Xing L, Geng Y, Li W, Lin L, Xu P
Department of Oral Implantology, Affiliated Stomatology Hospital of Guangzhou Medical University, Guangzhou Key Laboratory of Basic and Applied Research in Oral Regenerative Medicine, Guangzhou 510182, China.
Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Aug 31;41(9):1394-1399. doi: 10.12122/j.issn.1673-4254.2021.09.15.
To investigate the association of the expressions of RUNX2/LAPTM5 with osteogenesis and lysosomes in osteoblastic cells during mineralization induction.
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.
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 ( < 0.001). Overexpression and interference of RUNX2 obviously affected LAPTM5 expression in the cells ( < 0.05); modulation of LAPTM5 expression did not significantly affect RUNX2 expression but caused significant changes in ALP and OCN expressions ( < 0.01).
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.
探讨在矿化诱导过程中，成骨细胞中RUNX2/LAPTM5的表达与成骨及溶酶体的关联。
分别采用茜素红染色和碱性磷酸酶（ALP）染色，检测在成骨诱导培养基中培养的MC3T3-E1细胞的矿化及成骨分化情况。运用RT-qPCR和蛋白质免疫印迹法检测成骨诱导5天期间细胞中Runx2和LAPTM5的mRNA及蛋白表达。通过蛋白质免疫印迹法检测RUNX2/LAPTM5过表达和干扰对细胞中ALP和骨钙素（OCN）表达的影响。
在成骨诱导培养基中培养的MC3T3-E1细胞，随着时间推移矿化结节数量增加，且随着培养时间延长矿化结节尺寸增大；ALP染色的紫蓝色颗粒数量也随时间逐渐增加。RT-qPCR和蛋白质免疫印迹法显示，在成骨矿化过程中细胞中RUNX2和LAPTM5的表达逐渐增加（<0.001）。RUNX2的过表达和干扰明显影响细胞中LAPTM5的表达（<0.05）；LAPTM5表达的调节未显著影响RUNX2表达，但导致ALP和OCN表达发生显著变化（<0.01）。
RUNX2/LAPTM5可能参与成骨细胞分化的调控，且RUNX2可能参与LAPTM5表达的调控。RUNX2/LAPTM5可能在涉及溶酶体的成骨矿化过程中起介导作用。
