鸡生殖干细胞中Piwi基因的干涉效率

doi:10.3864/j.issn.0578-1752.2016.03.014

摘要/Abstract

摘要： 【目的】针对禽类干细胞研究中，存在转染效率与干涉效率低下的问题，通过比较不同干涉方式对鸡原始生殖细胞（primordial germ cells, PGCs）中Piwi基因的干涉效果，探讨提高禽类生殖干细胞干涉效率的有效方法。为进一步研究Piwi基因参与干细胞自我更新、RNA沉默以及转录后调控过程等生物学功能研究奠定基础。【方法】根据已建立的原代细胞分离技术，分别从健康鸡群的10日龄和4日龄鸡胚中分离成纤维细胞（chicken embryo fibroblast, CEF）与生殖脊，其中CEF作为PGCs细胞的饲养层，生殖脊经Trypsin-EDTA室温下消化获得PGCs细胞，并通过形态学、化学方法（PAS糖原染色、AKP 活性检测）和免疫学方法（TERT抗体、SSEA-1抗体）对其进行鉴定。根据Genbank公布的鸡Piwi基因的mRNA序列（NM_001098852），利用在线软件分别靶向不同位点设计合成得到3个小片段的stealth siRNAs，并将通用无义序列BLOCK-iT^TM Alexa Fluor® Red Fluorescent Oligo作为荧光素标记的阴性对照siRNA。同时，在线设计三条干扰序列和一条非特异性阴性对照序列，将其插入线性化空载体 pRNA-U6.1，构建不同的shRNA干涉载体。分别采用不同类型的转染试剂将构建好的siRNA和shRNA转染PGCs细胞。首先利用通用无义siRNA以及仅带有GFP荧光标记基因的shRNA载体作为阴性对照，检测siRNA和shRNA转染效率，确定最佳转染条件。其次，将试验组siRNA和shRNA在优化条件下转染PGCs细胞，分别收集转染了24、48、72和144 h这4个时间点的细胞，提取各个时间段的总RNA，采用实时荧光定量PCR技术检测Piwi基因mRNA表达水平，并用SPSS软件分析不同处理组的差异。【结果】在对原代分离的PGCs细胞进行了形态学、化学以及免疫学方法鉴定后，确认其所获得的细胞为PGCs细胞且状态良好。根据不同的转染试剂量与siRNA或shRNA载体量的配比，得出siRNA转染最优条件为siRNA﹕LipofectamineTM 2000=50 pmol﹕2 μL; shRNA转染最优条件为shRNA﹕X-Treme GENE HP DNA Transfection Reagent=500 ng﹕1.5 μL。在以上最优转染条件下，将试验组siRNA和shRNA分别转染第二代PGCs细胞。发现在siRNA干涉组中，阴性siRNA组和空白组相比，Piwi基因表达量在24、48、72和144 h差异均不显著（P＞0.05）；而3个试验组siRNA与阴性对照组相比，在24、48和72 h的Piwi基因表达量均呈现显著下降（P＜0.05），但在144 h时表达差异不显著（P＞0.05）。在shRNA干涉组中，空白组和阴性对照组相比，Piwi基因表达量在4个时间点上差异均不显著（P＞0.05）；而3个试验组与阴性对照组相比，Piwi基因表达量在4个时间点上均显著下降（P＜0.05）。与siRNA干涉试验组相比，shRNA 载体表现出更强及更长时间的稳定抑制Piwi基因的效果。【结论】siRNA与shRNA均能较好抑制目的基因的mRNA表达，不同干涉效果表明采用shRNA 载体具有更好及更长时间的抑制作用，这为RNAi技术在家禽生殖干细胞中的应用研究提供基础资料。

