Id1, NF-κB Affect On The CD133-Positive Cell Formation

Objective: To establish stable transfection cell line Rhek-Id1, Rhek-P65, and Rhek-IP,by lentivirus vectors which contained Id1, NF-κB and Id1- NF-κB complex respectively, based on and to compare the effect of Id1 and NF-κB on CD133 expression, and investigate whether these two genes will synergistically affect the expression of CD133, hoping to lay foundation for subsequent experiments and explore some new insights into the pathogenesis of cholesteatoma.Methods: Immunoblotting technology was used to measure the expression of Id1 and NF-κB in each cell line.Flow cytometry was used respectively to compare the expression difference of CD133 between different cell lines.Results:(1) With GAPDH as an internal reference, western blotting results showed that Id1 protein was stably expressed in Rhek-Id1 cells, while NF-κB protein was stably expressed in Rhek-P65 cells, and Id1 together with NF-κB protein was stably expressed in Rhek-IP cells.(2) FACS analysis indicated that CD133-positive cell ratio(%) was(x??S) 55.47?0.68、 50.85?0.58、66.80?0.80、70.14?1.17、67.74?1.13 in Rhek-1A cell line, Rhek-ev cell line, Rhek-Id1 cell line, Rhek-P65 cell line and Rhek-IP cell line respectively. Stastistical analysis showed that, compared with Rhek-ev cell line, the ratio of CD133-positive cells in Rhek-Id1 cell line, Rhek-P65 cell lines and Rhek-IP cell lines was increased; but compared with Rhek-Id1 cells, the ratio of CD133-positive cells in Rhek-IP cell lines wase not, with no obvious statistical significance(P?0.05), which could not explain synergistical effect of Id1 and NF-κB on expression of CD133.Conclusion:(1) Rhek-Id1 cell line, Rhek-P65 cell lines, Rhek-IP cell lines stably express Id1 protein, NF-κB(P65) protein, Id1 protein and protein NF-κB(P65) by themself.(2) Id1 and NF-κB can respectively increase the expression of CD133.