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高倩倩,葛剑平,琚燕琴,等.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[J].同济大学学报（医学版）,2015,36(2):24-28. [点击复制]
- GAO Qian-qian,GE Jian-ping,JU Yan-qin,et al.Effect of L-type calcium channels Cav 1.2 on odontoblasticdifferentiation in rat apical papilla cells[J].同济大学学报（医学版）,2015,36(2):24-28. [点击复制]
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| 摘要: |
| 目的 探讨L型钙离子通道Cav 1.2在大鼠根尖牙乳头干细胞向成牙本质细胞样细胞分化过程中的作用。方法 利用酶消化法分离大鼠根尖牙乳头干细胞,体外扩增,结晶紫染色检测牙乳头干细胞的克隆形成率；利用慢病毒转染系统介导pLVX-shRNA2质粒在牙乳头干细胞中表达,抑制牙乳头干细胞中Cav 1.2基因的表达。荧光显微镜下观察转染牙乳头干细胞绿色荧光蛋白的表达,RT-PCR及Western印迹法检测牙乳头干细胞中Cav 1.2基因沉默效率；矿化诱导体系下诱导Cav 1.2基因沉默牙乳头干细胞向成牙本质细胞样细胞分化,应用茜素红染色检测Cav 1.2对牙乳头干细胞成牙向分化的影响。结果 牙乳头干细胞的克隆形成能力为每200个细胞形成6~10个克隆；PCR及Western印迹法结果显示牙乳头干细胞Cav 1.2基因沉默效率达到70%；在牙乳头干细胞成牙向诱导第10天时,与对照组Luc-Cav 1.2相比,Cav 1.2基因沉默组成牙相关基因DSPP表达水平明显下降,同样茜素红染色结果显示Cav 1.2基因沉默牙乳头干细胞钙化结节形成也受到明显抑制。结论 L型钙离子通道Cav 1.2在大鼠根尖牙乳头干细胞向成牙本质细胞样细胞分化的过程中发挥着非常重要的作用。【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48h,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。 |
| 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 |
| DOI：10.16118/j.1008-0392.2015.02.006 |
| 投稿时间：2014-11-02 |
| 基金项目:国家自然科学基金(81070825,1)；上海市科委医学引导项目(10411964600) |
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| Effect of L-type calcium channels Cav 1.2 on odontoblasticdifferentiation in rat apical papilla cells |
| GAO Qian-qian,GE Jian-ping,JU Yan-qin,GAO Jian,WU Mei-sheng,ZHAO Shou-liang |
| (Dept. of Conservative Dentistry, Stomatology Hospital, Tongji University, Shanghai 200072, China) |
| Abstract: |
| Objective To investigate the role of L-type calcium channel-Cav 1.2 in odontoblastic differentiation of apical papilla cells in rat. Methods Rat apical papilla cells were isolated by enzyme digestion method and expanded in vitro, the colony-forming efficiency (CFE) of apical papilla cells was confirmed by crystal violet staining assay. Plasmid pLVX-shRNA2 was transferred into apical papilla cells by lentivirus expression system to obtain Cav 1.2 gene-silenced apical papilla cells. The fluorescent microscope was applied to detecting the expression of green fluorescent protein (GFP) in transfected apical papilla cells. Expression of dentin sialoph osphoprotein (DSPP) mRNA and proterin in apical papilla cells were measured by RT-PCR and Western blotting, respectively. The transfected apical papilla cells were induced with mineralization differentiation system, and the differentiated odontoblast-like cells were identified by Alizarin red S staining.Results The colony-forming efficiency of isolated apical papilla cells were 6 to 10 clones in 200 cells. The expression of Cav 1.2 in transfected apical papilla cells was markedly decreased compared with the Luc control. The mRNA and protein levels of DSPP gene were decreased at 10th day after the induction of odontoblastic differentiation in Cav 1.2-silenced apical papilla cells. The results of Alizarin red S staining were consistent with the results of DSPP gene expression. Conclusion L-type calcium channel may play an important role in odontoblastic differentiation of apical papilla cells in rats.【Abstract】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 48h 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. |
| Key words: apical papilla cells L-type calcium channels gene silencing odontoblastic differentiation |
