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.
1. 褚文辉，赵海平，杨福合，等.2009.梅花鹿Col Ⅹ基因的RNAi重组慢病毒载体的构建及鉴定[J].中国农业大学学报,14(4):29-34.(Chu W H, Zhao H P, Yang F H, et al.2009.Construction and characterization of lentiviral vector of RNA interference of collagen type Ⅹgene of sika deer[J]. Journal of China agricultural university,14(4):29-34.) 2. 郭倩倩，王大涛，褚文辉，等.2014. 利用慢病毒表达载体干扰梅花鹿角柄骨膜细胞P21基因[J].吉林农业大学学报,36(1):116-121.(Guo Q Q, Wang D T, Chu W H, et al.2014. RNA interference targeting P21 gene of pedicle periosteum cells from sika deer by shRNA lentivirus [J]. Journal of Jilin agricultural university, 36(1):116-121.) 3. 韩春梅，高庆华，李世军，等.2012. 原癌基因ｃ－ｍｙｃ在塔里木马鹿茸不同生长期的表达[J].中国兽医学报,32(10):1536-1541.(Han C M, Gao Q H, Li S J, et al.2012. Expression of proto-oncogene C-myc in the tarim deer antler in different growth stages[J]. Chin J Vet Sci,32(10):1536-1541. 4. 郝立宏，马坚伟，邵淑娟.2011.甘油醛－３－磷酸脱氢酶（ＧＡＰＤＨ）作为内参的质疑以及在肿瘤中的表达[J].中国组织化学与细胞化学杂志,20(5):496-498.( Hao L H, Ma S W, Shao S J.2011. 甘油醛－３－磷酸脱氢酶（ＧＡＰＤＨ）作为内参的质疑以及在肿瘤中的表达[J].Chinese journal of histochemistry and cytochemistry, 20(5):496-498.) 5. 何刚.1992. 鹿茸的药理作用与临床应用的研究进展[J]. 延边医学院学报，15(3):231-234. (He G. 1992.鹿茸的药理作用与临床应用的研究进展[J]. Journal of Yanbian Medical College,15(3):231-234.) 6. 蒋晓梅，张新全，严海东，等.2014. 柳枝稷根组织实时定量PCR分析中内参基因的选择[J].农业生物技术学报,22(1):55-63.( Jiang X M, Zhang X Q, Yan H D, et al.2014. Reference gene selection for real-time quantitative PCR normalization in Switchgrass (Panicum virgatum L.) rot tissue[J]. Journal of agricultural biotechnology,22(1):55-63.) 7. 秦晓艺，王杰.2015. 杏鲍菇采后木质化相关基因实时定量ＰＣＲ中内参基因的选择[J].西北农林科技大学学报（自然科学版）,43(7):221-227.(Qing X Y and Wang J.2015. Selection of reference gene for quantitative real-time PCR analysis of lignification related genes in postharvest Pleurptus eryngii[J].Journal of Northwest A&F University(Nat.Sci.Ed.),43(7):221-227.) 8. 孙亚丽，张德辉，赵亮，等.2014.铜胁迫下天蓝苜蓿根组织实时定量PCR内参基因的选择[J].农业生物技术学报,22(10):1223-1231.(Sun Y L, Zhang D H, Zhao L, et al.2014. Reference gene selection for real-time quantitative PCR in black medic (Medicago lupulina L.) root tissue under copper stress[J]. Journal of agricultural biotechnology, 22(10):1223-1231.) 9. 王继英，王彦平，郭建凤，等.2015.仔猪外周血中内参基因的筛选及细胞因子和受体的表达水平[J].中国农业科学,48(7):1437-1444.( Wang J Y, Wang Y P, Guo J F, et al.2015. Selection of Reference Genes and Determination of Cytokines and Receptor mRNA Expression in Peripheral Blood of Piglets[J].Scientia Agricultura Sinica,48(7):1437-1444.) 10. 谢红梅，孟祥宇，孙天霞，等.2016. siＲNA 沉默胸腺素β4 对鹿茸间充质干细胞增殖的影响[J].长春中医药大学学报,32(3):462-464.(Xie H M, Meng X Y, Sun T X, et al.2016.Effect of siRNA-mediated thymosin β4 silence on proliferation in mesenchymal stem cell[J].Journal of Changchun university of Chinese medicine,32(3):462-464.) 11. 张然然，刘华淼，邢秀梅，等.2015.鹿茸组织中内参基因的筛选和验证[J].中国畜牧兽医,42(4):883-889.(Zhang R R, Liu H M, Xing X M, et al. Selection and validation of reference genes in velvet antlers tissues[J].China animal husbandry &veterinary medicine ,42(4):883-889.) 12. 中国畜牧业协会鹿业分会.2015. 2014中国鹿业发展报告[C].第六届(2015)中国鹿业发展大会，中国辽宁西丰，中国畜牧业协会编著，页码:3-6. 13. Andersen C L, Jensen J L, ?rntoft T F. 2004. Normalization of Real- time quantitative reverse transcription- PCR data: A model- based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Research,64(15): 5245-5250. 