Myeloprotection Effect Of Sequential Transplantation Of Multidrug Resistance Gene And High-dose Chemotherapy

PART ONE RESEARCH ON THE TRANSDUCTING OFMULTIDRUG RESISTANCE GENE1ENCODED BYADENOVIRAL VECTOR INTO BONE MARROWMONONUCLEARS CELLS IN VITROObjective: In this study, recombinated adenoviral vector encodingMDR1(rAd-MDR1-GFP) was transducted into bone marrow mononuclearcells (BM-MNCs) of New Zealand rabbits in vitro to establish a stable andefficient method for the transduction of rAd-GFP-MDR1, to explore therelationship between transduction time, transduction efficiency and functionof MDR1to obtain potimal transduction conditions under which the highestcell transduction rate and the highest cell survival were ensured. Results ofthis part supplied basis for further study of the BM-MNCs-MDR1-GFPsequential transplantation in protecting bone marrow function oftumor-bearing animals from damage of high-dose pharmorubicin (EADM)in vivo.Methods:(1) Recombinated adenoviral supernatants were harvestedand concentrated by infection each other like Ping-Pong with package cell HEK293;(2) Fluorescence microscope was used to observe the transductedBM-MNCs which express green fluorescence protein (GFP). Flowcytometry (FCM) was used to detect the transduction efficiency;(3) Thetranscription of MDR1was detected by reverse transcription polymerasechain reaction (RT-PCR);(4) Immunofluorescence and Western blottinganalysis was used to detect the expression of P-glycoprotein (P-gp);(5) Thedrug-excretion activity of MDR1was determined by daunorubicin (DNR)efflux assay;(6) The drug sensitivity of BM-MNCs-MDR1-GFP topharmorubicin (EADM) and vincristine (VCR) were detected byMethyl-Thiazolyl-Tetrazolium (MTT) assay;(7) MTT assay was used todetect the survial rate of BM-MNCs-MDR1-GFP;(8) FCM was used todetect the cell cycle of BM-MNCs after been transfected withrAd-MDR1-GFP.(9) Methyl-cellulose Semi-solid medium Colony culturewas used to detect the colony-forming ability of hematopoietic stem cells inBM-MNCs-MDR1-GFP.Results:(1) The viral titers of rAd-MDR1-GFP was3.5×109plaqueforming units(PFU)/mL;(2) Successfully established a stable and efficientmethod for the transduction of rAd-MDR1-GFP into BM-MNCs and thetransduction efficiency was35%-45%. The survival station, survival rateand transfection efficiency of BM-MNCs were well at the4thtransfected day;(3) The transduction of rAd-MDR1-GFP could increase the expression ofMDR1significantly on day4revealed by RT-PCR, ICH and Western Blot;(4)DNR efflux assay confirmed that foreign MDR1had drug efflux pumpfunction;(5) The drug resistance index to EADM and VCR increased to21.1102-folds and8.4786-folds, respectively, for BM-MNCs-MDR1-GFP,compared with BM-MNCs-PBS and BM-MNCs-GFP;(6) The result ofMTT indicated that the transduction of rAd-MDR1-GFP did not influence the survival and proliferation of BM-MNCs;(7) The results of FCMrevealed that the transfection of rAd-MDR1-GFP did not influence the cellcycle of BM-MNCs;(8) The results of Methyl-cellulose Semi-solid mediumColony culture indicated that the transduction of rAd-GFP-MDR1did notinfluence the colony-forming ability of hematopoietic stem cells inBM-MNCs-MDR1-GFP.Conclusions: Recombinated adenoviral vector encoding MDR1couldbe transducted into BM-MNCs with a high transduction efficiency. ForeignMDR1could express in BM-MNCs functionaly, and the transduction ofrAd-GFP-MDR1did not influence the survival, proliferation andcolony-forming activity. All of the data above provides foundation andevidence for the following study of the BM-MNCs-MDR1-GFP sequentialtransplantation in protecting bone marrow function of tumor-bearinganimals from damage of high-dose EADM in vivo. PART TWO LONG-TERM MYELOPROTECTION OFBM-MNCS-MDR1-GFP