Study On Multidrug Resistance Gene1 (Mdr1) Mediated By Lentiviral Vector In Bone Marrow Mesenchymal Stem Cells And The Resistance To Chemotherapy Drug

Nowadays,malignant tumors have become the common diseases which are seriously threatening the health and life of human being.There are many therapies of malignant tumors.Chemotherapy is an irreplaceable function on the treatment of tumor.However,in the process of chemotherapy,it also has two side effect:multidrug resistance of tumor cells and arrest of bone marrow.Most of experiments have approved that the multidrug resistance genel(mdrl) and its p-gp expression in tumor cells are two main reasons caused fail of chemotherapy, because they can reduce concentration of drugs in tumor cells and causes drug resistance.Normal bone marrow cells have not express mdrl,it is very sensitive to chemotherapeutics so it will be damaged easily.To find an effective method to protect haemopoietic cells and improve the effect of treating tumor is very significative.Stem cells are a population of pluripotent cells that possess self-renewal, highly proliferative and multi-directional differentiating capacities,There are two kinds of stem cells in bone marrow,one is HSCs which manage function of hematopoiesis,the other is MSCs which possess multi-directional differentiating abilities.In the past researches,someone thansfected of multidrug resistance gene into haemopoietic stem cells in bone marrow,they wanted to protect haemopoietic stem cells and peripheral blood leucocytes against high concentration chemotherapeutics,and kill more tumor cells by the chemotherapeutics.But the low transfection efficiency of haemopoietic stem cells limits the development of this technique.It is considered that bone marrow derived mesenchymal stem cells(MSCs) is one of the best seed cell sources of both cell treatment and gene treatment,it is due to they have many biology characteristics,such as low immunogenicity.,transfection and express ex-gene easily,self-renewal and multi-directional differentiating abilities.In this experiment,Use a Lentiviral Vector taking mdr1 gene and GFP gene as report gene transfected MSCs,and then check up ability of MSCs against chemotherapeutics.To aim to provide rationale for cell therapy and gene therapy, at the same time,and bring some new ideas for against multidrug resistance.In the first place,we cultured and expended MSCs in vitro and observed its biology characteristics.we use Percoll separating medium by density gradient centrifugation to isolate human MSCs and amplify in vitro.In the same time, make graph of the growth curve of primary cells and passage cells.FACS observe symbol of MSCs.At last,the MSCs were induced into adipocytes in adipogenesis supplement medium.We found that the primary cells formed a monolayer in 18th day,after several passages,the MSCs had the uniform morphology and grew stably in certain conditions.FACS observed indicated that mature MSCs do not express CD₃₄,HLA-DR,however,almost all the cells express CD₄₄,CD₇₁,and had the potential of differentiation into other tissues.So they can be used as a rich resource of seed cells.Secondly,to detect the expression of objective gene after Lentiviral Vector taking mdr1 gene and GFP gene as report gene transfect MSCs.RT-PCR was used to determine the expression of mdr1 on mRNA level and Western Blot was used to the expression of P-glycoprotein encoded by mdr1 gene was detected.To detected transfected efficiency and rhodamin123(Rh-123) FACS was used to determine the function of p-gp.It is concluded that the expression of mdr1 is up-regulated in MSC transfected by lentiviral vector and there is no significant effect on MSCs.Thirdly,adding high concentration chemotherapeutics into transferred MSCs group and non-transferred group and cultured,then detecting the resistance of drug by MTT,we found that there was a significant increasing in the resistance factor of transferred MSCs compared with the non-transferred MSCs(P＜0.01). We can draw the conclusion that the transfection of mdr1 gene into human bone marrow MSCs could enhanced the resistance MSCs to chemotherapy,which provided a foundation for the application of high concentration anti-tumor agent.