14. Cegielski M, Izykowska I, Podhorska-Okolow M, et al. 2009. Histological studies of growing and mature antlers of red deer stags (Cervus elaphus)[J]. Anat Histol Embryol,38(3):184-8. 15. F Xie, P Xiao, D Chen, L Xu, B Zhang. 2012. miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant molecular biology 80 (1), 75-84. 16. Fior J. 2014. Salamander regeneration as a model for developing novel regenerative and anticancer therapies[J]. J Cancer, 5(8):715-9. 17. Huggett J, Dheda K, Bustin S, et al. 2005. Real-time RT-PCR normalisation; strategies and considerations[J]. Genes Immun,6(4):279-84. 18. Kierdorf U and Kierdorf H.2011. Deer antlers - a model of mammalian appendage regeneration: an extensive review[J]. Gerontology,57(1):53-65. 19. Li C and Chu W. 2016. The regenerating antler blastema: the derivative of stem cells resident in a pedicle stump[J]. Front Biosci (Landmark Ed),21:455-67. 20. Li C and Suttie JM. 2003. Tissue collection methods for antler research[J]. Eur J Morphol,41(1):23-30. 21. Li C, Yang F and Suttie J. 2011.Stem cells, stem cell niche and antler development[J]. Animal Production Science,51:267-276. 22. Li C, Zhao H, Liu Z, et al. 2014. Deer antler--a novel model for studying organ regeneration in mammals[J]. Int J Biochem Cell Biol,56:111-22. 23. Li C.2012.Deer antler regeneration: a stem cell-based epimorphic process[J]. Birth Defects Res C Embryo Today,96(1):51-62. 24. Li C.2013. Histogenetic aspects of deer antler development[J]. Front Biosci (Elite Ed),5:479-89. 25. Liu M, Yao B, Zhang H, et al.2014. Identification of novel reference genes using sika deer antler transcriptome expression data and their validation for quantitative gene expression analysis[J]. Genes & Genomics,36(5):573-582. 26. Lord EA, Martin SK, Gray JP, et al. 2007. Cell cycle genes PEDF and CDKN1C in growing deer antlers [J]. Anat Rec (Hoboken),290(8):994-1004. 27. Nguewa PA, Agorreta J, Blanco D, et al.2008. Identification of importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens[J]. BMC Mol Biol,9:103. 28. Pfaffl M W, Tichopad A, Prgomet C, et al. 2004.Determination of stable housekeeping genes,differentially regulated target genes and sample integrity: BestKeeper- Excel- based tool using pair- wise correlations[J]. Biotechnology Letters, 26(6): 509-515. 29. Pita-Thomas W, Fernández-Martos C, Yunta M,et al.2010. Gene Expression of Axon Growth Promoting Factors in the Deer Antler[J]. PLoS One,5(12):e15706. 30. Quackenbush J. 2002. Microarray data normalization and transformation[J]. Nature Genetics, 32: 496-501. 31. Radoni? A, Thulke S, Mackay IM, et al.2004. Guideline to reference gene selection for quantitative real-time PCR[J]. Biochem Biophys Res Commun,313(4):856-62. 32. Silver N, Best S, Jiang J, et al. 2006. Selection of housekeeping genes for gene expression studies in human reticulocytes using Real- time PCR[J]. BMCMolecular Biology, 7: 33. 33. Valasek MA and Repa JJ. 2005. The power of real-time PCR[J]. Adv Physiol Educ,29(3):151-9. 34. Vandesompele J, De Preter K, Pattyn F, et al. 2002. Accurate normalization of Real-time quantitative RT-PCR data by eometric averaging of multiple internal control genes[J].Genome Biology, 3(7): research0034.1-0034.11.
