PARP-1和氟诱导成骨细胞凋亡的相关性研究

Study on correlation between PARP-1 and fluorideinduced osteoblast apoptosis

目的探究染氟成骨细胞凋亡与多聚腺苷二磷酸核糖基聚合酶-l(PARP-1)蛋白的相关性;预先探索PARP-1在成骨凋亡过程中的作用,以期在氟骨症治疗中寻找新的靶点。方法采用人成骨肉瘤细胞系(Saos-2)建立体外染氟模型,即经体外细胞培养后分别加入如下浓度梯度氟化钠(NaF,0.0,2.5,5.0,10.0,20.0和40.0 mg/L)处理;在实验组中加入适量Nec-1抑制细胞程序性坏死,再以细胞毒性试验(CCK-8法)观察染氟成骨细胞在各时间点和实验组的细胞存活率;同时利用免疫印记法(Western blot)检测各组PARP-1[poly(ADP-ribose)polymerase-1],Bax(Bcl2-associated X protein)和Bcl-2(B-cell lymphoma-2)的蛋白含量。结果PARP-89 kD与氟浓度呈明显正相关趋势(r24 h=0.924,r48 h=0.927,P<0.01),PARP-113 kD与氟浓度呈明显负相关趋势(r24 h=-0.677,r48 h=-0.785,P<0.01),PARP-89 kD的转化率和氟浓度呈正相关(r=0.772,P<0.01)。氟浓度高于10 mg/L时,随氟浓度的增加Bax/Bcl-2也持续增长(r24 h=0.808,r48 h=0.948,P<0.01);氟浓度在5 mg/L以下时,加入NaF组的细胞存活率始终高于空白对照组,细胞存活率与氟浓度呈明显正相关(P<0.01);当氟浓度超过5 mg/L时,细胞存活率与氟浓度呈明显负相关(P<0.01)。结论提示PARP-1与染氟成骨细胞的凋亡密切相关。

Objective To explore the feasible correlation between PARP and fluoride-induced osteoblast apoptosis,discuss the role of PARP-1 in progress of osteogenic apoptosis in advance so as to find new target for skeletal fluorosis treatment.Methods A fluorosis model of human osteosarcoma cell line Saos-2 was established in vitro.That is,concentration gradient of NaF(0.0,2.5,5.0,10.0,20.0 and 40.0 mg/L)was added after cell culture in vitro.In the experimental group,proper concentration of Nec-1 was added to inhabit cell programmed necrosis.Then,CCK-8 was taken to test cell survival rate at the time point and in the experimental group.Protein contents of PARP-1[poly(ADP-ribose)polymerase-1],Bax(Bcl2-associated X protein)and Bcl-2(B-cell lymphoma-2)in all groups were detected with Western blot.Results PARP-89 kD was clearly related to the concentration of NaF(r24 h=0.924,r48 h=0.927,P<0.01),and PARP-113 kD had negative correlation with the concentration(r24 h=-0.677,r48 h=-0.785,P<0.01).When the fluoride concentration was over 10 mg/L,Bax/Bcl-2 increased continuously along with the concentration growth of NaF(r24 h=0.808,r48 h=0.948,P<0.01).When the concentration of NaF was under 5 mg/L,the cell survival rate in the fluoride group was higher than that in the control group,and positively correlated to the fluoride concentration(P<0.01),and the cell survival rate negatively correlated to the fluoride concentration(P<0.01).Conclusions These findings indicate that PARP-1 closely relate to osteoblasts apoptosis induced by fluoride.