目的:脂肪间充质干细胞(ADSCs)在软骨组织修复领域受到广泛关注。当归多糖(ASP)能促进间充质干细胞的增殖和分化。因此,我们拟探讨ASP对体外培养的ADSCs软骨分化的影响,并阐明潜在的机制。
方法:用不同浓度的ASP处理ADSCs以确定最佳浓度。使用Alcian蓝染色评估ADSC的软骨分化,qRT-PCR,westernblot,和IF染色。进行转录组测序以鉴定ASP治疗前后ADSCs的表达谱,其次是生物信息学分析,包括差异表达分析,富集分析,和PPI网络的构建,以鉴定与ASP和软骨分化相关的差异表达基因(DEGs)。
结果:通过CD44CD90CD45-CD106-鉴定分离的大鼠来源的ADSCs的表面标志物,并表现出脂肪生成的能力,成骨,和软骨分化。随着ASP处理浓度的增加,ADSCs的活性和酸性粘膜物质上调。Aggrecan的水平,COL2A1和Sox9在80µg/mlASP处理28天后显示ADSCs增加。转录组测序显示ASP相关DEGs调节细胞外基质合成,免疫反应,炎症反应,和细胞周期,并参与NF-κB,年龄-愤怒,和钙途径。此外,Edn1Frzb,Mdk,Nog,和Sulf1是DEG中的中心基因。值得注意的是,ASP上调ADSCs中MDK水平,而MDK的敲除减轻了ASP诱导的酸性粘膜物质升高,软骨分化相关标志物(Aggrecan,COL2A1和Sox9),和PI3K/AKT途径的活性。
结论:ASP通过激活MDK介导的PI3K/AKT通路增强ADSCs的增殖和软骨分化。
OBJECTIVE: Adipose-derived mesenchymal stem cells (ADSCs) have received significant attention in the field of cartilage tissue repair. Angelica sinensis polysaccharide (ASP) can enhance both the proliferation and differentiation of mesenchymal stem cells. Therefore, we intend to explore the effect of ASP on chondrogenic differentiation of ADSCs in vitro, and elucidate the underlying mechanisms.
METHODS: ADSCs were treated with different concentrations of ASP to determine the optimal concentration. The chondrogenic differentiation of ADSCs was evaluated using Alcian blue staining, qRT-PCR, western blot, and IF staining. Transcriptome sequencing was performed to identify the expression profiles of ADSCs before and after ASP treatment, followed by bioinformatic analyses including differential expression analysis, enrichment analysis, and construction of PPI networks to identify differentially expressed genes (DEGs) associated with ASP and chondrogenic differentiation.
RESULTS: Surface markers of isolated rat-derived ADSCs were identified by CD44+CD90+CD45-CD106-, and exhibited the capacity for lipogenic, osteogenic, and chondrogenic differentiation. With increasing concentration of ASP treatment, there was an upregulation in the activity and acidic mucosubstance of ADSCs. The levels of Aggrecan, COL2A1, and Sox9 showed an increase in ADSCs after 28 days of 80 µg/ml ASP treatment. Transcriptome sequencing revealed that ASP-associated DEGs regulate extracellular matrix synthesis, immune response, inflammatory response, and cell cycle, and are involved in the NF-κB, AGE-RAGE, and calcium pathways. Moreover, Edn1, Frzb, Mdk, Nog, and Sulf1 are hub genes in DEGs. Notably, ASP upregulated MDK levels in ADSCs, while knockdown of MDK mitigated ASP-induced elevations in acidic mucosubstance, chondrogenic differentiation-related markers (Aggrecan, COL2A1, and Sox9), and the activity of the PI3K/AKT pathway.
CONCLUSIONS: ASP enhances the proliferation and chondrogenic differentiation of ADSCs by activating the MDK-mediated PI3K/AKT pathway.