The AdLMP-1 transfection in two different cells; AF cells, chondrocytes as potential cell therapy candidates for disc degeneration.

Abstract

BACKGROUND: LMP-1 is known to increase proteoglycan production through the upregulating the BMPs and it is also known that BMP-2 acts on anulus fibrosus cells and chondrocytes to increase proteoglycan production.

METHOD: We carried out an experiment, the effect of AdLMP-1 transfection on AF cells and chondrocytes in the production of sulfated-glycosaminoglycans, mRNA expression (aggrecan, type I, II collagen, LMP-1, BMP-2, and BMP-7), and immunofluorescence staining. AF cells and chondrocytes were grown in monolayer and treated for 6 days with AdLMP1-green fluorescence protein (GFP) (10, 20, and 30 multiplicity of infection [MOI]). After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining. The mRNA expression was measured with real-time PCR after 20 MOI infection of AdLMP1-GFP. The each cells treated with 20 MOI infection of AdGFP was used as a control group for the mRNA expression. The each cell group was immunofluorescence stained with each antibodies in the chamber slide at 3 x 10(4) cells/chamber.

FINDINGS: 1) The sGAG production was maximum in 20 MOI AdLMP1-GFP infection on the AdLMP-1 treatment for both of AF cells and chondrocytes. 2) The mRNA expression of aggrecan, type I collagen, type II collagen, LMP-1, BMP-2, and BMP-7 is increased in both AF cells and chondrocytes in 20 MOI AdLMP1-GFP infection. 3) On the immunofluorescence staining results, the positive immunofluorescence stained cell numbers are increased after 20 MOI AdLMP1-GFP infection concordant with upregulation of mRNA expression.

CONCLUSIONS: The AdLMP-1 treatments in AF cells and chondrocytes may be useful for cell transplantation therapy in disc degeneration