雌激素受体α-shRNA慢病毒载体对根尖牙乳头干细胞成牙/成骨向分化的影响

›› 2015, Vol. 35 ›› Issue (5): 341-344.

雌激素受体α-shRNA慢病毒载体对根尖牙乳头干细胞成牙/成骨向分化的影响

马姝¹,闫明²,吴锦涛²,王子露³,张光东³,于金华⁴

1. 江苏省南京医科大学
2. 南京医科大学口腔医学研究所?附属口腔医院牙体牙髓病科
3. 南京医科大学口腔医学院
4. 江苏省口腔医院

收稿日期:2014-12-16 修回日期:2015-02-13 出版日期:2015-05-28 发布日期:2015-05-13
通讯作者: 于金华 E-mail:yujinhua@njmu.edu.cn
基金资助:
国家自然科学基金;江苏省自然科学基金;江苏高校优势学科工程

Effects of ERα gene silencing by ERα-shRNA mediated with lentiviral vector on theodonto /osteogenic differentiation of stem cells from apical papilla

Received:2014-12-16 Revised:2015-02-13 Online:2015-05-28 Published:2015-05-13

摘要/Abstract

摘要： 目的构建雌激素受体α（estrogen receptor α,ERα）短发夹RNA（short hairpin RNA,shRNA）慢病毒表达载体，探讨其对根尖牙乳头干细胞（stem cells from apical papilla,SCAPs）体外成牙/成骨向分化的影响。方法根据shRNA设计原则，合成用于体内干扰ERα的两条shRNA序列，重组入慢病毒载体PLKO.1。将PLKO.1对照组与ERα-shRNA (shERα-1和shERα-2)实验组分别感染SCAPs，western blot检测ERα的表达及其对SCAPs成牙/成骨向分化的影响。结果测序结果证实重组质粒构建成功；体外实验表明，SCAPs转染ERα-shRNA后ERα蛋白表达水平降低，其中shERα-2组对ERα的下调作用更明显，两组ERα-shRNA中核心结合蛋白因子2（runt-related transcription factor-2,RUNX2）、成骨细胞特异性转录因子（osterix,OSX）、牙本质涎蛋白（dentin sialoprotein,DSP）和骨钙素（osteocalcin,OCN）的表达均有不同程度下调，与对照组相比具有显著性差异（P<0.05）。结论慢病毒介导的ERα-shRNA成功在体外抑制了目的基因的表达，并显著影响了根尖牙乳头干细胞的成牙/成骨向分化。

关键词: 短发夹RNA, 慢病毒载体, 雌激素受体α, 根尖牙乳头干细胞

Abstract: 目的构建雌激素受体α（estrogen receptor α,ERα）短发夹RNA（short hairpin RNA,shRNA）慢病毒表达载体，探讨其对根尖牙乳头干细胞（stem cells from apical papilla,SCAPs）体外成牙/成骨向分化的影响。方法根据shRNA设计原则，合成用于体内干扰ERα的两条shRNA序列，重组入慢病毒载体PLKO.1。将PLKO.1对照组与ERα-shRNA (shERα-1和shERα-2)实验组分别感染SCAPs，western blot检测ERα的表达及其对SCAPs成牙/成骨向分化的影响。结果测序结果证实重组质粒构建成功；体外实验表明，SCAPs转染ERα-shRNA后ERα蛋白表达水平降低，其中shERα-2组对ERα的下调作用更明显，两组ERα-shRNA中核心结合蛋白因子2（runt-related transcription factor-2,RUNX2）、成骨细胞特异性转录因子（osterix,OSX）、牙本质涎蛋白（dentin sialoprotein,DSP）和骨钙素（osteocalcin,OCN）的表达均有不同程度下调，与对照组相比具有显著性差异（P<0.05）。结论慢病毒介导的ERα-shRNA成功在体外抑制了目的基因的表达，并显著影响了根尖牙乳头干细胞的成牙/成骨向分化。

Key words: Short hairpin RNA, Lentiviral vector, Estrogen receptor α, Stem cells from apical papilla

中图分类号:

R780.2

马姝闫明吴锦涛王子露张光东于金华. 雌激素受体α-shRNA慢病毒载体对根尖牙乳头干细胞成牙/成骨向分化的影响[J]. 口腔医学, 2015, 35(5): 341-344.

参考文献

1. Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, Huang GT. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 2008; 34: 166-171.
2. Vidal O, Kindblom LG, Ohlsson C. Expression and localization of estrogen receptor-beta in murine and human bone. J Bone Miner Res 1999; 14: 923-929.
3. 岑洪, 林茂芳, 黄河, 蔡真. 雌激素受体α和β在间充质干细胞体外成骨、成脂肪分化中的表达变化. 广西医科大学学报 2005; 22: 883-885.
4. Wang SN, Mu JQ, Fan ZP, Yu Y, Yan M, Lei G, Tang CB, Wang ZL, Zheng YY, Yu JH, Zhang GD. Insulin-like growth factor 1 can promote the osteogenic differentiation and osteogenesis of stem cells from apical papilla. Stem Cell Research 2012; 8: 346-356.
5. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806-811.
6. Elbashir SM, Harborth J, Weber K, Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 2002; 26: 199-213.
7. Liu L, Zhang N, Liu J, Min J, Ma N, Liu N, Liu Y, Zhang H. Lentivirus-mediated siRNA interference targeting SGO-1 inhibits human NSCLC cell growth. Tumour Biol 2012; 33: 515-521.
8. Riggs BL, Khosla S, Melton LJ, 3rd. Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev 2002; 23: 279-302.
9. Notelovitz M. Estrogen therapy and osteoporosis: principles & practice. Am J Med Sci 1997; 313: 2-12.
10. Frasor J, Danes JM, Komm B, Chang KCN, Lyttle CR, Katzenellenbogen BS. Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: Insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 2003; 144: 4562-4574.
11. Bonnelye E, Aubin JE. Differential expression of estrogen receptor-related receptor alpha and estrogen receptors alpha and beta in osteoblasts in vivo and in vitro. Journal of Bone and Mineral Research 2002; 17: 1392-1400.
12. 侯宁宁, 朱依敏, 黄荷凤. 雌激素受体α和β在不同雌激素干预大鼠骨代谢中的表达. 分子细胞生物学报 2006; 39: 289-295.
13. Sonoyama W, Liu Y, Fang DAJ, Yamaza T, Seo BM, Zhang CM, Liu H, Gronthos S, Wang CY, Shi ST, Wang SL. Mesenchymal Stem Cell-Mediated Functional Tooth Regeneration in Swine. PLoS One 2006; 1
14. Huang GT, Sonoyama W, Liu Y, Liu H, Wang S, Shi S. The hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. J Endod 2008; 34: 645-651.
15. Bakopoulou A, Leyhausen G, Volk J, Koidis P, Geurtsen W. Effects of resinous monomers on the odontogenic differentiation and mineralization potential of highly proliferative and clonogenic cultured apical papilla stem cells. Dent Mater 2012; 28: 327-339.

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