Adenoviral-mediated Expression Of PTEN CDNA And Its Biological Activities In Endometrial Carcinoma Cells

Adenoviral-mediated Expression of PTEN cDNA and its biological activities in endometrial carcinoma cells Endometrial carcinoma (EC) is one of the common malignant tumors in women.According to recent report, the incidence rate of EC has been risen yearly, and it has beenthe fourth commonest malignant tumor in women. A great stride has been made in thetherapy of EC, but problems still exist. For example, there still are poor therapeuticefficacy, high rate of recurrent, metastasis formation and low survival rate in the patientswith high grade and advanced stage. The developing of normal endometrium to EC costsa long period for about 10 years. To find the early gene mutations is critical and effectualto EC. Since PTEN was reported as a new anti-oncogene by three research groups in1997, it has been confirmed mutation in many malignant tumors, and it is the mostimportant anti-oncogene after p53. The mutation rate of PTEN is 26% -80% in EC and18% -55% in endometrial precancerous lesion, and it is thought that mutation of PTEN isan early molecule event and plays an important role in the development of EC. Theobjective of this study is to construct PTEN cDNA recombinant adenovirus and to studythe biological activities of PTEN cDNA recombinant adenovirus in the EC cells in vitro.The research results could provide a new strategy to cure EC in clinic. Up to now, wecould not find out any domestic papers, which are concerned with recombinantadenovirus as a vector to express PTEN cDNA in EC cell lines and studying its biologicalactivities the same with our research project, had published. This research describes (1) Construction of recombinant adenovirus vectorcontaining PTEN cDNA. (2) Production of recombinant adenovirus encoding PTENcDNA and estimating its titer. (3) Expression of PTEN cDNA in EC (RL95-2) cells afterinfected by the recombinant adenovirus containing PTEN cDNA. (4) Assessment ofbiological activities of PTEN cDNA recombinant adenovirus in the RL95-2 cells in vitro.Methods: 6第二军医大学 硕士研究生毕业论文 1.The restrictive endonuclease Kpn Ⅰand XhoⅠcutting sites at the 5′and 3′terminal of the fragment of PTEN cDNA in plasmid pcDNA3.0 PTEN cDNA and thenthe cDNA fragment obtained from digestion was subcloned into pShuttle-CMV vectorby T4 DNA ligase to create a new shuttle vector pShuttle-CMV-PTEN cDNA. 2. PmeⅠdigested and dephosphorylated DNA of pShuttle-CMV-PTEN cDNA washomologically recombinated with DNA of pAdeasy-1 within BJ5183 cells and plasmidDNA of recombinant adenovirus vector, pAdEasy-PTEN cDNA (pAd-PTEN cDNA), wasextracted from both DNA co-transformed BJ5183 cells. 3. After PacⅠ digestion of plasmid DNA of pAdeasy-PTEN cDNA, 30kb fragment,which included adenovirus genomic DNA fragment (except adenovirus E1, E3 gene) andPTEN cDNA fragment, was purified from an agarose gel, and then transfected byliposome into adenovirus packaging cell, HEK293. The recombinant adenovirusAd-PTEN, a replication deficient-recombinant adenovirus, was obtained from the lysatesof HEK293 pellet by repeat froze-melt procedure. And estimate the titer of it. 4. Confirming the expression of PTEN cDNA in RL95-2 cells after been infected byAd-PTEN by the way of RT-PCR and Western Blotting. Then estimate the transfectionefficiency of the recombinant adenovirus use the method of X-gal dyeing. 5. The biological activities of recombinant adenovirus Ad-PTEN was assessed asfollowing: (1) cell proliferation curve was draw after trypan blue stain and cell numbercounting. In the MTT assay, light absorb degree was measured after infecting Ad-PTEN,Ad-CMV and medium alone. Further analysis with propidinm iodide stained cells byFCM showed the number of cells in the G0-G1, S and G2-M phase. (2) Flipping ofmembrane PS was determined by FCM after immunostaining for Annexin Ⅴ and PI.Identify Caspase 3 by FCM after immunostain...