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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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

Received: 16 January 2017 Published: 02 May 2017

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	ZHANG Wei
	LIU Jia
	DONG Zhen
	LI Chun-Xi

Cite this article:

ZHANG Wei,LIU Jia,DONG Zhen, et al. Reference Gene Selection for Antler Stem Cells and Antler Growth Center Cells for qRT-PCR[J]. , 2017, 25(5): 851-860.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2017/V25/I5/851

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