Studies On The Canceration Mechanism Of Gastric Mucosa In The SV40T Transgenic Mice

SV40is one kind of DNA viruses, in which SV40Tag is an important protein responsible to regulate the cell cycle progression and result in malignant transformation from normal cells in multiple tissues in animals or human beings. Since the SV40Tag is capable to induce oncogenesis in multiple tissues of the transgenic mice in vivo, the established transgenic animals are performed by use of SV40Tag which contributes to oncogenic transformation in cultured cells in vitro as well. The underlying mechanism of oncogenesis induced by SV40Tag is interacted with tumor suppressor genes, Rb and wp53, mediated via T antigen binding domains with Rb or wp53to diminish tumor suppressor gene character, such as inhibiting cell cycle progression, facilitating proliferation and resulting in mutagenesis. Generally the T antigen can activate E2F-dependant translation to enter S phase through inactivating pRb, p130or p107. Concomitantly. the cell cycle arrest and apoptosis induced by T antigen through inactivating wp53.In preliminary study of this subject, we have established the antigen transgenic mice,in which gastric parietal cell can express SV40T specially, by using the microinjection legal system.We got10strains of GO positive transgenic mice:2#、4#8#、16#、24#、51#、57#、61#、68#and73#, SV40T can stable express in mice after nine heritable passage generations.The gastric mucosa hyperplasia in all positive transgenic mice. In order to study the mechanism of gastric mucosa from hyperplasia to cancer,we study the expression level of Rb gene and its pathway-realated members (E2F-1、Cyclin D1and CDK4) in the24#(mucosal hyperplasia) and73#(gastric cancer)strain transgenic mice by using the RT-PCR and Western blotting,so as to explore the role and mechanism of SV40Tag and Rb in the process of gastric canceration.Methods1. HE staining of spleen, lung, liver, small intestine, large intestine, testis, stomach separately from six24#strains、six73#strains male transgenic mice and six normal male mice to observe the pathological changes in various organs of mice.2. Detect the RNA expression levels of the Rb gene and its pathway related genes (E2F-1、Cyclin D1and CDK4) in gastric mucosa and testis of six24#strain and six73#strain male transgenic mice use RT-PCR.3. Detect the protein expression levels of the Rb gene and its pathway related genes (E2F-1、Cyclin D1and CDK4) in gastric mucosa and testis of six24#strain and six73#strain male transgenic mice by Western blotting.4. Detect the SV40Tag in the testes of73#strain male transgenic mice by using immunohistochemical staining.Results1. HE staining shows that the gastric mucosa hyperplasia can be seen in mice of24#strain male transgenic mice; earlysymptoms of cancer can be seen in the73#strain male transgenic mice, its testis reveals cancerous.2. RT-PCR results showed that in the presence of SV40Tag, The mRNA expression levels of Rb gene was significantly lower than the control (P<0.05);The mRNA expression levels of E2F-1、Cyclin D1and CDK4was significantly increased (P <0.05).3. Western blotting shows that in the presence of SV40Tag,the protein expression level of Rb gene is significantly lower,(P<0.05);Rb pathway-related gene of E2F-1is significantly increased(P<0.05), compared with the control, but the protein expression level of Cyclin D1and CDK4was detected out.4. The immunohistochemistry result shows that SV40Tag expression was detected in the testis of73#strain of transgenic mice.Conclusions1. The expression levels of E2F-1, Cyclin D1and CDK4were up-regulated through down-regulating the expressions mRNA and protein of Rb gene by SV40Tag in 73strain transgenic mice, which suggested that SV40Tag may be involved in the transgenic mice pathogenesis through inhibiting the tumor suppressor Rb signaling cascade pathway.2Testicular cancer of73#strain transgenic mice is associated with the expression of SV40Tag.