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Abstract
Objectives
To evaluate the mRNA expressions of matrix components, and analyze the cellular proliferation and proteoglycan synthesis of human intervertebral disc cells in response to dexamethasone and TGF- β1
Summary of Literature Review
Corticosteroids are responsible for the regulation of a diverse range of biological processes through modulation of the expression of target genes. The direct injection of methylprednisolone to the intervertebral disc (IVD) has been shown to cause degeneration and calcification of the disc in rabbits. Systemic administration of hydrocortisone induced degeneration of notochordal cells, which accelerated the aging process of the disc in mice. Transforming growth factor beta- 1(TGF- β1) is known as a potent agent for the proliferation, differentiation and matrix synthesis of IVD.
Materials and Methods
IVD cells were isolated from ten patients, and subsequently cultured. Various doses of dexamethasone (DEX) and/or TGF- β1 were administered to the IVD cultures. DNA and proteoglycan syntheses were measured by the incorporation of [3H]- thymidine and [35S]- sulfate, respectively. RT-PCRs were performed for the expressions of aggrecan, collagen types I and II, and osteocalcin mRNA.
Res ults
Cultures with DEX showed increased cellular proliferation and decreased proteoglycan synthesis (p<0.05). TGF- β1 potentiated the proliferative effect of DEX, but failed to stimulate proteoglycan synthesis in the cultures containing DEX. There were no recognizable changes in the mRNA expressions of aggrecan, collagen types I and II, and osteocalcin in response to DEX and TGF- β1.
Conclusions
DEX demonstrated a proliferative effect on human IVD cells, with the combination of DEX and TGF- β1 showing potentiation of the proliferative effect, while at high doses(100 and 1000nM, the DEX was shown to down- regulate the proteoglycan synthesis. Caution should be exercised in the use of corticosteroid in the therapeutic approaches for the treatment of disc disease or in the regenerative matrix of the IVD.
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Fig. 1.
Survival of intervertebral disc cells with various concentration of dexamethasone(0.1, 1, 10, 100, 1000nM). There is no significant cytotoxicity of dexamethasone with dexamethasone compared to control.
Fig. 2.
[3H]-thymidine incorporation with various dose of dexamethasone and TGF-β1(10 ng/ml). White bar denotes culture with TGF- β1. Dexamethasone with a concentration of 10 nM, 100 nM, and 1000 nM renders increased DNA synthesis compared to control(p<0.05). Culture with dexamethasone and TGF-β1(10 ng/ml) showed increased DNA synthesis from the 1nM of dexamethasone and also demonstrated synergistic effect in DNA synthesis. ∗ p<0.05
Fig. 3.
[35S]-sulfate incorporation with various dose of dexamethasone and TGF-β1(10 ng/ml). White bar denotes culture with TGF-β1. Dexamethasone with a concentration of 10 nM, 100 nM, and 1000 nM renders decreased proteoglycan synthesis compared to control (p<0.05). Culture with dexamethasone and TGF- β1(10 ng/ml) showed further decrease in proteoglycan synthesis at 1nM of dexamethasone. ∗ p<0.05
Fig. 4.
Expression of aggrecan, type I collagen, and type II collagen mRNA measured by reverse transcriptase poly-merase chain reaction. Cultures with various dose of dexamethasone showed no significant changes in the expression of aggrecan, type I collagen and type II collagen mRNA expression. Densitometric date was nor-malized by β-actin.
Fig. 5.
Expression of aggrecan, type I collagen, and type II collagen mRNA measured by reverse transcriptase poly-merase chain reaction. Cultures with various dose of dexamethasone and with TGF-β1(10 ng/ml) showed down-regulation of aggrecan mRNA expression while unchanged pattern in type I collagen and type II collagen mRNA expression. Densitometric date was normal-ized by β-actin.
Table 1.
Sequences of the RT-PCR Primers Used
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