关键词: 鸡, 家禽干细胞, 生殖干细胞（PGCs）, Piwi 基因, siRNA, shRNA, RNAi

Abstract: 【Objective】Solving the low transfection efficiency and interference efficiency in poultry stem cell is a big challenge so far. This study was designed to compare the interference effects on Piwi gene expression in chicken primordial germ cells (PGCs) using different ways, aiming to explore effective methods to interfere the poultry stem cells, which is a fundamental work for the study of the self-renewal of stem cell, RNA silencing and post-transcriptional regulation. 【Method】According to the techniques of the separation of primary germ cell, the authors selected 10th and 4th days fertilized eggs post incubation to separate the chicken embryo fibroblast cells(CEF) and germinal ridge, respectively; Herein, the CEF cells were taken as a feeder layer and the PGCs cells were treated by Trypsin-EDTA. The PGCs cells were further identified by the morphology, chemical method and immunology assay. According to the mRNA sequence of chicken Piwi gene published by Genbank (NM_001098852), three positive siRNAs were chemically synthesized and the BLOCK-iT™ Alexa Fluor® Red Fluorescent Oligo was as a negative control. Simultaneously, three interfering vectors of shRNA were constructed using the free carrier of pRNA-U6.1, and an empty vector with GFP fluorophore was as a negative control. Afterwards, the transfection efficiency was detected by the siRNA and shRNA negative control using different types of transfection reagent to optimize the condition, and extracted the RNA of PGCs cells at 24, 48, 72 and 144 h post transfection and analyzed differential expressions of Piwi gene with RT-PCR techniques. All data were analyzed by the SPSS software.【Result】With the premise of well identification of the PGCs cells, siRNA and shRNA were successfully transfected into the cells under the optimized conditions with 50 pmol siRNA and 2 μL Lipofectamine TM 2000 for siRNA transfection group and 500 ng shRNA and 1.5 μL X-Treme GENE HP DNA Transfection Reagent for shRNA transfection group. Compared to the blank group, the Piwi expressions of the negative control had no significant difference at the four time points (24,48,72 and 144 h) in siRNA and shRNA intervention group (P＞0.05); Compared to the negative control and the blank group, Piwi gene expressions were significantly down-regulated in experimental groups at the first three time points in siRNA intervention group, while were significantly down-regulated at all of time points in the shRNA intervention group (P＜0.05).【Conclusion】Collectively, the interference effects on Piwi gene expression in chicken PGCs were compared by siRNA and shRNA RNAi technology, and indicated that the shRNA vector had higher and more stable interference efficiency, which help to provide some basic information for future research of RNAi technology.

Key words: chicken, poultry stem cell ; primordial germ cells(PGCs), Piwi gene, siRNA, shRNA, RNAi

李志腾，常国斌，徐璐，马腾，陈静，陈蓉，王洪志，刘璐，徐琪，陈国宏. 鸡生殖干细胞中Piwi基因的干涉效率[J]. 中国农业科学, 2016, 49(3): 563-472.

LI Zhi-teng, CHANG Guo-bin, XU Lu, MA Teng, CHEN Jing, CHEN Rong, WANG Hong-zhi, LIU Lu, XU Qi, CHEN Guo-hong. Interference Efficiency of Piwi Gene Expression in the Chicken Germ Stem Cells[J]. Scientia Agricultura Sinica, 2016, 49(3): 563-472.

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参考文献

[1] Cox D N, Chao A, Baker J, Chang L, Qiao D, Lin H. A novel class of evolutionarily conserved genes defined by Piwi are essential for stem cell self-renewal. Genes & development, 1998, 12(23):3715-3727.

[2] Cox D N, Chao A, Lin H. Piwi encodes a nucleoplasm factor whose activity modulates the number and division rate of gremlins stem cells. Development, 2000, 127(3):503-514.

[3] 李碧春, 施青青, 孙敏. 鸡生殖干细胞研究现状与展望. 中国家禽, 2011, 33(11):1-6.

Li B C, Shi Q Q, Sun M. The current status and prospects in germline stem cell. China Poultry, 2011, 33(11):1-6.(in Chinese)

[4] 陈胜锋, 王丙云, 计慧琴.鸡胚胎干细胞及其应用研究进展.动物医学进展, 2006, 27(1):9-13.

Chen S F, Wang B Y, Ji H Q. Applications and advances in chicken embryonic stem cells. Progress in Veterinary Medicine, 2006, 27(1): 9-13.(in Chinese)

[5] Peters L, Meister G. Argonaute proteins: mediators of RNA silencing. Molecular Cell, 2007, 26(5):611-623.

[6] Seto A G, Robert E, Lau N C. The coming of age for Piwi proteins. Molecular Cell, 2007, 26(8):603-609.

[7] 谢小燕, 裴雪涛. Piwi家族:干细胞分裂中的重要调控基因.生理科学进展, 2003, 34(2):139-141.

Xie X Y, Pei X T. Piwi family: An important gene regulate the stem cell division. Progress of Physiological Sciences, 2003, 34(2): 139-141. (in Chinese).

[8] Kuramochi-miyagawa S, Kimura T, Ljirit W, Isobe T, Asada N, Fujita Y, Ikawa M, Iwai N, Okabe M, Deng W, Lin H, Matsuda Y, Nakano T. Mili, a mammalian member of Piwi family gene, is essential for spermatogenesis. Development, 2004, 131:839-849.

