L型钙离子通道Cav 1.2在大鼠根尖牙乳头干细胞成牙向分化中的作用  

Effect of L-type calcium channels Cav 1. 2 on odontoblastic differentiation in rat apical papilla cells

作　　者：高倩倩[1] 葛剑平[1] 琚燕琴[1] 高健[1] 吴美生 赵守亮[1] 

机构地区：[1]同济大学附属口腔医院牙体牙髓科,上海200072

出　　处：《同济大学学报（医学版）》2015年第2期24-28,共5页Journal of Tongji University(Medical Science)

基　　金：国家自然科学基金(81070825;81170951);上海市科委医学引导项目(10411964600)

摘　　要：目的利用慢病毒转染系统构建Cav 1.2基因沉默的大鼠根尖牙乳头干细胞（stem cells from apical papilla,SCAPs）,探讨Cav 1.2在大鼠牙乳头干细胞成牙向分化中的作用。方法取3周龄SD大鼠,采用酶消化法获取大鼠根尖牙乳头干细胞,取第二代大鼠根尖牙乳头干细胞进行克隆平板实验鉴定其增值能力;采用慢病毒转染的方法构建Cav 1.2基因沉默Sh-Cav 1.2牙乳头干细胞及对照组Luc-Cav 1.2牙乳头干细胞,RT-PCR及Western印迹法鉴定其转染效率,并诱导其向成牙本质细胞样细胞分化;在成牙向诱导分化第10天,RT-PCR及茜素红S染色观察Cav 1.2对大鼠根尖牙乳头干细胞成牙向分化的影响。结果分离获得较强增殖能力的大鼠牙乳头干细胞,其中每200个细胞可形成6~10个克隆;荧光倒置显微镜下转染48 h,Sh-Cav 1.2及Luc-Cav 1.2均呈现绿色荧光,转染效率均达到90%;在两组细胞成牙向诱导过程中,实验组Sh-Cav 1.2牙乳头干细胞成牙相关基因DSPP的表达及钙化结节的形成均明显低于对照组Luc-Cav 1.2。结论 L型钙离子通道Cav 1.2对大鼠根尖牙乳头干细胞成牙向分化有重要作用。Objective The stem cells from apical papilla（ SCAPs） which Cav 1. 2 gene was silenced with a Lentivirus-mediated gene knock down system were constructed to clarify the effect of L-type calcium channel-Cav 1. 2 in the process of the odontoblastic differentiation of the apical papilla cells in rats. Methods The apical papilla cells were isolated from 3-weeks-old SD rats by enzyme digestion methods. Then the proliferation ability were measured by calculating the colony forming efficiency in vitro. The SCAPs were transfected with a Sh-Cav 1. 2 Lentivirus vector,while the Luc-Cav 1. 2 vector as a negative control. The expression of Cav 1. 2 gene in differentially treated SCAPs was determined by RT-PCR and Western blotting. The odontoblastic differentiation of differentially treated SCAPs was evaluated via Alizarin red S staining and detecting the expression of dentin sialoph osphoprotein（ DSPP） in SCAPs. Results The SCAPs obtained from SD rats had a strongly proliferation ability,and the colony forming efficiency of isolated SCAPs was 6 to 10 clones in 200 cells. The expression of green fluorescent protein in differentially treated SCAPs were both 90% at 48 h after transfection.Besides,the expression of DSPP gene and the formation of calcific nodules were dramatically decreased in the Cav 1. 2 gene silenced SCAPs compared with the control group. Conclusion L-type calcium channel-Cav 1. 2 has a critical role in the odontoblastic differentiation of SCAPs.

关 键 词：根尖牙乳头干细胞 L型钙离子通道 基因沉默 成牙向分化 

分 类 号：R78[医药卫生—口腔医学]