Preliminary Study On Nanog And Cancer Cells Supernatant Induced Cancer Stem Cells

Nanog was one of the marker genes and ubiquitous regarding embryonic stem cells.It has fundamental significance in many research field such as induced pluripotent stem cells(iPSCs)and cancer research.Induced pluripotent stem cells and cancer stem cells(CSCs)have numerous common metabolic pathways.The latest research indicated mice induced pluripotent stem cells can differentiate into cancer stem cells under the induction of lung cancer cells supernatant,which demonstrated that it was probable somatic cells could directly differentiated into cancer stem cells by induction.In order to study of the regulation function of Nanog in induced pluripotent stem cells in-depth,and explored the feasible methods that somatic cells could directly differentiated into cancer stem cells by induction in vitro.We transfered Nanog gene into the NIH/3T3 cells of mice applying Tet-on 3G induced expression system.With the induction of Doxycycline,the expression of Nanog can be tightly regulated.Then we were anticipated to get induced cancer stem cells along with the induction of 4 kinds of cancer cells supernatant conditioned medium and provide an in vitro study system for illustrating the formation and occurrence mechanism of cancer.The main results of this study were as follows:1.Constucting pTRE-mcherry-Nanog expression vector,then transferred it into the NIH/3T3 cells of mice successfully through the Xfect Polymer.Confirming the optimal condition of exogenous gene transfection and purpose gene expression in NIH/3T3 cells as follows:(1)5 p.g plamid,(2)the concentration of doxycycline was 400 ng/ml,(3)cell density was 50%.The positive clone cells(red fluorescence cells)ratio was more than 20%.2.Detecting the relative expressions of related gene(Nanog,Oct3/4,Caspase 3,p38)through doxycycline induction and real time PCR.The expressions of Nanog and Oct3/4 improved significantly.The expressions of cell apoptosis related gene Caspase 3 declined,while cell cycle related gene p38 improved.3.Applying four kinds of cancer cells supernatant(A172,HCC 1937,HL-60,Vcap)in the induction of Nanog recombinant NIH/3T3 cells.With the induction of Vcap cancer cells supernatant,cell morphology characteristics were altered obviously especially at the time of 24 hours after the induction.Partial Nanog recombinant NIH/3T3 cells displayed similar characteristics with Vcap cancer cells in cellular morphology and cell adherent time increased.4.With the induction of Vcap cancer cells supernatant,the expressions of Nanog were improved relatively,While it was reduced in other cancer cells(A 172,HCC 1937,HL-60)supernatant.In addition,the expressions of Caspase 3 all declined.