adenovirus expressing the target gene be generated.Results: (1) Cloned the MDR1 gene in vitro and verified the true result by gene screening, the recombination plasmid pAdTrack-CMV were constructed successfully. (2) Intergrated the MDR1 into the plasmid vecter of adenovirus and result the recombinant ad-plasmid Adeasy-MDR1, Transfected the Adeasy-MDR1 into the package cell HEK293 and generated the high titer adenovirus expressing the MDR1. (3) The titre was 8.3×10¹¹pfu/ml.Conclusion: Cloned the exact MDR1 gene and generated the recombianant adenovirus expressing the target gene MDR1, made a real good basic for further research. Objective: Explore a perfect, stable and efficient method for exogenous mdr1 transduction to mononuclear cells of mouse. 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 chemotherapeutics agents.Methods: Viral supernatants were harvested and concentrated by infection each other like ping-pong. The foreign mdr1 gene was transferred into the bone marrow mononuclear cells of mouse of C57 and Balb/c by pretreatment with suitable TNF-α. The functional expression of mdr1 gene in bone marrow mononuclear cells were detected by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and daunorubicin (DNR) efflux assay in the different aspects. The relationships among transferring time, transduction efficiency and functional expression were researched .Results: (1) A stable and efficient method of transferring mdr1 gene into bone marrow mononuclear cells of mouse has been established successfully,(2) The transduction efficiency to bone marrow mononuclear cells of mouse was 26.26% tested by flow cytometry(FCM),(3)It was confirmed by DNR efflux assay that the product (P-glycoprotein) of transferred mdr1 gene maintained its biological function,(4) The efficient expression of mdr1 gene in bone marrow mononuclear cells can be tested by RT-PCR method, (5)Expression of P-glycoprotein was up obviously after transduction detected by Western-blot.Conclusions: The exogenous mdr1 gene could be transferred into bone marrow mononuclear cells of mouse efficiently by vecter of adenovirus 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 chemotherapy drugs in vivo . Objective: To research on the myeloprotection and curative effects in over-dosage chemotherapy of exogenous mdr1 gene after it was transferred into the bone marrow mononuclear cells which were transplanted into C57 with Lewis lung cancer and Balb/c with 4T1 breast cancer, Moreover, the function of the exogenous mdr1 gene in receptor was detected.Methods:The bone marrow mononuclear cells of mouse had been co-cultured with the concentrated recombinant adenoviruses supernatant which contained a full-length cDNA of human mdr1 gene for 4 days, After the C57 with Lewis lung cancer and Balb/c with 4T1 breast cancer had been pretreated by 60Co-γray, the transferred cells were transplanted by intra–bone marrow injection(IBMI). In over-dosage chemotherapy experiment with pharmorubicin, the myeloprotection of mdr1 gene and the curative effects to C57 with Lewis lung cancer and Balb/c with 4T1 breast cancer were detected.The functional expression of mdr1 gene in bone marrow mononuclear cells of receptors were detected by RT-PCR and Western blot in the different aspects. Results:(1) The animal model of C57 with Lewis lung cancer and Balb/c with 4T1 breast cancer were established successfully by tumor cells planting method, (2) The mdr1-transferred bone marrow mononuclear cells were transplanted into C57 with Lewis lung cancer and Balb/c with 4T1 breast cancer by intra–bone marrow injection(IBMI), and the functional expression of the exogenous mdr1 gene were detected in bone marrow mononuclear cells of receptors, (3) In chemotherapy experiments, the mouse in mdr1-transferring group could be survival after treating with 3-fold dosage chemotherapy, and the white blood cell count in peripheral blood was in normal range. Meanwhile, the tumor were treated effectively, and the survival time and quality in the group were improved (P﹤0.05). The mouse in control groups were died of the severe bone marrow depression caused by over dosage chemotherapy or deterioration of the tumor.Conclusions:The bone marrow of the receptor could be protected from toxic effects of chemotherapy after transplantation of bone marrow mononuclear cells transferred with mdr1 gene, and functional expression of the mdr1 gene could be detected, And the malignant tumor cells could be killed effectively by over dosage chemotherapy.