Suppression of discoidin domain receptor 1 expression enhances the chondrogenesis of adipose-derived stem cells
Suppression of discoidin domain receptor 1 expression enhances the chondrogenesis of adipose-derived stem cells
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DOI：
10.1152/ajpcell.00398.2014
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发表时间：
2015-05-01
影响因子：
5.5
通讯作者：
Wang, Chau-Zen
中科院分区：
文献类型：
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作者：
Wu, Shun-Cheng;Hsiao, Hsu-Feng;Wang, Chau-Zen
有效地引导脂肪干细胞（ADSC）的软骨形成以工程化关节软骨是基于ADSC的关节软骨组织工程的重要挑战。盘状结构域受体1（DDR 1）已被证明会影响软骨稳态，然而，很少有人知道DDR 1在ADSC软骨形成的作用。本研究采用三维培养颗粒模型结合软骨诱导技术，探讨DDR 1在人脂肪干细胞（hADSCs）软骨诱导分化中的作用。实时荧光定量PCR和Western blot检测DDRs和软骨形成基因的表达。硫酸化糖胺聚糖（sGAG）通过阿辛蓝和二甲基亚甲基蓝（DMMB）测定法检测。末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色用于评估细胞死亡。在hADSCs的软骨形成过程中，DDR 1的表达显著升高，而DDR2的表达不显著。使用短发夹RNA消耗hADSC中的DDR 1表达增加了软骨形成基因（SOX-9、II型胶原蛋白和聚集蛋白聚糖）和软骨基质沉积（II型胶原蛋白和sGAG）的表达，并且仅略微增加了细胞死亡（2-8%）。hADSC中DDR 1过表达降低了软骨形成基因（SOX-9、II型胶原和聚集蛋白聚糖）和sGAG的表达，并提高了hADSC的存活率。此外，DDR 1缺失的hADSC显示终末分化基因Runx 2和基质金属蛋白酶13的表达降低。这些结果表明，DDR 1抑制可能通过增强软骨形成基因的表达和软骨基质沉积来增强ADSC软骨形成。我们提出，抑制ADSC中的DDR 1可能是优化基于ADSC的关节软骨组织工程的遗传修饰的候选策略。
Effectively directing the chondrogenesis of adipose-derived stem cells (ADSCs) to engineer articular cartilage represents an important challenge in ADSC-based articular cartilage tissue engineering. The discoidin domain receptor 1 (DDR1) has been shown to affect cartilage homeostasis; however, little is known about the roles of DDR1 in ADSC chondrogenesis. In this study, we used the three-dimensional culture pellet culture model system with chondrogenic induction to investigate the roles of DDR1 in the chondrogenic differentiation of human ADSCs (hADSCs). Real-time polymerase chain reaction and Western blot were used to detect the expression of DDRs and chondrogenic genes. Sulfated glycosaminoglycan (sGAG) was detected by Alcian blue and dimethylmethylene blue (DMMB) assays. Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to assess cell death. During the chondrogenesis of hADSCs, the expression of DDR1 but not DDR2 was significantly elevated. The depletion of DDR1 expression in hADSCs using short hairpin RNA increased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and cartilaginous matrix deposition (collagen type II and sGAG) and only slightly increased cell death (2-8%). DDR1 overexpression in hADSCs decreased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and sGAG and enhanced hADSC survival. Moreover, DDR1-depleted hADSCs showed decreased expression of the terminal differentiation genes runt-related transcription factor 2 (Runx2) and matrix metalloproteinase 13 (MMP-13). These results suggest that DDR1 suppression may enhance ADSC chondrogenesis by enhancing the expression of chondrogenic genes and cartilaginous matrix deposition. We proposed that the suppression of DDR1 in ADSCs may be a candidate strategy of genetic modification to optimize ADSC-based articular cartilage tissue engineering.