Publications (2)2.08 Total impact
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Article: Intermittent Cyclic Mechanical Tension-Induced Calcification and downregulation of ankh gene expression of end plate chondrocytes.
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ABSTRACT: Intermittent Cyclic Mechanical Tension (ICMT) was applied to end plate chondrocytes by using an FX-4000T Flexercell Tension Plus unit (Flexcell International Corporation, Hillsborough, NC). Changes of end plate chondrocytes were observed after ICMT stimulation. To investigate the relationship between mechanical stimulation and calcification of end plate chondrocytes. Previous study showed that end plate calcification was related to mechanical stress, but there was no clear evidence to indicate whether or not mechanical stimulation could induce calcification of end plate chondrocytes in vitro. Rat end plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) was applied for 25 days, 4 hours a day and continued to culture for 5 days. End plate chondrocytes were incubated for 12 hours in the presence or absence of 10 ng/mL of transforming growth factor-β1 (TGF-β1) (prepared from a stock solution at 10 μg/mL in 2 mM citric acid containing 2 mg/mL bovine serum albumin) in MEM/F-12 containing a final concentration of 1% FCS. End plate chondrocytes calcification was stained by alizarin red S (AR-S). End plate chondrocytes viability was examined by LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, CA). Related gene expression was examined by reverse transcription-polymerase chain reaction and Western blot. LIVE/DEAD assay verified that the nonloading (NC) group and the ICMT group end plate chondrocytes remained adherent, with no change in viability after the application of ICMT. Alizarin red staining showed that ICMT induced the calcification of end plate chondrocytes. Real-time reverse transcription-polymerase chain reaction showed that mRNA expression of endogenous TGF-β1 decreased and mRNA expression of type I, type X, osteocalcin and osteopontin increased after ICMT. The ankh gene expression of both mRNA and protein levels decreased in the ICMT stimulation. The ankh gene expression of both mRNA and protein levels increased in TGF-β1 stimulation. Compared with NC group, the alkaline phosphatase activities significantly increased in ICMT group. Our results directly showed that ICMT induced the calcification and downregulation of ankh gene expression of end plate chondrocytes, which may be caused by the endogenous TGF-β1.Spine 06/2012; 37(14):1192-7. · 2.08 Impact Factor -
Article: [Altered expression of endogenous transforming growth factor β1 and early calcification related genes in rat endplate].
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ABSTRACT: To explore the relationship between endogenous transforming growth factor (TGF)-β1 and calcification-related genes through an in vitro degeneration model by propagating rat endplate chondrocytes during a natural degeneration process. Endplate chondrocytes were extracted from rat lumbar vertebrae, isolated by enzyme digestion and P2 and P4 generations selected for a 6-day in vitro culture. The specimens were photographed microscopically to observe the cellular differences by alizarin red staining. Type II collagen marker gene, transcription factor SOX-9 gene and metabolism-related genes proteoglycan. matrix metalloproteinase (MMP)-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 were detected by RT-PCR to verify the degeneration model. Based on this model, the changes of growth factor TGF-β1 and calcification-related genes ankyrin (ANK), ectonucleotide pyrophosphatase (ENPP), tissue-nonspecific alkaline phosphatase (TNAP) were continuously tested. Compared with P2 cells, P4 cells tended to assume a spindle-shaped morphology. And alizarin red staining showed no change between them. The level of transcription factor SOX-9 of P4 cells (P4/P2 = 0.0690, P = 0.0489) was significantly lower than that of P2 cells. Type II collagen (P4/P2 = 0.0535, P = 0.009) and proteoglycan (P4/P2 = 0.2672, P = 0.0343) were also significantly lower than those of P2 cells. No significant changes were observed in other metabolism-related genes. TGF-β1 (P4/P2 = 0.5934, P = 0.0482) was significantly lower. The expressions of TNAP (P4/P2 = 0.0385, P = 0.0139) and ANK (P4/P2 = 0.2121, P = 0.0009) were significantly lower. But ENPP showed no significant change. P4 endplate chondrocytes undergo natural degeneration in vitro with the rising passage number. Type II collagen, SOX-9 and proteoglycan are significantly reduced. Endogenous TGF-β1 gene and calcification-related genes are down-regulated. The decrease of ANK gene may be caused by the down-regulation of endogenous TGF-β1. Modulating the expression of endogenous TGF-β1 gene in endplate chondrocytes may become a new therapeutic approach for the degeneration of intervertebral disc.Zhonghua yi xue za zhi 08/2011; 91(31):2181-5.
