|
Objective: To study adhesins 5 N terminal CED-4 (cadherin extracellular domain-4) gene by recombinant retrovirus -mediated transfection of human breast cancer cells MDA-MB-435 induced apoptosis of MDA-MB-435 cells . The molecular mechanism . Methods : preliminary work has been completed the cloning and identification of CED-4 , and transformed into E. coli cloning vector , access to copy plasmid . Undertake the preparatory work of the experiment , the main experimental methods : ⑴ restriction endonuclease plasmid : restriction endonuclease BspHI I Bg1II Afl Ⅱ respectively digested the the plasmid pVSV - G the pGAG -POL pMSCV , pMSCV - CED4 , electrophoresis identification of plasmids. ⑵ 293 packaging cell packaging containing the gene produce recombinant retrovirus : liposome -mediated plasmid pVSV-G, pGAG-POL, pMSCV, pMSCV-CED4, into 293 packaging cells by G418 , 293 cells produce and without target gene recombinant retrovirus . ( 3 ) virus infection of target cells MDA-MB-435 cells : two sets of recombinant retrovirus ( the experimental group and the experimental control group ) were infected with the MDA-MB-435 cells , as enhanced green fluorescent protein (enhanced green fluorescent protein, EGFP) gene transfection positive cells mark the third day, the green fluorescent closely observed changes in cell morphology after 2 weeks . ⑷ apoptosis detection : optical microscope under microscopic observation of apoptotic morphology , Annexin V - BIOTIN apoptosis detection kit cell staining , flow cytometry apoptosis ; ⑸ RT- PCR : extract the experimental group , the experimental control group and blank the three groups of cells of the control group overall RNA, primers were designed by RT-PCR amplification of target gene CED -4 mRNA integrin beta1 subunit mRNA, the proto-oncogene c -fos mRNA , agarose gel electrophoresis ; ⑹ Westhen blot detection of c-fos protein expression. Results : ⑴ plasmid DNA size normal : digested plasmid , electrophoresis results showed that , the the plasmid pVSV - G , pGAG - POL pMSCV , pMSCV - CED4 normal in size , not degradation . ( 2 ) 293 cells produce recombinant retrovirus packaging : the plasmid was transformed into 293 cells, and 48 hours later, visible green fluorescence under a fluorescence microscope . ( 3 ) recombinant retrovirus infected MDA-MB-435 cells: retroviral infection of MDA-MB-435 cells after 48 hours under a fluorescence microscope visible green fluorescence , suggesting that the virus-mediated gene transfection success. ⑷ RT-PCR detected the target gene CED-4 base
|