The Experimental Studies Of Recombinant Adenovirus-Mediated Wild Type P53 Gene On Human Colorectal Cancer Cell Lines With Different P53 Status

Objective: We utilize a replication-deficient adenovirus 5 vector encoding a wild-type p53(wtp53) to transfer colorectal carcinoma cell lines with different p53 gene status and then examine the inhibiting effection on those colorectal cancer in vitro. We also investigate the distinction in sensibility to the ectogenesis wtp53 among cell lines with different p53 gene status. With obtained results, the role of wild type p53 tumor suppressor gene in occurrenc and progress of malignant tumor could be deepen research. Methods: The whole studies involved three parts. In the first part, we focus on confirming that three cell lines selected bear different p53 status. H.E. stain and cell morphology observation is used to get the message of the form of various kinds cells. Irnmunocytochemical stain is used to detect three cell lines' endogenous p53 protien expression. PCR-SSCP andsilver stain is applicated to evaluate the status of endogenous p53 gene 5-8 exons in those cell lines. As the final, three colorectal cancer cell lines' p53 gene status (mutation pattern, deletion pattern or wild pattern) would be confirmed. In the second part, Ad-wtp53 with gradient titres will be used in transferring three cell lines and MTT process will be taken to choose optimal transfection titre. And then, Immunocytochemical stain and PCR technique will be adopt to evalute the transfection effect. In the third part, three kinds cell lines will be transferred by Ad-wtp53 in optimal titre and Ad-LacZ be transferred as control. Cell morphologyo observation, growth curve and colony forming experiment will be put into practice, so that the inhibition effect on cell growth and cellproliferation could be appraisal. Flow cytometry (FCM) detect transferred cells (PI monochromatic) to analysis cell cycle changes. Result: Consequence coming from the 1st part experiment make it clear that: ?Three cell lines (THC-8908, HT-29 and Lovo) all belong to epithelial cells, but with different microscopic shape ;(2)Different p53 protein expression ability was presented in those cell lines. THC-8908 negative, HT-29 32.0% and Lovo 54.0%; (3) Distinct p53 gene status were detected in three cell lines. THC-8908 cell line shew p53 allele deletion, Lovo cell line took on wild type p53 gene and HT-29 cell line existed multiple mutation sites. Result obtained from the 2nd part experiment show that: ㏕here is a positive correlation relation between growth inhibition effect and transfection titres of Ad-wtp53 in a certain titres range. IDso about three cell lines is 10 MOI, arid maximal inhibiting effect on cell growth occurred at 1 OOOMOI titre. (2) Recombinant adenovirus-mediated wild type p53 gene can be introducted into the nucleus of three colorectal carcinoma cell lines successfully and expressed p53 protein in nucleus further. In the 3rd part, some cell growth inhibiting experiment outcomes have been gained : 〢s the result of transfecting the Ad-wtp53, three cell lines growth was inhibited seriously. Cell morphology appeared markedchange: cells shape rounded, distribution single and diffuse, and a serial of apoptosis character manifestated such as chromatin stain deepen, nuclear body separated and nucleus fragmentated. (2) After being transfected Ad-wtp53, three cell lines growth inhibition rate were over 65% altogether. THC-8908 cell line have the highest sensitivity, inhibition rate achieved 89.08%. On the other hand, three cell lines growth normal after being transfected Ad-LacZ as control. (3) Comparing with the Ad-LacZ transfected groups, Three cell lines' colony formation rates of the Ad-wtp53 transfected groups notable descended. THC-8908 cell line's colony formation rates of the Ad-wtp53 transfected groups was 0%. ?Cell cycle analysis shew Go/Gj phase cell amount ascend in all Ad-wtp53 transfected groups. This findings suggest that Ad-wtp53 acted on the G]-S transition period, blocked cell cycle in Go/Gi phase. Conclution: Recombinant adenovirus-mediated wild type p53 gene observably inhibited three colorectal carcinoma cell lines growth and...