RUNX2/LAPTM5在小鼠颅骨前成骨细胞矿化诱导中的表达

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。

Expression of RUNX2 /LAPTM5 inMC3T3-E1osteoblastic cellswith inducedmineralization

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.