甲状旁腺激素通过非依赖PLC的PKC途径抑制成骨细胞的凋亡

摘要

Objective To investigate the effect of the non-PLC-dependent protein kinase C (PKC) pathway of parathyroid hormone (PTH) on the apoptosis and proliferation of osteoblast MC-3T3E1 cells. Methods MC-3T3E1 cells were seeded in 96-well plates at the density of 1.5 × 104 cells/mL and incubated for 3 day. The cells were then exposed to 100 nmol/L of [Gly1, Arg19]hPTH(1-28), 100 nmol/L of [Gly1, Arg19]hPTH(1-34), 100 nmol/L of [Gly1, Arg19]hPTH(1-34)+1 μmol/L Go6983, 1 μmol/L Go6983, or deionized water (control) for 1, 24 or 48 h. After the treatments, cell counting kit-8 (CCK-8) and Caspase-Glo® 3/7 Assay (Caspase-3) were used to examine the proliferation and apoptosis of MC3T3-E1 cells. Results CCK-8 results showed that hPTH(1-34) increased the number of MC3T3-E1 cells compared with hPTH(1-34)+Go6983 at 1 h and 24 h, but this difference was not statistically different. At 48 h, treatment with hPTH(1-34), as compared with hPTH(1-28), significantly increased the number of MC3T3-E1 cells (P<0.05), and this effect was blocked by the PKC inhibitor Go6983 (P<0.05). hPTH(1-34) did not result in significant inhibition of MC3T3-E1 cell apoptosis at 1 h and 24 h as compared with hPTH(1-34)+Go6983, but significantly inhibited the cell apoptosis as compared with hPTH(1-28) (P<0.05);this inhibitory effect was blocked by Go6983 (P<0.05). Conclusions A relatively long time (for 48 h) of exposure to PTH can inhibit apoptosis and promote the proliferation of MC3T3-E1cells through a non-PLC-dependent PKC pathway.%目的：观察甲状旁腺激素的非依赖PLC的PKC转导通路（PTH/nonPLC/PKC）是否会对成骨细胞（MC3T3-E1）的凋亡以及细胞数量具有影响。方法培养MC3T3-E1细胞，以1.5×104密度接种到96孔板，然后置于培养箱培养3 d直到细胞达到汇合状态，随机分为5组：按100 nmol/L[Gly1, Arg19]hPTH（1-28）；100 nmol/L[Gly1, Arg19]hPTH（1-34）；100 nmol/L[Gly1, Arg19]hPTH （1-34）+1μmol/L Go6983，1μmol/L Go6983；空白对照组加入等体积的去离子水，分别刺激细胞1、24、48 h，然后使用细胞计数试剂盒（CCK-8）和Caspase-Glo®3/7试剂盒（caspase-3）检测细胞的细胞数量与凋亡。结果 CCK-8检测结果显示，[Gly1, Arg19] hPTH（1-34）组与[Gly1, Arg19]hPTH（1-34）+Go6983组相比，在1 h和24 h具有提高细胞数量的趋势，但结果并没有统计学差异，48 h[Gly1, Arg19]hPTH（1-34）组与[Gly1, Arg19]hPTH（1-28）组相比，可以明显提高细胞的数量（P<0.05）。进一步的研究发现在[Gly1, Arg19]hPTH（1-34）组添加抑制剂Go6983后，细胞数量增多的效应消失（P<0.05）。Caspase-3检测凋亡结果显示，[Gly1, Arg19]hPTH（1-34）组与[Gly1, Arg19]hPTH（1-34）+Go6983组相比，在1 h和24 h具有抑制细胞凋亡（细胞凋亡受到抑制），但是差异无统计学意义。48 h检测细胞凋亡显示，[Gly1, Arg19]hPTH（1-34）组与[Gly1, Arg19]hPTH（1-28）组相比，前者可以明显抑制细胞凋亡（P<0.05），给予PKC抑制剂Go6983后，其抑制凋亡现象消失。结论 PTH的nonPLC/PKC信号转导通路可能在长时间（48 h）作用于MC3T3-E1细胞时，可抑制细胞凋亡，提高细胞的数量。