| Objective (1) To investigate whether the multidrug resistance gene-1(mdr-1) in the Ad-EGFP-MDR1transferred bone marrow cells (BMC) hadfunctional expression in vitro, and whether the cells could tolerateoverdose mycophenolate mofetil (MMF), so as to provide foundation andevidence to the next study in vivo.(2) To found a way to establish the livertransplantation (LT) and acute rejection (AcR) model liver cancer modeland to investigate whether the mdr-1gene the Ad-EGFP-MDR1transferredbone marrow cells (BMC) could stably express mdr1gene in it.(3) Toprove after the transplantation of the mdr-1gene transferred BMC to thismodel, the recipients could burden the overdose MMF therapy, the growthof the tumor was inhibited.Methods (1) First, we transferred the Ad-EGFP-MDR1to the bonemarrow mononuclear cell (BMNC) of the Brown Norway (BN) rats. Then,we detected the survival rat of the BMMC; using RT-PCR, Western-blot and daunorubicin (DAM) methods to detect the functional expression ofmdr1gene; At last, we detected the survival rat of the transferred BMMCtreated by different dose MMF by MTT.(2) Transplant the mdr1genetransferred BMNC to the BN recipients three days before livertransplantation. Before this operation, we used60Co-γradiation exposureto empty the bone marrow niche.(3)Three days after the transplantation ofBMC, we found the Lewis→BN LT and acute rejection AcR rat model;Meanwhile, we planted the H22cells under the capsule of transplant liver.(4) Divide BN rats into5groups at random: A untreated groups (no BMC,no MMF); B routine dose groups (no BMC, routine dose MMF); C overdose groups (no BMC, over dose MMF); D BMC transplantation and overdose groups (BMC but without mdr1, over dose MMF); E mdr1-BMCtransplantation and overdose groups (mdr1-BMC transplantation and overdose MMF).(5)1month after the BMC transplantation, we used RT-PCR,Western-blot and DAM methods to detect the functional expression ofmdr1gene in the BMC of the survival rats.(6) Collecting the peripheralvein blood of the survival rats, then counted the red blood cell (RBC),white blood cell (WBC) and platelet (PLT), to judge the hemopoiesisability of the bone marrow.(7) Observing the survival condition of the ratsto draw the survival curve; measuring the mix and max diameter tocalculate the tumor weight.(8)3,5and7days after the operation, wecollected the venous blood and the fresh liver tissue of the survival rats, to analysis the alanine aminotransferase (ALT), aspartate aminotransferase(AST) and total bilirubin (TBIL), to judge the liver function. The livertissues were treated by H-E staining to observe their pathology changes,and then we used Banff RAI criteria to judge the degree of the acutereaction (AcR).Results (1) The transfection efficiency of the virus was30.14%, andthe survival rate of the BMC was89.25%.(2) There were obviousfunctional expression of the mdr1gene in the mdr1gene transferred BMC,but none of other groups; the P-gp expressed by mdr1gene has pumpingfunction.(3) The result of the MTT demonstrated that the BMC transferredby mdr1gene can bear the burden of the different dosage of MMF, therewere no obvious difference of their survival rates (P>0.05).(4) There stillhad functional expression of the mdr1gene in the BMC in the E groups,but didn't found any expression in other groups.(5) The combination ofLewis→BN rats LT and the meanwhile plantation the tumor cells underthe skin can help us establish a correct and steady LT and AcRtumor-bearing rat model.(6) The average live time of the E group(42.20±12.44days) was obviously longer than the other groups'(P<0.05);The average live time of the C and D group were the shortest one(8.60±5.72,10.20±4.28days).(7) The tumor weight of the C, D and Egroup were lighter than other groups'(P<0.05).(8) The WBC, RBC andPLT average counting number of the E group were obviously higher than A and B groups'(P<0.01), and after the termination of the chemotherapy, ithad a tendency to normal.(9) The ALT, AST and TBIL of thechemotherapy (B, C, D and E) groups were higher than A group's (P<0.05),and the over dosage groups'(C, D and E) were higher than B group's(P<0.05).(10) There were no obvious difference of the RAI value of A, B,and E groups (P>0.05) at three days after LT; But the RAI value of E and Bgroups were higher than A group's (P<0.05) at five and seven days after LT,and the E group's was lower (P<0.05); the RAI value of A group had atendency to rise, but nor with the E group's.Conclusions (1) The exogenous mdr1gene could be transferred intoBMC of BN rats efficiently by vecter of adenovirus in vitro, and there werefunctional expression of mdr1gene in these cells; these transferred BMCscan tolerate the influence of over dosage MMF in vivo.(2) Sequential bonemarrow transplantation can transplant the BMC in to BN rats successfully;The combination of Lewis→BN rats LT and the meanwhile plantation thetumor cells under the skin, can help us establish a correct and steady LTand AcR tumor-bearing rat model; The expression of mdr1gene can betested in the rats of the above two models combination.(3) Thehemopoiesis ability of the receptors' bone marrow could be protected fromtoxic effects of over dosage MMF after transplantation of bone marrowmononuclear cells transferred with mdr1gene, and the live time of thereceptors could be extended; AcR and the growth of the tumor can be inhibited by short term over dosage MMF chemotherapy. |
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