RUNX2/LAPTM5在小鼠颅骨前成骨细胞矿化诱导中的表达

Expression of RUNX2/LAPTM5 in MC3T3-E1 osteoblastic cellswithinducedmineralization

目的探讨RUNX2/LAPTM5在矿化诱导过程中的表达与成骨及溶酶体的相关性。方法矿化诱导MC3T3-E1,对照组不做处理,茜素红染色检测矿化情况,碱性磷酸酶染色检测成骨分化情况。RT-qPCR及Western blot检测分化0-5 d RUNX2及LAPTM5的基因及蛋白表达。过表达与干扰RUNX2/LAPTM5的表达后,Western blot检测RUNX2、LAPTM5的表达。过表达与干扰LAPTM5的表达后,Western blot检测成骨相关基因碱性磷酸酶、骨钙素的表达。结果倒置显微镜下观察,茜素红染色矿化结节计数随时间变化逐渐增多,矿化结节的大小也逐渐变大;碱性磷酸酶染色蓝紫色颗粒计数随时间逐渐增加。RT-qPCR及Western blot结果显示RUNX2及LAPTM5的表达,其在成骨矿化过程中呈上升趋势(P<0.001)。过表达与干扰RUNX2影响LAPTM5表达(P<0.05);过表达与干扰LAPTM5对RUNX2的影响不显著。过表达与干扰LAPTM5影响了成骨的表达(P<0.01)。结论RUNX2/LAPTM5可能参与了成骨细胞分化调节,RUNX2可能参与LAPTM5的表达调控。RUNX2/LAPTM5可能在溶酶体参与成骨矿化的过程中起到桥梁作用。

Objective To investigate the association of the expressions of RUNX2/LAPTM5 with osteogenesis and lysosomes in osteoblastic cells during mineralization induction.Methods MC3T3-E1 cells cultured in osteogenic induction medium was examined for mineralization and osteogenic differentiation using Alizarin red staining and alkaline phosphatase(ALP)staining,respectively.RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Runx2 and LAPTM5 in the cells during osteogenic induction for 5 days.The effects of overexpression and interference of RUNX2/LAPTM5 on the expressions of ALP and osteocalcin(OCN)in the cells were examined with Western blotting.Results MC3T3-E1 cells cultured in osteogenic induction medium showed an increased number of mineralized nodules over time,and the size of the mineralized nodules increased as the culture time extended;the number of purple-blue granules stained by ALP also increased gradually with time.RT-qPCR and Western blotting showed that the expressions of RUNX2 and LAPTM5 in the cells increased progressively during osteogenic mineralization(P<0.001).Overexpression and interference of RUNX2 obviously affected LAPTM5 expression in the cells(P<0.05);modulation of LAPTM5 expression did not significantly affect RUNX2 expression but caused significant changes in ALP and OCN expressions(P<0.01).Conclusion RUNX2/LAPTM5 may participate in the regulation of osteoblast differentiation,and RUNX2 may be involved in the regulation of LAPTM5 expression.RUNX2/LAPTM5 may play a mediating role in the process of osteogenic mineralization involving lysosomes.