A novel culture platform for fast proliferation of human annulus fibrosus cells.
A novel culture platform for fast proliferation of human annulus fibrosus cells.
复制标题
一个新型的培养平台，用于快速的人环纤维细胞增殖。
DOI：
10.1007/s00441-016-2497-4
复制
发表时间：
2017-02
影响因子：
3.6
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Xiao L;Ding M;Saadoon O;Vess E;Fernandez A;Zhao P;Jin L;Li X
组织工程学为椎间盘退变的治疗提供了一种有希望的方法，这通常需要大量的种子细胞。一个简单可靠的体外培养系统，以扩大种子细胞在及时的时尚是必要的，以实现临床应用。在这里，我们试图建立一个具有成本效益的培养系统，用于扩大人纤维环（AF）细胞使用细胞外基质（ECM）蛋白作为培养基质。将细胞培养在涂有各种类型ECM的塑料表面上，所述ECM包括纤连蛋白、玻连蛋白、I型胶原、明胶和由人髓核（NP）细胞沉积的无细胞基质。通过显微镜、实时RT-PCR、蛋白质印迹、酶谱、免疫荧光染色和生化测定评估AF细胞形态、生长、粘附和表型（合成代谢和分解代谢标志物）。纤维连接蛋白、胶原和明胶以剂量依赖方式促进细胞增殖和粘附。纤连蛋白提高蛋白多糖的mRNA表达，并增强糖胺聚糖（GAG）的生产。胶原和明胶增加II型胶原蛋白的表达。与增加的细胞粘附一致，胶原蛋白和纤连蛋白促进了细胞-基质连接中粘着斑复合物的形成，表明肌动蛋白网络与ECM底物的结合增强。另一方面，纤维连接蛋白、胶原和明胶降低基质金属蛋白酶-2和基质金属蛋白酶-9在培养基中的表达。最后，纤连蛋白（1.7 μg/mL）和胶原蛋白（1.3 μg/mL）的混合物被确定为促进增殖和维持合成代谢-分解代谢平衡的最有前途的体外培养基质系统。我们的方法为椎间盘研究中的组织工程应用提供了一个简单且具有成本效益的平台。
Tissue engineering provides a promising approach to treat degenerative disc disease, which usually requires a large quantity of seed cells. A simple and reliable in vitro culture system to expand seed cells in a timely fashion is necessary to implement the application clinically. Here, we sought to establish a cost-effective culture system for expanding human annulus fibrosus (AF) cells using extracellular matrix (ECM) proteins as culture substrates. Cells were cultured onto a plastic surface coated with various types of ECMs, including fibronectin, vitronectin, collagen type I, gelatin, and cell-free matrix deposited by human nucleus pulposus (NP) cells. AF cell morphology, growth, adhesion, and phenotype (anabolic and catabolic markers) were assessed by microscopy, real-time RT-PCR, western blotting, zymography, immunofluorescence staining and biochemical assays. Fibronectin, collagen and gelatin promoted cell proliferation and adhesion in a dose-dependent manner. Fibronectin elevated mRNA expression of proteoglycan and enhanced glycosaminoglycan (GAG) production. Both collagen and gelatin increased protein expression of type II collagen. Consistent with increased cell adhesion, collagen and fibronectin promoted formation of focal adhesion complexes in the cell-matrix junction, suggesting enhanced binding of actin network with both ECM substrates. On the other hand, fibronectin, collagen and gelatin decreased expression of matrix metalloproteinase-2, and matrix metalloproteinase-9 in media. Finally, a mixture of fibronectin (1.7 μg/mL) and collagen (1.3 μg/mL) was identified as the most promising in vitro culture substrate system in promoting proliferation and maintaining anabolic-catabolic balance. Our method provides a simple and cost-effective platform for tissue engineering applications in intervertebral disc research.