软骨细胞体外增殖去分化的拉曼光谱分析

Raman Spectroscopic Analysis of Chondrocyte Dedifferentiation during in vitro Proliferation

基于显微拉曼光谱技术,对组织工程的软骨种子细胞在传代增殖过程中的去分化进行单细胞分析。首先,对体外单层培养的第1-4代(P1-P4)大鼠软骨细胞样本进行了单细胞拉曼光谱检测,由此识别出软骨细胞中各种碱基、糖基、氨基酸等主要物质分子结构的特征峰集合。随后,分析拉曼光谱中若干重点特征峰强度随细胞传代次数的变化,发现软骨细胞体外增殖过程中核酸(789、1094、1576 cm^(-1))含量降低、Ⅱ型胶原(特异组分为羟脯氨酸,1207 cm^(-1))和蛋白聚糖(特异组分为糖胺聚糖,1042、1063、1126、1160 cm^(-1))合成下降、脂质(1304 cm^(-1))及磷酸盐(957 cm^(-1))含量增加等分子水平变化,从而在活体单细胞层次初步揭示了去分化引起软骨细胞增殖变缓、分泌减弱、形态纤维化等现象的分子机制。

Seeded chondrocytes play a crucial role in current cartilage tissue engineering, yet both the quality and quantity of these cells could be impaired owing to cell dedifferentiation during in vitro proliferation. Here, we used micro-Raman spectroscopy to investigate changes in cellular components upon monolayer culturing of primary rat chondrocytes through multiple passages. Based on the average spectral profiles, we detected a series of Raman peaks and recognized related radicals such as nucleobases, pyranose rings, sulfate, tyrosine, proline, and amides at the single-chondrocyte level. Quantitative analysis of the Raman peak intensities showed that nucleic acids （at 789, 1094, 1576 cm-~） decreased significantly from passage 1 （P1） to passage 4 （P4）, whereas lipids （at 1304 cm-1） and phosphate （at 957 cm-1） increased significantly. Moreover, the syntheses of two major hyaline cartilage-associated proteins, aggrecan and type-2 collagen, were impeded, as indicated by the marked decline in the levels of their specific components （glycosaminoglycan at 1042, 1063, 1126, 1160 cm-1, andhydroxyproline at 1207 cm-1）. Taken together, these features reveal the diminished propagation and secretion abilities of passaged chondrocytes needed for matrix-induced implantation, and shed light on the molecular mechanism of chondrocyte dedifferentiation.