| Burkitt Lymphoma(BL)is a highly invasive B-cell Lymphoma derived from the germinal center.Short course of treatment and high-dose combined chemotherapy can significantly improve the cure rate of children with Burkitt lymphoma.However,adult and advanced patients,especially the age group over 40 years old,the central nervous system is often violated by BL.Moreover,chemotherapy has serious side effects and prognosis is poor,so it is necessary to find a treatment that is low in toxicity and effective.Many studies have shown that the mutation of PTEN is closely related to the occurrence of a variety of malignant tumors.To explore the biological effects and pathogenesis of PTEN on Burkitt lymphoma cell line CA46 cells and RAJI cells,to reveal the relationship between PTEN and Burkitt lymphoma,and provide new ideas about develop new antitumor drugs and new gene-targeted therapies for the clinical diagnosis and treatment of Burkitt lymphoma.The research of this topic is mainly divided into the following three parts:Part Ⅰ Construction of PTEN overexpression and silencing recombinant lentiviral vectorObjective:Constructing of PTEN overexpression and silencing lentivirus to infect stablely the Burkitt lymphoma cells.Methods: 1.PTEN c DNA was obtained by PCR and the PTEN-sh RNA was designed and synthesized;2.PTEN c DNA/PTEN-sh RNA was ligated with the vector PLV-CMVFLAG/vector PLKO.1-PURO after the same restriction endonuclease digestion;3.The transformation of the ligation product;4.the PLV-CMV-FLAG-PTEN vector/PLKO.1-sh PTEN vector was verified by enzyme digestion and sequencing after plasmid extraction;5.The PTEN-sh RNA was transfected into Burkitt lymphoma cells by Lipofectamine LTX,The total protein was extracted,the silencing efficiency was verified by Western blot,and the sequence with lower silencing efficiency was selected for subsequent virus construction.Results:1.The PTEN overexpression recombinant lentiviral vector plasmid PLV-CMVFLAG-PTEN was successfully constructed by enzyme digestion and sequencing;2.The PTEN silencing recombinant lentiviral vector plasmid PLKO.1-PURO-sh PTEN was successfully constructed,and PLKO.1-PURO-sh PTEN#1,PLKO.1-PURO-sh PTEN#2 with high silencing efficiency were screened for subsequent virus construction.Conclusion:PTEN overexpression and silencing vector plasmids were successfully constructed.Part Ⅱ Establishment of PTEN overexpressing and silencing Burkitt lymphoma stable cell linesObjective:To screen out PTEN overexpressing and silencing Burkitt lymphoma stable cell lines in preparation for exploring the biological effects of PTEN on Burkitt lymphoma.Methods:1.Three plasmids were co-transfected with 293 FT to construct lentivirus;2.Enrichment of lentivirus by ultra-high speed centrifugation;3.Determination of virus titer by flow cytometry and Real-time PCR;4.The lentivirus infects CA46 and RAJI cells and were screened for stable cell lines by puromycin;5.To verify whether the stable strains were successfully screened,the expression of EGFP protein was observed by fluorescence microscope,the expression of PTEN m RNA was detected by Real-time PCR,and the expression PTEN protein was detected by Western blot.Results:1.Successfully constructe PTEN overexpression and silencing lentivirus,and completed virus titer determination;2.EGFP expression was observed by fluorescence microscopy,Real-time PCR detected up-regulation of PTEN m RNA expression in PTEN overexpressing Burkitt lymphoma cell line,The expression of PTEN protein in PTEN overexpressing Burkitt lymphoma cells was up-regulated by Western blot;3.Real-time PCR detected down-regulation of PTEN m RNA expression in PTEN-silencing Burkitt lymphoma cell line,The expression of PTEN protein in PTEN-silencing Burkitt lymphoma cells was down-regulated by Western blotPart Ⅲ The biological effects and pathogenesis of PTEN on Burkitt lymphoma cell linesObjective:To explore the biological effects and pathogenesis of PTEN on Burkitt lymphoma cell linesMethods:1.The effect of PTEN on the growth and proliferation of CA46 cells and RAJI cells was analyzed by CCK-8 assay;2.The apoptosis was detected by Hoechst 33342 and PI double staining assay;3.The cell cycle distribution was analyzed flow cytometry;4.The migration and invasion were detected by transwell experiment;5.Western blot was used to detect the related proteins’ expression of PTEN/AKT signaling pathway,cell cycle,migration and invasion in CA46 cells and RAJI cells,the effect of PTEN expression on biological characteristics of Burkitt lymphoma cells was observed.Rusults:1.PTEN down-regulated p AKT expression inhibited Burkitt lymphoma cell proliferation;2.PTEN up-regulated Bad and Bax induced apoptosis in Burkttt lymphoma cells;3.PTEN up-regulated P53 and P21 expression,down-regulated CDK4,CDK6,Cyclin D3,and Cyclin H expression to blocking the cell cycle progression of Burkitt lymphoma;4.PTEN up-regulates the expression of E-cadherin,down-regulates the expression of N-cadherin,β-catenin,TCF-8,Vimentin,Slug,Snail,and inhibits the migration and invasion of Burkitt lymphoma cells;5.Silencing PTEN promotes proliferation,migration and accelerated cell cycle progression of Burkitt lymphoma cells.Conlusion:PTEN can inhibit the proliferation and migration of Burkitt lymphoma cells,induce apoptosis and cycle arrest.From what has been discussed above,PTEN plays an important role in the development of Burkitt’s lymphoma,which is hopeful to become a clinical target for the treatment of Burkitt’s lymphoma.. |
