摘要：

目的：验证遗传性釉质发育不全（amelogenesis imperfecta，AI）相关基因FAM83H突变（c.1354C>T, p.Gln452*）后的功能。方法：构建FAM83H突变（c.1354C>T, p.Gln452*）和空白对照野生型慢病毒载体；应用转染技术感染人胚肾上皮细胞（HEK293T），对转染后的细胞形态进行观察；用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测细胞中FAM83H信使RNA（mRNA）的表达水平；细胞免疫荧光技术（immunofluorescence technique）检测转染后的细胞中FAM83H蛋白细胞定位的改变。结果：慢病毒转染HEK293T细胞后，细胞形态无明显变化，FAM83H突变细胞中，mRNA表达低于对照组；细胞免疫荧光结果显示，FAM83H突变后，胞内定位改变，主要在胞核表达，少量表达于细胞质，提示FAM83H胞内作用位点的改变可能导致釉质钙化不全。结论：突变FAM83H改变了其蛋白在HEK293T细胞中的表达及定位，为进一步理解FAM83H突变导致AI的发病机制提供了依据。

关键词: 遗传性釉质发育不全, FAM83H, 慢病毒载体, 亚细胞定位

Abstract:

Objective: To explore the regulatory function of FAM83H mutant (c.1354C>T, p.Gln452*) in amelogenesis imperfecta(AI). Methods: We constructed FAM83H mutant plasmid: mutation(c.1354C>T, p.Gln452*), then carried the lentivirus package for the mutation and control. Infection of human embryonic kidney epithelial cell line(HEK293T) by transfection technique. The cell morphology changes were observed. The expression level of FAM83H mRNA was detected by real-time quantitative polymerase chain reaction(RT-qPCR). The subcellular localization of FAM83H protein in transfected cells were detected by immunofluorescence technique. Results: After lentivirus transfection into HEK293T cells, there was no significant cell morphologic changes. The expression of mRNA in FAM83H mutant cells was lower than that in control group. The results of cellular immunofluorescence showed that the intracellular location of FAM83H changed after mutation. Mutant FAM83H was mainly expressed in the nucleus and a small amount in the cytoplasm, which suggested that the change of intracellular action site of FAM83H may lead to incomplete enamel calcification. Conclusion: Mutant FAM83H changes the expression and localization of its protein in HEK293T cells, which provides a basis for further understanding the pathogenesis of AI caused by FAM83H mutation.

Key words: amelogenesis imperfecta, FAM83H, lentivirus transfection, subcellular localization