星孢菌素对山羊颞下颌关节盘细胞自组装基体收缩的影响

Effects of staurosporine on the contraction of self-assembled constructs of goat temporomandibular joint disc cells

目的探索不同浓度星孢菌素对山羊颞下颌关节盘细胞自组装基体收缩性及细胞外基质合成的影响。方法单层培养山羊颞下颌关节盘细胞至P3代,将5.5×10 ^6个细胞在不同浓度(0、0.1、1、10、100 nmol·L ^-1)星孢菌素的完全培养基中分别接种于三维柱状琼脂糖井;每天每井换液,培养1周后观察自组装基体的形态变化;冰冻切片染色显示自组装基体细胞外基质及α-平滑肌动蛋白(α-SMA)的分布;酶联免疫吸附测定(ELISA)和Blyscan试剂盒检测基体细胞外基质含量的变化。结果山羊颞下颌关节盘细胞接种4 h后,星孢菌素组和对照组细胞均在琼脂糖井内自组装,形成一圆盘状基体,之后变成球形。各组自组装基体在1周时天狼星红染色可见基体内大量胶原纤维分布;番红染色显示基体内纤维软骨样细胞明显分泌糖胺多糖(GAGs);Ⅰ型胶原(ColⅠ)在免疫组织化学染色中呈强阳性;α-SMA免疫组织化学染色为弱阳性。星孢菌素组自组装基体内ColⅠ和GAGs较高,其中10 nmol·L^-1组最明显(P<0.01)。结论星孢菌素对自组装基体的收缩有一定干预效果,但不是非常突出;它更有利于细胞外基质的合成。

Objective The effects of the staurosporine on contraction of self-assembled constructs and extracellular matrix syntheses of goat temporomandibular joint discs were investigated. Methods Goat temporomandibular joint disc cells were isolated and cultured to P3, and 5.5×10^ 6 cells were combined with different concentrations of staurosporine (0, 0.1, 1, 10, 100 nmol·L^-1) in agarose wells and cultured for one week. The samples were frozen and sectioned. Safranin-O, Picro-sirius red and immunohistochemical staining were performed to observe the distributions of the extracellular matrix and the expression of alpha-smooth muscle actin (α-SMA). Enzyme linked immunosorbent assay (ELISA) and Blyscan kits were utilized to quantitatively detect the contents of type Ⅰ collagen (ColⅠ) and glycosaminoglycans (GAGs). Results Each group of goat temporomandibular joint disc cells in the agarose wells were gathered to self-assemble into a disc-shaped base for 4 hours and then to gradually contract into a round shape. The Picro-sirius red staining was strong and indicated collagen distribution. The Safranin-O staining observed GAGs throughout the entire construct. The expression of ColⅠ was strongly positive in the staurosporine groups;however, the expression of α-SMA was weak. ColⅠ and GAGs contents in the staurosporine groups were greater than that of the control group, especially in the 10 nmol·L ^-1 group (P<0.01). Conclusion Staurosporine has a certain effect on the shrinkage of self-assembled constructs;however, such effect is not prominent. Staurosporine contributes to the construction synthesis of extracellular matrix.