p38β在矿化诱导液诱导的人牙髓细胞成牙本质向分化中的作用

doi:10.3877/cma.j.issn.1674-1366.2015.01.001

中华口腔医学研究杂志(电子版) ›› 2015, Vol. 09 ›› Issue (01) : 1 -6. doi: 10.3877/cma.j.issn.1674-1366.2015.01.001

所属专题：文献；

基础研究

p38β在矿化诱导液诱导的人牙髓细胞成牙本质向分化中的作用

刘鹏程¹, 秦伟¹, 黄舒恒¹, 陈玲玲¹, 邢泉¹, 林正梅¹^,^†(

)

^1. 510055　广州，中山大学光华口腔医学院·附属口腔医院，广东省口腔医学重点实验室

收稿日期:2014-11-21 出版日期:2015-02-01
通信作者: 林正梅
基金资助:
国家自然科学基金(81271124); 广东省科技计划国际合作项目(2012B050300027); 广东省医学科研基金(A2014255、B2013152)

The role of p38β in odontoblastic differentiation of human dental pulp cells

Pengcheng Liu¹, Wei Qin¹, Shuheng Huang¹, Lingling Chen¹, Quan Xing¹, Zhengmei Lin¹^,^†()

^1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China

Received:2014-11-21 Published:2015-02-01
Corresponding author: Zhengmei Lin
About author:
Corresponding author: Lin Zhengmei, Email: linzhm@mail.sysu.edu.cn, Tel: 020-83741753

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目的

研究p38β丝裂原激活的蛋白激酶（MAPK）对矿化诱导液诱导的人牙髓细胞（hDPC）成牙本质向分化的影响。

方法

体外培养hDPC，Western blot检测hDPC矿化诱导0、3、7、14 d后p38β蛋白表达情况；构建3条慢病毒载体p38β-shRNA并分别转染hDPC，Western blot及实时荧光定量聚合酶链反应（PCR）检测p38β蛋白及基因表达；设立空白对照组、矿化诱导（OM）组、OM+空载体（NC）组、OM+sh-p38β组共4组。成牙本质向矿化诱导1、7、14 d，实时荧光定量PCR检测成牙本质向分化指标牙本质涎磷蛋白（DSPP）、碱性磷酸酶（ALP）的基因表达；Western blot检测成牙本质向分化指标DSPP蛋白的表达；成牙本质向矿化诱导14 d，茜素红染色检测矿化结节的形成情况。

结果

Western blot结果表明正常牙髓组织中存在p38β蛋白；成功构建p38β稳定低表达的hDPCs（实时荧光定量PCR显示三条序列干扰效率分别为55.4%、68.3%、54.2%）；实时荧光定量PCR显示沉默p38β的hDPCs经矿化诱导后，成牙本质向分化指标DSPP、ALP基因表达水平显著降低，Western blot结果显示成牙本质向分化指标DSPP蛋白表达水平显著降低；茜素红染色结果显示，矿化诱导+sh-p38β组与空白对照组较另外两组的矿化结节数量明显减少。

结论

p38β在OM诱导的hDPC成牙本质向分化中发挥作用。

关键词: 丝裂原激活的蛋白激酶, 牙髓细胞, 成牙本质向分化

Objective

To investigate the role of p38β MAPK in odontoblastic differentiation of human dental pulp cells (hDPCs) .

Methods

HDPCs obtained from the healthy third molars or premolars were cultured. Western blot were used to detect the expression of p38β in hDPCs after OM induced for 0, 3, 7 and 14 days. Lentivirus shRNA were constructed and transfected into hDPCs, The protein and mRNA levels of p38β were detected by western blot and quantitative real-time PCR. Then hDPCs were treated with odontogenic medium for 1, 7 and 14 days. The DSPP protein and mRNA levels were analyzed by western blot and quantitative real-time PCR, and the mRNA levels of ALP were also analyzed by quantitative real-time PCR. The formation of mineralized nodules were examined by Alizarin red staining.

Results

Western blot analysis showed that the expression of p38β can be detected in hDPCs. Quantitative real-time PCR showed that DSPP and ALP mRNA levels were obviously decreased after stable low expression of p38β, the levels of DSPP protein also decreased. Mineralization nodules were less observed in p38β-shRNA group after the induction of odontogenic differentiation of HDPCs for 14 days.

Conclusion

P38β MAPK is involved in OM-induced odontoblastic differentiation of HDPCs.

Key words: Mitogen-activated protein kinases, Dental pulp cell, Odontoblast differentiation

表1 实时荧光定量PCR引物序列

基因	引物序列	大小（bp）
DSPP	F：5 ′ -TGGAGCCACAAACAGAAGCAACAC-3 ′	105
?	R：5 ′ -TCTTGGACAACAGCGACATCCTCA-3 ′	?
ALP	F：5 ′ -CCACGTCTTCACATTTGGTG-3 ′	196
?	R：5 ′ -AGACTGCGCCTGGTAGTTGT-3 ′	?
p38β	F：5 ′ -AGAAGGTGGCGGTGAAGAAG-3 ′	121
?	R：5 ′ -TCCAGAAGCCCGATGACG-3 ′	?
GAPDH	F：5 ′ -TCTCCTCTGACTTCAACAGCGACA-3 ′	126
?	R：5 ′ -CCCTGTTGCTGTAGCCAAATTCGT-3 ′	?

表1 实时荧光定量PCR引物序列

基因	引物序列	大小（bp）
DSPP	F：5 ′ -TGGAGCCACAAACAGAAGCAACAC-3 ′	105
?	R：5 ′ -TCTTGGACAACAGCGACATCCTCA-3 ′	?
ALP	F：5 ′ -CCACGTCTTCACATTTGGTG-3 ′	196
?	R：5 ′ -AGACTGCGCCTGGTAGTTGT-3 ′	?
p38β	F：5 ′ -AGAAGGTGGCGGTGAAGAAG-3 ′	121
?	R：5 ′ -TCCAGAAGCCCGATGACG-3 ′	?
GAPDH	F：5 ′ -TCTCCTCTGACTTCAACAGCGACA-3 ′	126
?	R：5 ′ -CCCTGTTGCTGTAGCCAAATTCGT-3 ′	?

图1 正常人牙髓细胞矿化诱导培养后p38β蛋白表达情况

图2 p38β-shRNA转染人牙髓细胞后p38β蛋白及基因表达情况

图3 p38β-shRNA转染人牙髓细胞48 h后荧光表达情况

图4 p38β对牙髓细胞DSPP、ALP基因水平的影响

图5 p38β对牙髓细胞DSPP蛋白水平的影响

图6 各组hDPC矿化诱导14 d后茜素红染色情况

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