In Vitro Experiment Of Gene Therapy For Ovarian Cancer With Suicide Mediated By A Non-viral GE7 Delivery System

Basic strategies for gene therapy on tumor are mainly classfied into four kinds: mutation compensation ,molecular chemotherapy ,immunopotentiation and alteration of drug resistance. Herps simplex thymid kidnase/ganciclovir system(HSV1-TKIGCV) belongs to molecular chemotherapy,it has positive therapeutic effect on malignant brain tumor,meanwhiles,it is a only treatment which is admitted to go into clinical trail in our country. HSV-TKIGCV gene therapeutic system is currently regarded as one of the most prospective strategies in clinical application .Its therapeutic mechanisms can be sumnirized as follows: HSV-Tk gene can express the HSV1-Tk after transferred into corresponding selective tumor cell mediated by delivery system.When treated by GCV, HSV-Tk can phosphorylatilize GCV, and made it to be cellular toxic substance which is capable of inhibiting DNA synthesis,and at last ,lead cells to death.Furthermore ,its so-called "bystander effect"can kill HSV-TK negative cell nearby. GE7 delivery system is non-viral gene transferring system which is constructed by jianren Gu experiment and can carry exogenous gene into cell mediated by EGF receptor.Expression of exogenous gene tranducted by this way is transient ,and don not worry about "gene disease"hich arise from gene transferation. GE7 delivery system is effective ,safe and targeting delivery system .So we performed a serious experiments to explore whether HSV-Tk suicide gene mediated by GE7 delivery system have good effect on ovarian cancer. 1. Construction of GE7/pcmv-tk and GE7/pcmv-β-galactosidase 2. Transduction of β-galactosidase gene into ovarian cancer cell mediated by GE7 delivery system To observe transducting efficiency of GE7 delivery system,β-galactosidasegene,as reporter gene,is cAned by GE7 delivny System into ovarian cancer cell .Thex-gal staining results show:there are 75% x-gal stalning posive cell in Caov3 anddisply blue sedng .unexPectely,SkOV3 cell have only 3% posivecell.ActUrally,both the Caov3 cell and the SkOV3 cell have sAnilar percen of theEGFR posive cell. The one is 88%,the other is 9I%.3. Tran8duction of HSVTK gene into ovarian cancer mediated by GE7tran8fcrring sy8temTO determin Whther HSV-Th gene is transducted into ovarian cancer cellmediated by GE7 delivery system, NOrthem blot is emPloied to detect correspondingWA .According to presention of posive scrip,We infer expression of HSV-TK.We fOund oot posive scrip in CaOV3 cell thesducted HSV-Th gene,bu foundnothing in SKOV3 cell,neither cell trAnsducted HSV-TK gene nor cell untransductedHSV-Th gene.4. The bioIogical function of ovarian cancer ceII after transducted HSVTK genemediated by GE7 delivery systemTo observe in vitro effection of GCV on SKOV3 cell. CaOV3 cell which havetrallsducted HSV-TK gene,and to search for the mechanisms of its working,wemeasure it grOwth-inhbiting rate by MTT assay under differellt GCV concentrationand in different time .We found tha corresPonding to effect of GCV the growth-iwhbiting fate presellt time and dose-depent, Whn GCV concentration is l0ug/ml,thepeak of the growth-inhibiting rate can Anve at 95%,in genneral,it laSt 3day from 3thto 5th dny after transduCtd. ComPared with SKOV3 cell untransducted,SKOV3 celltransducted HSV-TK have scarcely variation. Fndermore,FACS is aPplied detect thepercent of cell differen phases in cell cycle and the ratio of aPopsis.Percent of Sphase is significen elevated in 48h after transducted,5-7th later,it begin to decline,sois the ratio of aPoPsis.It is inferrd that GCV chang into cellular eoxic subhance afterHSV-TK trDeducted into cell,and blocked synthsis of the DNA,and inducedaPopsis.and arrected cell in S sphas...