利用qRT-PCR技术筛选鹿茸生长中心细胞与鹿茸干细胞内参基因

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摘要摘要合适的内参基因是利用qRT-PCR准确分析基因相对表达量的先决条件。为了对鹿茸生长中心细胞和鹿茸干细胞间的差异表达基因进行准确定量分析，本研究筛选了TATA盒结合蛋白(TATA box binding protein, TBP)、β-肌动蛋白(actin beta, ACTB)、微管蛋白1α(tubulin alpha 1a, TUBA1A)、葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase, G6PDH)、3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)及β2-微球蛋白(β2-microglobulin, B2M)这6个基因作为内参基因，利用qRT-PCR技术分别检测这些基因mRNA在鹿茸生长中心细胞及鹿茸干细胞中的表达情况，并对其表达的稳定性进行了综合评估。运用Delta-Ct method，geNorm(ver.3.5)、Bestkeeper(ver.1.0)和NormFinder(ver.0.953)和RefFinder软件综合分析表明，在鹿茸干细胞之间，ACTB和TUBA1A基因表达稳定性最高；在鹿茸生长中心细胞之间，B2M和ACTB基因表达稳定性最高；在所有鹿细胞系之间，B2M和ACTB基因表达稳定性最高。因此，B2M、ACTB以及TUBA1A候选基因可作为研究鹿茸生长中心细胞及鹿茸干细胞基因差异表达的内参基因，本研究为进一步研究鹿茸生长发育机制和开展鹿茸模型应用于生物医学研究提供了一定的理论依据。

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	李春义

关键词 ：鹿茸, 干细胞, 生长中心, qRT-PCR, 内参基因

Abstract：Abstract Annual full regeneration of deer (Cervidae) antlers has been proved to be a stem cell-based process, and antler stem cells reside in both antlerogenic periosteum (AP) and pedicle periosteum (PP). The extraordinary growth rates of antlers provide us a rare system in which rapid cell proliferation is elegantly regulated without becoming cancerous. Thus deer antlers are unique mammalian organs that have potential to be a valuable model for biomedical research, such as the areas of stem cell, organ regeneration, bone development and growth control. qRT-PCR is one of the most common-used techniques in molecule biology study of deer antler. Appropriate internal reference genes are prerequisites for the accurate analysis of gene expression in antler research by qRT-PCR. In order to perform accurate quantitative analysis for differentially expressed genes between antler growth center cells and antler stem cells. In this study, antler stem cell tissue samples including AP, PP and facial periosteum (FP) (as a perfect negative control in antler stem cells research) were collected using surgical operation in a relative sterile environment. Antler growing center tissue samples, including reserve mesenchymal (RM), precartilage (PC), transition zone (TZ) and cartilage (C) were isolated from growing antler tips. Primary culture of these cells were carried out after releasing cells from these tissue types using typeⅡcollagenase (150 U/mL) and cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal calf serum (FBS) in 37 ℃ and 5% CO2. Stability of mRNA expression and levels of 6 candidate reference genes, including actin beta (ACTB), tubulin alpha1a (TUBA1A), TATA box binding protein (TBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PDH) and beta-2-microglobulin (B2M) in antler growth center cells and antler stem cells were measured by through qRT-PCR method. Software, which are Delta-Ct method, geNorm (ver.3.5), Bestkeeper (ver.1.0), NormFinder (ver.0.953) and RefFinder, were used to analyze all the acquired data. The results showed that expressions of ACTB and TUBA1A genes were the most stable in antler stem cells; B2M and ACTB were the same in antler growth center cells. The results of comprehensive analysis using the software indicated that B2M, ACTB and TUBA1A genes were the most stable reference genes, and suitable for antler research. The present study provides a foundation for accurate quantification of differential gene expression for antler research and mechanistic study of this fascinating model for mammalian organ regeneration.

Key words： Deer antler Stem cells Growth center qRT-PCR Reference genes

收稿日期: 2017-01-16 出版日期: 2017-05-02

基金资助:枯草芽孢杆菌纤维素酶系表达系统的构建;枯草芽孢杆菌纤维素酶系表达系统的构建;中国农业科学院科技创新工程项目

通讯作者: 李春义 E-mail: lichunyi1959@163.com

引用本文:

张伟刘佳董振李春义. 利用qRT-PCR技术筛选鹿茸生长中心细胞与鹿茸干细胞内参基因[J]. , 2017, 25(5): 851-860.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I5/851

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