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


METHODS
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.


RESULTS
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.
Kuh
S U
SU
Department of Neurosurgery, The Spine and Spinal Cord Institute, Yonsei University Medical College, Seoul, Korea.
Zhu
Y
Y
Li
J
J
Tsai
K-J
KJ
Fei
Q
Q
Hutton
W C
WC
Yoon
S T
ST
D048868:Adaptor Proteins, Signal Transducing; D053674:Aggrecans / Q000235:genetics; D055396:Bone Morphogenetic Protein 2; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D017690:Cell Transplantation; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology / Q000637:transplantation*; D024042:Collagen Type I / Q000235:genetics; D024043:Collagen Type II / Q000235:genetics; D003598:Cytoskeletal Proteins; D005786:Gene Expression Regulation / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D007403:Intervertebral Disc / Q000378:metabolism* / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D060588:LIM Domain Proteins; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D013122:Spinal Diseases / Q000628:therapy*; D014162:Transfection / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D053674:Aggrecans; C525718:BMP2 protein, human; C525819:BMP7 protein, human; D055396:Bone Morphogenetic Protein 2; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D024043:Collagen Type II; D003598:Cytoskeletal Proteins; D006025:Glycosaminoglycans; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C484380:PDLIM7 protein, human; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C013786:A73025