[9] Deng W, Lin H. miwi, a murine homolog of Piwi, encodes a cytoplasmic protein essential for spermatogenesis. Developmental Cell, 2002, 2(6):819-830.

[10] Shen X Y, Liu Y X, Chen H, Zhu S, Zhong X. Correlation between PIWI proteins and prognosis of Colon cancer. Chinese Journal of Gastrointestinal Surgery,2013, 18(9):530-535.

[11] Paddison P J, CaudyA, Hannon G J. Stable suppression of gene expression by RNAi in mammalian cells. Science, 2002, 99(3): 1443-1448.

[12] Elbashir S M, Martinez J, Pakaniowska A, Lendeckel W, Tuschl T. Functional anatomy of siRNA for mediating efficient RNAi in Drosophila Melanogaster embryolysate. Embo Journal, 2001, 20: 6877-6888.

[13] Holen T, Amarzguioui M A, Wiiger M T, Babaie E, Prydz H . Postional effect of short interferencing RNAs targetingthe human coagulation trigger tissue factor. Nucleic Acid Research, 2002, 30(8): 1757-1766.

[14] 秦玉新, 蒙凌华, 丁健. RNA干扰技术的研究进展. 中国药理学通报, 2007, 23(4):421-424.

Qin Y X, Meng L H, Ding J. Research progresses of RNA interference. Chinese Pharmacological Bulletin, 2007, 23(4):421-424. (in Chinese)

[15] Tolia N H, Joshua T L. Slicer and the argonautes. Nature Chemical Biology, 2007, 3(1):36-43.

[16] Hamburger V, Hamilton H L. A series of normal stages in the development of the chick embryo. Developmental Dynamics,1992, 195(4):231-272.

[17] Li B C, Tian Z Q, Sun M, Xu Q, Wang X Y, Qin Y R. Directional differentiation of chicken primordial germ cells into adipocytes, neuron-like cells, and osteoblasts. Molecular Reproduction and Development, 2010, 77(9):795-801.

[18] 李建超, 张颖, 戴爱琴, 王洪志, 翟飞, 华登科, 夏明秀, 常国斌, 陈国宏. 体外抑制Piwi基因对鸡原始生殖细胞相关基因表达的影响.畜牧兽医学报, 2014, 45(6):871-878.

Li J C, Zhang Y, Dai A Q, Wang H Z, Zhai F, Hua D K, Xia M X, Chang G B, Chen G H. In Vitro inhibition of Piwi gene expression and the influence on related genes in primordial germ cells. Acta Veterinarian et Zootechnical Sinica, 2014, 45(6):871-878.(in Chinese)

[19] Aravin A A, Hannon G J, Brennecke J. The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science, 2007, 318 (5851): 761-764.

[20] 任小青. RNA 干扰技术及其应用. 天津农学院学报, 2009, 16(1): 45-48.

Ren X Q. RNA Interference and its application. Journal of Tianjin

Agricultural University, 2009, 16(1): 45-48. (in Chinese)

[21] 房宝英, 何冬梅, 张洹. siRNA及其导入体内外方法的研究进展. 国际病理科学与临床杂志, 2007, 27(1):44-47.

Fang B Y, He D M, Zhang Y. SiRNA and its delivery in vivo and vitro. International Journal of Pathology and Clinical Medicine, 2007, 27(1):44-47. (in Chinese)

[22] Ren Z, Li S, Wang Q L, Xiang Y F, Cui Y X, Wang Y F, Qi R B, Lu D X, Zhang S M, Zhang P Z. Effect of siRNA on HSV-1 plaque formation and relative expression levels of RR mRNA. Virologica Sinica, 2011, 26(1):40-46.

[23] Paddison P J, Candy A A, Bernstein E, Bernstein E, Hannon G J, Conklin D S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cell. Genes & Development, 2002, 16: 948.

[24] 王燕丽, 郝柱, 李艳玲, 彭静, 沈一飞.梅岭土鸡原始生殖细胞的生物学特性. 农业生物技术学报, 2012, 20(4):397-403.

Wang Y L, Hao Z, Li Y L, Peng J, Shen Y F. Biological characterization of primordial germ cells from Meiling chicken (Gallus domesticus). Journal of Agricultural Biotechnology, 2012, 20(4):397-403.(in Chinese)

[25] 王红刚, 林波, 汪文利. 不同转染条件影响质粒转染效率的探讨. 河南大学学报, 2012, 31(2):111-118.

Wang H G, Lin B, Wang W L. Study for the influence of different transfected condition on plasmid transfected efficiency. Journal of Henan University, 2012, 31(2):111-118.(in Chinese)