The relation between Sox9, TGF-β1, and proteoglycan in human intervertebral disc cells
The relation between Sox9, TGF-β1, and proteoglycan in human intervertebral disc cells
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DOI：
10.3340/jkns.2008.43.3.149
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发表时间：
2008-03-01
影响因子：
1.6
通讯作者：
Heo, Su-Halk
中科院分区：
文献类型：
--
作者：
Lee, Yong-Jik;Kong, Min-Ho;Heo, Su-Halk
目的：本研究旨在阐明转化生长因子- β (tgf - β)1和l -抗坏血酸对椎间盘细胞中蛋白多糖合成的影响，以及Sox9、蛋白多糖和tgf - β 1三者之间的关系。方法：将人椎间盘组织在DMEM/F-12培养基中依次消化至0.2% pronase和0.025%胶原酶，提取的细胞于37℃，5% CO2培养箱中培养。当用tgf - β 1或l -抗坏血酸培养椎间盘细胞时，用二甲基亚甲基蓝（DMMB）法测定硫酸糖胺聚糖（sGAG）的产生水平。RT-PCR和Western blot检测各组细胞tgf - β 1介导的Sox9 mRNA和蛋白表达水平的变化。结果：sGAG含量随培养时间的延长而增加，颗粒培养细胞的sGAG含量远高于单层培养细胞。单层和颗粒原代培养的椎间盘细胞经tgf - β 1 20 ng处理后，实验组的sGAG含量均显著高于对照组。l -抗坏血酸系列浓度（50 ~ 300 ug/ml）可显著提高单层培养椎间盘细胞sGAG含量。tgf - β 1和l -抗坏血酸共处理比单独处理增加了更多的sGAG产量。tgf - β 1处理后，与对照组相比，椎间盘细胞中Sox9 mRNA和蛋白的表达率显著升高。结论：本研究提示tgf - β 1可通过顺序上调Sox9 mRNA和蛋白的表达来增加硫酸氨基糖聚糖（sGAG）和versican等蛋白聚糖的表达。
Objective : The aim of this study is to elucidate the effects of transforming growth factor-beta (TGF-beta)1 and L-ascorbic acid on proteoglycan synthesis, and the relationship between Sox9, proteoglycan, and TGF-beta 1 in intervertebral disc cells.Methods : Human intervertebral disc tissue was sequentially digested to 0.2% pronase and 0.025% collagenase in DMEM/F-12 media and extracted cells were cultured in 37 degrees C, 5% CO2 incubator. When intervertebral disc cells were cultured with TGF-beta 1 or L-ascorbic acid, the production level of sulfated glycosaminoglycan (sGAG) was estimated by dimethyl methyleneblue (DMMB) assay. The changes of Sox9 mRNA and protein levels via TGF-beta 1 were detected by RT-PCR and Western blot analysis in each.Results : The amount of sGAG was increased with the lapse of time during incubation, and sGAG content of pellet cultured cells was much larger than monolayer Culture. When primary cultured intervertebral disc cells in monolayer and pellet cultures were treated by TGF-beta 1 20 ng, sGAG content of experimental group was increased significantly compared to control group in both cultures. L-Ascorbic acid of serial concentrations (50-300 ug/ml) increased sGAG content of mono layer cultured intervertebral disc cells significantly in statistics. The co-treatment of TGF-beta 1 and L-ascorbic acid increased more sGAG production than respective treatment. After treating with TGF-beta 1, Sox9 mRNA and protein expression rates were significantly increased in disc cells compared with the control group.Conclusion : This study suggests that TGF-beta 1 would increase sulfated glycosaminoglycan (sGAG) and other proteoglycans such as versican by elevating Sox9 mRNA and protein expressions in order.