摘要
目的探讨FTY720-P对MC3T3-E1细胞分化成熟的作用。方法取MC3T3-E1细胞,分为实验组和对照组。实验组以400 ng/mL FTY720-P(以氯仿为溶剂)作用于细胞,建立体外诱导成骨分化模型;对照组培养基中仅加入氯仿。培养48 h后,倒置相差显微镜观察两组细胞形态;免疫荧光染色检测两组成骨细胞相关蛋白Ⅰ、Ⅲ型胶原蛋白的表达;ALP染色及茜素红染色观察两组成骨细胞形成情况及矿化结节形成情况;TUNEL荧光法检测两组细胞凋亡情况。结果培养48 h后,可见两组细胞均已长满瓶底,呈梭形,细胞形态无明显差异。免疫荧光染色显示,实验组Ⅰ型胶原蛋白表达呈阳性,对照组呈弱阳性;实验组的Ⅰ型胶原蛋白积分吸光度(IA)值为187 600±7 944,显著高于对照组的14 230±1 070,差异有统计学意义(t=43.680,P=0.001)。实验组和对照组Ⅲ型胶原蛋白表达均呈弱阳性,两组Ⅲ型胶原蛋白IA值比较差异无统计学意义(t=1.976,P=0.119)。实验组细胞ALP染色和茜素红染色均呈阳性,而对照组均呈阴性。实验组TUNEL染色呈阳性,对照组呈阴性;实验组TUNEL染色细胞阳性率为35.82%±2.99%,显著高于对照组的2.28%±0.51%(t=23.420,P=0.002)。结论 FTY720-P可以促进MC3T3-E1细胞成骨分化,加快细胞外基质成熟和矿化,且能影响细胞凋亡。
Objective To investigate the effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells. Methods The MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/m L FTY720-P（chloroform as solubilizer） in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein（collagen typeⅠ and collagen type Ⅲ） was detected by immunofluorescence staining; the alkaline phosphatase（ALP） staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis. Results After 48 hours of culture, the cells of 2 groups had grown into slender fusiform at the bottom of the bottle, and there was no significant difference in cell morphology between 2 groups. Immunofluorescence staining showed that the expression of collagen type Ⅰ was positive in the experimental group and weakly positive in the control group; the integrated absorbance（IA） value of the experimental group was 187 600±7 944, which was significantly higher than that of the control group（14 230±1 070）（t=43.680, P=0.001）. The expression of collagen type Ⅲ was weakly positive in the experimental group and the control group, and there was no significant difference in IA value between 2 groups（t=1.976, P=0.119）. ALP staining and alizarin red staining were positive in the experimental group and negative in the control group. TUNEL staining was positive in the experimental group and negative in the control group; the rate of TUNEL staining positive cells in the experimental group was 35.82%±2.99%, which was significantly higher than that in the control group（2.28%±0.51%）（t=23.420,P=0.002）. Conclusion FTY720-P can promot
作者
郑力彬
黄东
ZHENG Libin;HUANG Dong(Guangdong Medical University, Zhanjiang Guangdong, 524000, P.R.China;Department of Trauma Orthopedics, Guangdong Second Provincial Central Hospital, Guangzhou Guangdong, 510000, P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2018年第3期285-290,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
广东省科技发展专项基金(2016A020215124)~~
