1. The Safety Research Of Retroviral Vector Which Carried MDR1 Gene 2. Experimental Research On The Over Dose Adriamycin Chem Combined With All-Tran-Ratinoic Acid To Mice With Liver Cancer

THE SAFETY RESEARCH OF RETROVIRAL VECTOR WHICH CARRIED MDR1 GENEObjective : To detecte wether there have producted replication -competent retroviral before or after the MDR1 gene which carried by retroviral vector transfect into BM-MNCs,and the BM-MNCs transfected with MDR1 gene import into mice, and observe wether the BM-MNCs transfected with MDR1 gene have oncogenicity,in order to offer a experimental support for retrovirol as a vector of gene therapy .Method: (1)Collected and concentrated the supernatant fluid of PA317-MDR1/A cell, detect the expression of MDR1 gene and env gene by RT-PCR;(2)after transfected mouse bone marrow mononuclear cells (BM-MNCs) with concentrated supernatant of retroviral, detect the expression of MDR1 gene and env gene by RT-PCR;(3)BM-MNCs transfected with MDR1 gene input into BALB/c mouse through vena caudalis primed by 60CO-γ,the control group input tales dose physiological saline, each group have 10 mouses, monitor the white blood cells'(WBC) change, detect the expression of MDR1 and env gene in bone marrow and peripheral blood mononuclearcells;(4)BM-MNCs transfect with MDR1 gene growth in axil subcutaneous, the control group inject physiological saline, each group have 10 mouse,observe wether there have oncologesis; survey wether there have abnormality change by electron microscope; monitor the WBC change; detect the expression of MDR1 and eenv gene in subcutaneous connective tissue of axil of athymic mouse,bone marrow and peripheral blood mononuclear cells.Result: (1)the MDR1 gene in concentrated supernanant fluid can be detected by RT-PCR assay, but no env gene detected;(2)MDR1 gene was detected in BM-MNCs transfected with MDR1gene by RT-PCR assay, but no env gene detected;(3)the white cell count of experimental group decreased slowly than the control group's, and there are difference between two groups after 12 days;MDR1 gene was detected in bone marrow and peripheral blood mononuclear cells of the experimental group,there was no env gene expression;(4)The white cell count were no difference between the two group of nude mouse(P<0.05);there were no MDR1 or env gene express in axil subcutaneous connective tissue,bone marrow and peripheral blood mononuclear cells of nude mouse detected by RT-PCR assay; rrtroviral particle can not be detected in axil subcutaneous connective tissue. There was no retroviral particle in oxter connective tissue of node mouse detected by transmission electron microscope;the morphous and structure of the connective tissue have no abnormalities detected by HE staining,and there had no patho-caryocinesia in the connective tissue.Conclusion : the env gene can not be detected before or after BM-MNCs which carried MDR1 gene import into mouse;the BM-MNCs that transfected MDR1 gene have no oncogenicity EXPERIMENTAL RESEARCH ON THE OVER DOSE ADRIAMYCIN CHEM COMBINED WITH ALL-TRAN- RATINOIC ACID TO MICE WITH LIVER CANCERObjective:Retinoid compound have many effectiveness to kinds of malignant tumour ,such as induce differentiation,suppress proliferation,derivn apoptosis,et al,it is an antitumor drug. Our research used BM-MNCs transfected with MDR1 gene import into mouse with liver cancer,to observe the growth of liver cancer,the count of WBC in peripheral blood,the express of P-gp,proliferation and apoptosis factor in liver cancer,the distribute of MDR1 gene in vivo.Method: Collected and condensed the virus supernatant liquid of incasing cell PA317-HaMDR1/A which product retroviral,using virus supernatant liquid to transfect the BM-MNCs of BALB/c mouse,detected the expression of MDR1 gene in BM-MNCs by RT-PCR; 40 BALB/c mousse with liver cancer divided into 5 groups randomly: A control group(nor exposure or import), B blank(exposure and impore physiological saline),C nagtive control(exposure and impore BM-MNCs non-transfected MDR1 gene)D1 transfected group(exposure and impore BM-MNCs transfected MDR1 gene), D2 transfected group(exposure and impore BM-MNCs transfected MDR1 gene),each group has 8 mouses. B,Cand D1 group treated with Over dose ADM chem Combined with ATRA , D2group treated with over dose ADM chem only, monitor the size of liver cancer,the count of peripheral white blood cells every three days, detect the expression and distribute of MDR1 gene in tumor and the important organs by RT-PCR and FISH,detect the expression of P-gp,proliferation and apoptosis factor in liver cancer every week by immunohistochemical method。Result:(1) It's confirm by RT-PCR that exogenous MDR1gene can integrate and express in BM-MNCs of mouse;the expression of MDR1 gene can be detected in peripheral blood and BM-MNCs of liver cancer mouse in both the drug combination D1 and the ADM group D2. (2) the weight of the tumor in the drug combination group D1 and the ADM group D2 lighter than the control groups 18 days after chem (P<0.05), the weight of liver cancer in mouse of D1 group lighter than the D2 group,but the difference has no statisticsal significance (P> 0.05).(3) White cell count in peripheral blood of the mouse in MDR1 transferring group D1 and D2 groups are more than the control groups, There was statistically significant between the two groups 2 weeks later after chemotherapy (P<0.01);but the count of perithal blood whith cell in the drug combination group D1 compared with the ADM D2 group's have no significant difference (P>0.05);(4)there were no MDR1 gene expression in important organ and tumor detected by FISH and RT-PCR assay;(5) following the increase of the chem dose, the expression of P53,bax,PCNA and Ki-67 in liver cancer between D1 and D2 group have no difference(P>0.05); the expression of P-gp in liver cancer of the drug combination group D1 was higher than the ADM group D2 after chem two weeks(P<0.05); the expression of Bcl-2 in liver cancer of the drug combination D1 group was higher than the ADM group D2 on every week (P<0.05).Conclusion: (1) The exogenous MDR1 gene can be transfected into BM-MNCs of mouse through retroviral vector, and it can express in BM and PB-MNCs of mouse after transfection;(2) ATRA combined with ADM can't enhance or attenuate the curative effect of ADM chem.,usingADM combined with ATRA have no synergistic or rivalry effect;(3) ATRA have no influence on the haemopoiesis of bone marrow;(4) there were no extrogen MDR1 gene expressed in important organ and tumor;(5) ATRA can increase the expression of P-gp and reinforce the drug resistance in liver cancer, we suppose that maybe related with up-regulation the expression of Bcl-2, but may not related with the expression of PCNA,Ki-67,P53 and bax.