SEQUENTIAL TRANSPLANTATIONTO TUMOR-BEARING RABBITS UNDERGOING HIGH-DOSECHEMOTHERAPYObjective: Exploring the long time bone marrow protection ofBM-MNCs-MDR1-GFP sequential transplantation to the VX2hepatocarcinoma bearing New Zealand rabbits undergoing high-dosechemotherapy.Methods:(1) The animal model with VX2hepatocaricoma wasestablished by implantation of VX2block in liver. The echo, size andmetastasis of tumor were monitored by Ultrasound examination regularly;(2)BM-MNCs-MDR1-GFP was transplanted into VX2hepatocarcinomaanimal model successively, which had been pretreated by high-dosechemotherapy of cyclophosphamide (CTX), after beening co-cultured withthe concentrated adenoviral supernatant and cytokines for4days, whichcontained a full-length cDNA of human MDR1gene;(3) High-dosechemotherapy of EADM was executed after each transplantation ofBM-MNCs-MDR1-GFP, and the hemogram of VX2hepatocarcinomaanimal model was monitored every week after high-dose chemotherapy ofEADM to observe the myeloprotection of the extogenic MDR1. Ultrasonicexamination was executed every week to observe the killing function of high-dose chemotherapy to malignant tumor cells and to observe the harm ofhigh-dose chemotherapy to non-hematopoietic systems after high-dosechemotherapy;(4) Bone marrow and peripheral blood MNCs of testedanimal model were got to detect the functional expression of extogenicMDR1by RT-PCR and FCM after each transplantation ofBM-MNCs-MDR1-GFP.Results:(1) The VX2hepatocarcinoma animal model was establishedsuccessfully and the rate of which was100%. The growth and developmentregularity of VX2hepatocarcinoma was mastered.(2)BM-MNCs-MDR1-GFP was transplantated into tested tumor bearinganimal model successively and successfully by scheduled bone marrowtransplantation protocols;(3) In experiments of high-dose chemotherapy ofEADM, the white blood cell count of peripheral blood of rabbits in testedgroup could be maintained on a higher level, the tumor cells and cancerstem cells could be killed effectively (P﹤0.05), the survival time and thehealing rate of the tested group were significantly improved compared withthe controll group (P﹤0.05). And the rabbits in control groups werepredominantly died from severe bone marrow depression caused byhigh-dose chemotherapy and/or deterioration of tumor. However, the severelesions to non-hematopoietic systems caused by high-dose chemotherapywere observed;(4) The results of RT-PCR and FCM indicated that theextogenic MDR1encoded by recombinated adenoviral vector could implant into bone marrow and express functionally for about5-6weeks.Conclusions:Sequential Transplantation of BM-MNCs modified byadenoviral vector encoding extogenic MDR1could protect thehematopoietic function of bone marrow of tumor bearing animal enduringhigh-dose chemotherapy for a long-term effectively. Key Words: multidrug resistance gene1; bone marrow sequentialtransplantation; high-dose chemotherapy; myeloprotection; cancer stemcell. PART THREE THE KILLING FUNCTION OF HIGH-DOSECHEMOTHERAPY ON CANCER STEM CELLSObjective: Exploring the killing function of high-dose chemotherapyon cancer stem cells.Methods: The amount of cancer stem cells was detected in the residualtumor tissue after the death of the tested hepatocarcinoma animal byimmunohistochemistry (IH), immunofluorescence (IF) and confocal laserscanning microscope.Results:The results of IH and IF indicated that high-dosechemotherapy could kill cancer stem cells significantly compared withgeneral-dose chemotherapy (P﹤0.01).Conclusions: High-dose chemotherapy could kill cancer stem cellssignificantly and this may be one of the mechanism of high-dosechemotherapy in preventing the relapse and the metastasis of malignanttumors more effectively.