The Base Research Of Gene Therapy Of Haemopoietic Stem Cell (HSC)-GSTπ In Gynecologic Malignant Tumor

Objective:Patients with ovarian malignant tumor are almost in late stage and need to receive surgery and chemotherap.The drug resistant is one of cause to lead to failure of chemotherap and poor prognoses.The study showed that the resister of ovarian malignant tumor is associated to multidrug resistance gene ,such as highly express GSTπ in ovarian malignant tumor. In order to overcome drug resistant, patient usually need high dosage of drug, but bone marrow depression always followed HDC.The research is plan to transfer GSTπ into HSC and improve the tolerance of Hematopoietic system to chemotherapy. The main objecte of the study is to recombine the retroviral of pLXSN-GSTπ and to construct the packaging cell line PA317-HGSTπ with highest tite, then, to transfer resistant gene GSTπ into the K562 cell line using retroviral ,to study the relation of GSTπ and CDDP,ADM,CTX,to detect the infective rate of the retroviral containing the gene of GSTπ infecting the k562. and to establish base for application of GSTπ in Hematopoietic system .Methods: retrivoral vector pLXSH-GSTπ and pLXSN were cut respectively with restriction endonucleses BamhⅠ,HpaⅠ,the small fragment of GSTπ and the big fragment of pLXSN were ligased to pLXSN-GSTπ using T4 ligase.the pLXSN-GSTπ was transfected PA317 and then G418 resistant colonies were isolated.The titer of retroviral were estimated by Hela. To colture the cells of PA317/HGSTπ/B3-6,to collecte the virus,then to infect K562 and to detecte the express of GSTπ using SP and to detecte the sensitivity of the transfected K562 to CDDP,ADM and CTX using MTT.Result: 1 The small fragment of GSTπ can be seen in gel electrophoresis. 2 The PA317/HGSTπ/B3-6 with the highest titer was constructed and the titer is 2.2×103 CFU/ml. 3 The ectogenesis resistant gene ofGSTπ were introducted into the K562 four times successive by retrovirus and the infect rate is 29% using SP. 4 The infected K562 had the ablity of multidrug resistance,the resistant rate of infected K562 to CDDP,ADM and CTX is 2.49,3.00 and 3.19 respectively.Conclusions: 1 The multidrug resistant gene GSTπ is associated with CDDP,ADM and CTX. 2 The study of transfection of GSTπ into K562 using retroviral show that transfection of GSTπ into HSC using retroviral transfection system is a kind of feasibility methode of gene therapy.It would help us to transfer GSTπ into patien to protecting HSC and a large amounts of chemotherapy could be used in ovary maligant tumor case.There was important significance for practice GSTπ gene therapy to improve the therapeutic efficacy of the ovary cancer in the futer...