目的:建立并评价脂多糖(LPS)诱导的成牙本质细胞(OB)凋亡模型。方法:采用梯度LPS(0、0.1、1、10、100tzg/mL)刺激法诱导小鼠OB细胞系MDPC-23细胞凋亡;分别于诱导后24、48、72、96h各时间点,以MTT法检测细胞增殖活力,流式细胞术检测细胞凋亡比例,确定合适的诱导浓度和时间后,建立OB细胞凋亡模型,并通过Hoechst染色观察细胞形态和凋亡比例。结果:0.1μg/mL和1μg/mL的LPS作用24h可明显促进MDPC-23细胞增殖,72h后则明显抑制细胞增殖(P〈0.05);当LPS浓度大于1μg/mL时,对细胞的影响以毒性作用为主。在各个时间点不同浓度的LPS均可诱导MDPC-23细胞凋亡,其中以0.1μg/mL的LPS诱导MDPC-23细胞凋亡的效果较理想。Hoechst染色显示0.1μg/mL LPS可诱导细胞出现典型的凋亡形态学特征,且随着LPS作用时间的延长,凋亡细胞比例逐渐增高(P〈0.05),但96h时有大量细胞死亡。结论:LPS诱导MDPC-23细胞凋亡的合适浓度为0.1μg/mL,合适作用时间为24—72h。
AIM: To establish and assess cell apoptosis model of odontoblastic MDPC-23 cells induced by lipopolysaccharide (LPS) in vitro. METHODS: E. eoli LPS at 0 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL and 100 μg/mL was used to induce apopotosis of MDPC-23 cells for 24 h, 48 h, 72 h and 96 h respectively. MTT assay was employed to observe the cell proliferation. Flow cytometry (FCM) and Hoechst staining were used to detect cell apoptosis ratio. RESULTS : 0.1 μg/mL and 1 μg/mL LPS promoted cell proliferation in 24 h exposure, but after 72 h exposure, cell proliferation was significantly inhibited (P 〈 0.05). LPS with higher concentration ( 〉 1 Izg/mL) mainly exhibited toxic effects on MDPC-23 cells. At various time points (24, 48, 72 and 96 h) , all concentrations of LPS induced apoptosis of MDPC-23 cell cells. However, 0.1 μg/mL LPS showed appropriate apoptosis rate. Hoechst33258 fluorescent staining confirmed that MDPC-23 cell induced by LPS exhibited specific morphological features of apopto- sis, such as pyknosis, nuclear fragmentation and apoptotie bodies. LPS-induced MDPC-23 cell apoptosis showed a time-dependent manner. However, a great number of MDPC-23 cell died after 96 h exposure. CONCLUSION: Ex- posure of 0.1 μg/mL LPS to MDPC-23 cells for 24-72 h is appropriate for the apoptosis induction of the cells.