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目的观察甲状旁腺激素的非依赖PLC的PKC转导通路(PTH/non PLC/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]h PTH(1-28);100 nmol/L[Gly1,Arg19]h PTH(1-34);100 nmol/L[Gly1,Arg19]h PTH(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]h PTH(1-34)组与[Gly1,Arg19]h PTH(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]h PTH(1-34)组与[Gly1,Arg19]h PTH(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]h PTH(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]h PTH(1-34)组与[Gly1,Arg19]h PTH(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]h PTH(1-34)组与[Gly1,Arg19]h PTH(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non PLC/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。
Objective To investigate whether PTH / non PLC / PKC, a parathyroid hormone-dependent PKC pathway, affects the apoptosis and cell number of osteoblasts (MC3T3-E1). Methods MC3T3-E1 cells were cultured and seeded into 96-well plates at a density of 1.5 × 104. The cells were cultured in an incubator for 3 days until the cells reached the confluent state and were randomly divided into 5 groups: 100 nmol / L [Gly1, Arg19] h PTH (Gly1, Arg19] h PTH (1-34) + 1 μmol / L Go6983, 1μmol / L Go6983; 100 nmol / The blank control group was added with equal volume of deionized water and the cells were stimulated for 1, 24 and 48 h respectively. Then the cell count (CCK-8) and Caspase-Glo (3/7) Cell number and apoptosis. Results The results of CCK-8 assay showed that cells in [Gly1, Arg19] h PTH (1-34) group had higher cell numbers at 1 h and 24 h than those in [Gly1 and Arg19] h PTH (1-34) However, the results showed no significant difference. Compared with the [Gly1, Arg19] h PTH (1-28) group, the number of cells in 48 h [Gly1, Arg19] h PTH (1-34) P <0.05). Further study found that after the addition of inhibitor Go6983 in [Gly1, Arg19] h PTH (1-34) group, the effect of increasing cell number disappeared (P <0.05). The results of Caspase-3 assay showed that the cells in [Gly1, Arg19] h PTH (1-34) group had inhibitory activity at 1 and 24 h compared with [Gly1 and Arg19] h PTH (1-34) + Go6983 group Apoptosis (inhibition of apoptosis), but the difference was not statistically significant. 48 h detection of apoptosis, the former can significantly inhibit the apoptosis of [Gly1, Arg19] h PTH (1-34) group compared with [Gly1, Arg19] h PTH (1-28) , After given PKC inhibitor Go6983, its inhibition of apoptosis disappeared. Conclusion The non-PLC / PKC signal transduction pathway of PTH may inhibit cell apoptosis and increase the number of cells when acting on MC3T3-E1 cells for a long time (48 h).