Research Of Human Double-Mutant Dihydrofolate Reductase And Cytidine Deaminase Gene Transfected To Murine Bone Marrow Cell

IntroductionChemotherapy is one of significance methods to malignant tumour. Dosage multiplication of chemicals would eliminate undoubtedly and more availably tumor cells and its tiny residue in paracmasis. Thus , disease free survival was raised. But severe infection,hemorrhage originated by Myelosuppression induced by dosage multiplication of chemicals became a major factor of death. Therefore, increasing resistant of myeloid element to chemicals bocame a focus problem recently .For this reason , Resistant drug of bone marrow cells to chemicals obtained from resistant drug phenotype created by drug resistance gene( mdr1 ) transfected to bone marrow cells. some of uncertain problem limited clinical practice generally but it potentiality. glycosylated transmembrane protein encoded by multidrug resistance gene can pump chemotherapeutics off cells and then cytotoxicity diminish resulting from decreasing chemicals concentration . chemo protection of myeloid element transfected by mdrl was animal experiment and was not ideal in clinical practice.and bone marrow hyperplasy syndrome of mouse transfected by mdr1 became a big problem.So many drug resistance genes of chemicals were discoveried recently, such as : aldehyde dehydrogenase gene resist to cyclophosphamide,6-O-methyl guanine-DNA-methyl transferases gene resist to aldyl agent,thymine synase gene resist to 5-fluorouracil,dihydrofolate reductase gene resist to methotrexate,cytidine deaminase gene resist to arabinosylcytosin etc and having conclusion followed: ideal drug resistance gene should be provided with sorts of drug resistance no one single,unimmunogenicity,short theoretical gene fragment,little toxicity of chemicals corresponding to drug resistance gene,no mutating and cancerating of gene transfected. dihydrofolate reductase gene and cytidine deaminase gene to be provided with above-mentioned condition have been conformed.For the solution of myelosuppression caused by high dose chemicals ,Murine bone marrow cells transfected with DHFR and DHFR-CD passing incasing cellss PA317 , and Murine bone marrow cells transfected with DHFR-CD was transplanted to mouse and whose hemogram,body mass,survival rate,colony-forming unit-granulocyte macrophage and expression of drug resistance genes of mmurine bone marrow cells following treatment of high dose chemicals were obtained. Its objective is to evaluate resistance and chemo protection of murine bone marrow cellss transfected with DHFR-CD to high dose chemicals.Providing a animal experiment model of double drug resistence genes in clinical application in future.Methods1,Plasmid amplificationCompetent cells,plasmid of DHFR and DHFR-CD respectively and common nutritive medium mingled and shaked in centrifuge tube were spread on agar plate containing antibiotic, and then, placed upside down the agar plate after fluid absorbed. Its colony was inoculated in culture fluid to stay overnight,and then collecting bacterium sedimentum .Extraction and purification of plasmid was performed according to reagent instruction.2,Culture of cellsPA317 of Ambiphag incasing cells and NIH3T3 of murine fibroblast cells were cultured in DMEM containing 10% calf serum within 37℃,5%CO₂.PA317 will be used for transfection; NIH3T3 was used for titration of virus and detection of helper virus.3,Cells' transfection,clone's bolting and amplificationDrug resistance clone gained by drug bolting with diversity level of methotrexate or/and cytarabine from PA317 transfected by drug resistance gene containing DHFR or DHFR-CD with LipofectamineTM2000 and amplifiy drug resistance cells quantity. Addition to control transfection group without plasmid.4,Preparation of murine bone marrow cells5-Fu was injected to Balb/c mouse from vena caudalis, dying and soaking it with alcohol three days latter, suspension was made from murine bone marrow cells collected from femoral bone and shin bone of mouse, mononuclearcells separated with lymphocyte isolation medium was suspensed in plastic materia culture flask stuffing with 20%FCS RPMI1640,cultivating two hours in 37℃,5%CO₂, collecting suspended murine bone marrow cells.5,Infection of murine bone marrow cellsCounting murine bone marrow mononuclearcells and making the proportion of nucleated cells and virus production cells transfected is 1:1, culturing it within 10%WEHI containing 20% calf serum and 1% glutamine;control group with PA317 of transfection group without plasmid. Cells suspension made of murine bone marrow cells collected after co-culturing forty-eight hours in 37℃,5%CO₂ was used for colony forming unit-granulocyte macrophage cultivation.6,Cultivation of CFU-GM and detection of drug resistance gene of