Experimental Studies On Therapy For Endometrial Carcinoma With Recombinant Adenovirus Carrying Wild Type PTEN Gene(Ad-PTEN)

Endometrial carcinoma, one of the most common malignancies of the femalegenital tract , is the fourth most common cancer in women and the incidence has beenriseing yearly. Effective treatment for advanced endometrial cancer remains elusive.Although 75% of patients deagnosed with this type of malignance are found to haveStage Ⅰdisease which is treatable by surgery and /or radiotherapy, there are only afew effective options for treating advanced disease. Paients with advancd or recurrentdisease have a response rate of approximately 25% with hormone therapy or about28% with chemotheray. With the great development of molecular biology, genetherapy for cancer is from bench to bed. PTEN is a tumor suppressor gene located on10q23.3, encodeing a multifunctional phosphatase , and alterations of this gene havebeen identified in a large fraction of cancers including endometrial carcinoma. Theincidence of PTEN mutations(36%-83%) in endometrial carcinoma is one of thehighest among analyzed tumors. PTEN is the most commonly mutated gene identifiedin endometrial carcinoma. Additionally, the mutations were also seen in about 20% ofcases of endometrial hyperplasia, a precussor of endometrial carcinoma. Recentreseach reveald that PTEN mutations were equally frequent in both early (Stage Ⅰ/GradeⅠ )and more advanced(Stage Ⅳ/GradeⅢ)endometrial carcinomas. It is alsoreported that PTEN expression is associated with prognosis for patients withadvanced endometrial carcinoma undergoing postoperative chemotherapy. Thesurvival rate for PTEN-positive patients was significantly higher than that forPTEN-negative or –mixed patients with advanced endometrial carcinoma whounderwent postoperative chemotherapy. Introduceing the losing tumor suppressorgene into tumor cells to control them and raising the chemosensitivity of it is animportant strategy in tumor theatment. In the present study , we constructed a recombinant adenoviral vector containingwide type PTEN cDNA through a bacterial homologous recombinant system. Theeffects of adenoviral transgene expression of PTEN on the endometrial carcinomacells(RL95-2) was determined both in vitro and in vivo experiment. Our findings may 9第二军医大学 英文摘要 博士学位论文provide new insights into endometrial carcinoma treatment with gene therapy. Theexperiments consists of three parts as follows: Ⅰ. Construction of recombinant adenovirus vector carring wild type PTEN cDNA(Ad-PTEN) The fragment of PTEN cDNA in plasmaid pcDNA3.0 PTEN was cut off withrestrictive endonuclease Kpn Ⅰand XhoⅠat the 5′and 3′ terminal site and thenthe cDNA fragment obtained from digestion was subcloned into pShuttle-CMVvector by T4 DNA ligase to create a new shuttle vector pShuttle-CMV-PTEN cDNA.PmeⅠ digested and dephosphorylated DNA of pShuttle-CMV-PTEN cDNA washomologically recombinated with DNA of pAdeasy-1 within BJ5183 cells andplasmid DNA of recombinant adenovirus vector, pAdeasy-PTEN cDNA, wasextracted from both DNA co-transformed BJ5183 cells. After PacⅠ digestion ofplasmid DNA of pAdeasy-PTEN cDNA, 30kb fragment, which included adenovirusgenomic DNA fragment (without adenovirus E1, E3 gene) and PTEN cDNA fragment,was purified from an agarose gel, and then transfected by LipognenTM into adenoviruspackaging cell, HEK293. The recombinant adenovirus Ad-PTEN, a replicationdeficient-recombinant adenovirus, was obtained from the lysates of HEK293 pellet byrepeat froze-melt procedure and the titer of it was estimated. The expression of PTENcDNA in RL95-2 cells was Conformed after being infected by Ad-PTEN by the wayof RT-PCR and Western Blotting. Then the transfection fficiacy of the recombinantadenovirus was estimated using the method of X-gal dyeing. The cloning sites and directions of insert of PTEN cDNA in the structure ofpShuttle-CMV-PTEN cDNA were exactly confirmed by digestion of two restrictiveendonucleas...