murine bone marrow cells transfected with DHFR andDHFR-CDFor detection of CFU-GM and drug resistance gene of metainfective murine bone marrow cells,which was divided two groups: experiment group with chemicals; control group without chemicals, the colony counted was analyzed after cultivating about twelve days; Integration of drug resistance gene was detected with PCR to DHFR group and RT-PCR to DHFR-CD group which extraction of RNA,reverse transcription of cDNA,amplification and electrophoresis was progressed according to agent instruction.7,Experiment of murine bone marrow cells transfected with DHFR-CD in vivoBalb/c mouse exposured to fatal dose cobalt-60 was divided to two groups randomly according to body mass: metainfective bone marrow cells was injected to experiment group mice through vena caudalis; control group mouse with murine bone marrow cells infected of control group in coordination amount. MTX and Ara-C was injected to intraperitoneal injection of mice every day for 4 days 4 weeks latter of transplantation, variation of hemogram,body mass and survival rate were obtained; One mouse of two groups respective was put to death after 5 weeks, the cultivation of CFU-GM and detection of drug resistance gene were done with the bone marrow cells.Results1,Stable virus producing cellsDrug fast clone had emerged in culture of transgenic PA317 by drug screening and its drug resistance gene was positive after plasmid amplification; NIH3T3 is used as target cells to determine retrovirus titer :virus titer of DHFR and DHFR-CD is 3.21×10⁴CFU / mL,3.35×10⁴CFU / mL respectively to 0.01 mL virus stock suspension; virus titer of DHFR and DHFR-CD was 1.43×10⁵CFU / L,1.54×10⁵CFU / L respectively to 0.10mL virus stock suspension;replicated virus was not found in detection of helper virus; It indicated that the precipitated retrovirus or PA317 transfected can be used to infect target cells.2,Experiment of murine bone marrow cells transfected withDHFR in vitro(1),Drug fast clone of experiment group and control group is 287 and 273 rspectively without MTX; the data is 42(14.6%) and 0 respectively with it in clone formation of drug resistance of murine bone marrow cells, clone frequency is significant between two groups(P<0.05),it demonstrated that murine bone marrow cells transfected with DHFR have resistance to MTX.(2),Electrophoresis of PCR product in experiment group was shown and control group was not. It indicated that the DHFR transfected had been integrated. 3,Experiment of murine bone marrow cells transfected withDHFR-CD in vitro(1),Drug fast clone of experiment group and control group is 307 and 296 respectively without MTX and Ara-C; the data is 47(15.3%) and 0 with them respectively, in clone formation of drug resistance of mouse bone marrow cells, clone frequency is significant between two groups(P<0.05),it demonstrated that mouse bone marrow cells transfected with DHFR-CD have resistance to MTX and Ara-C.(2),Electrophoresis of RT-PCR product in experiment group was shown and control group was not. It indicated that the DHFR-CD transfected had been expressed in molecular biology.4,Experiment of murine bone marrow cells transfected withDHFR-CD in vivo(1),Following high dose MTX and Ara-C to transgenic mice,whose data of body mass,white blood cell a began to drop in and have lower lamplitude change than control group; whose data of white blood cell recover to normal six weeks latter; whose survival rate was significant between experiment group 66.7% and control group 0,(P<0.01).(2),Drug fast clone of experiment group and control group is 167 and 173 respectively without MTX and Ara-C; two drugs;the data is 42(25.1%) and 0 with them respectively in clone formation of drug resistance of murine bone marrow cells, clone frequency is significant between two groups(P<0.05),it demonstrated that murine bone marrow cells transfected with DHFR-CD have resistance to drugs.(3),Electrophoresis of RT-PCR product in transgenic murine bone marrow cell group was shown and control group was not. It indicated that the drug resistance gene transfected had been expressed in molecular biology.Conclusions1,Stable cells line of produce viral can be obtained from PA317 transfected of plamid containing DHFR or DHFR-CD with engineering of liposome transfection.2,Drug resistance gene can be transfected to murine bone marrow cells by mean of co-culture and it can be expressed availably and present satisfactory resistance to drugs.3,Drug resistance gene transfected can be detected in murine bone marrow cells which was obtained from the mouse which had received murine bone marrow cells of transgenic mice by intravenous infusion and survival rate of the mice was higher than control mice, It indicated that the genes owned protection to chemotherapy in vivo of mice.