兔原始生殖细胞体外培养的研究

doi:10.11843/j.issn.0366-6964.2020.10.012

Abstract

Abstract: The purpose of the present study was to explore the optimal condition for the isolation and culture of rabbit primordial germ cells(PGCs) in vitro, and to establish a mature and stable method for the isolation and culture of rabbit PGCs in vitro. Firstly, trypsin digestion and mechanical methods were used to explore the best method for isolating PGCs from 14- to 18-day-old fetal rabbits in vitro, and trypsin digestion, mechanical method and digestion with the feeder layer were used to explore the optimal way to passage the isolated PGCs. Then, 4 different culture media(A, B, C and D (control)) were used to explore the effect of different concentrations of cytokines on the morphological changes and colony formation of rabbit PGCs. Secondly, rabbit PGCs were identified by alkaline phosphatase staining (AKP), and the expression of transcription factor Oct-4 was detected by real-time PCR (RT-PCR). The results showed that the number of rabbit PGCs colonies isolated by mechanical method was 2.2 times higher than those isolated by trypsin digestion method. Rabbit embryo fibroblasts (REF) were passaged to the second generation (P2) using trypsin digestion method and digestion with the feeder layer method, and REF were passaged to the fourth generation (P4) using the mechanical method. The largest number of colonies and better colony morphology were observed when PGCs were cultured in medium B(basic medium＋10% fetal bovine serum (FBS) ＋2 ng·mL^-1 of transforming growth factor-β1 (TGF-β1) ＋4 ng·mL^-1of basic fibroblast growth factor (bFGF) ＋10 ng·mL^-1leukemia inhibitory factor (LIF)). Rabbit PGCs remained undifferentiated for a longer time when cultured in medium B. AKP staining showed that the isolated PGCs was positive in red and black, and expression of Oct-4 gene was also detected in the isolated PGCs by RT-PCR. The results showed that mechanical isolation method and mechanical passage method were the optimal ways to isolate and passage rabbit primary PGCs, respectively. Supplementation with appropriate concentration of cytokines into culture medium was beneficial for rabbit PGCs to maintain a large number of colonies and undifferentiated state for a longer time in vitro. In the present study, the culture methods of rabbit PGCs in vitro were screened and optimized, which would lay a technical foundation for the further establishment of a stable and mature rabbit PGCs cell line.

Key words: Oryctolagus cuniculus, primordial germ cells (PGCs), isolation methods, passage methods, cytokines

CLC Number:

S829.1

Lü Haimiao, ZHU Hanyu, PENG Zhan, YAN Chenbo, YANG Dexin, HU Wenju, DING Xuefen, WANG Xinzhuang. Study on the Culture of Rabbit (Oryctolagus cuniculus) Primordial Germ Cells in Vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2443-2452.

References 41

[1]	SAITOU M,MIYAUCHI H.Gametogenesis from pluripotent stem cells[J].Cell Stem Cell,2016,18(6):721-735.
[2]	TAN H H,TEE W W.Committing the primordial germ cell:An updated molecular perspective[J].Wiley Interdiscip Rev Syst Biol Med,2019,11(1):e1436.
[3]	TANG W W C,KOBAYASHI T,IRIE N,et al.Specification and epigenetic programming of the hu-man germ line[J].Nat Rev Genet,2016,17(10):585-600.
[4]	BLITZ I L.Primordial germ cell transplantation for CRISPR/Cas9-based leapfrogging in Xenopus[J].J Vis Exp,2018(132):56035.
[5]	GROSS-THEBING T,YIGIT S,PFEIFFER J,et al.The vertebrate protein dead end maintains primordial germ cell fate by inhibiting somatic differentiation[J].Dev Cell,2017,43(6):704-715.e5.
[6]	OHTA H,KURIMOTO K,OKAMOTO I,et al.In vitro expansion of mouse primordial germ cell-like cells recapitulates an epigenetic blank slate[J].EMBO J,2017,36(13):1888-1907.
[7]	JIANG Z Q,WU H Y,TIAN J,et al.Targeting lentiviral vectors to primordial germ cells (PGCs):An efficient strategy for generating transgenic chickens[J].Zool Res,2020,41(3):281-291.
[8]	MATSUI Y,ZSEBO K,HOGAN B L.Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture[J].Cell,1992,70(5):841-847.
[9]	HYLDIG S M W,OSTRUP O,VEJLSTED M,et al.Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos[J].Biol Reprod, 2011, 84(6):1087-1093.
[10]	TODARO F,CAMPOLO F,BARRIOS F,et al.Regulation of kit expression in early mouse embryos and ES cells[J].Stem Cells,2019,37(3):332-344.
[11]	DE FELICI M,DOLCI S,PESCE M.Cellular and molecular aspects of mouse primordial germ cell migration and proliferation in culture[J].Int J Dev Biol,1992,36(2):205-213.
[12]	LI C P,LAI W Y,WANG H L.An alternative culture method to maintain genomic hypomethylation of mouse embryonic stem cells using MEK inhibitor PD0325901 and vitamin C[J].J Vis Exp,2018(136):56391.
[13]	FISCHER B,CHAVATTE-PALMER P,VIEBAHN C,et al.Rabbit as a reproductive model for human health[J]. Reproduction,2012,144(1):1-10.
[14]	GRAUR D,DURET L,GOUY M.Phylogenetic position of the order Lagomorpha (rabbits,hares and allies)[J]. Nature, 1996,379(6563):333-335.
[15]	FAN J L,KITAJIMA S,WATANABE T,et al.Rabbit models for the study of human atherosclerosis:from pathophysiological mechanisms to translational medicine[J].Pharmacol Ther,2015,146:104-119.
[16]	TIAN J W,HU S N,SUN Y L,et al.A novel model of atherosclerosis in rabbits using injury to arterial walls induced by ferric chloride as evaluated by optical coherence tomography as well as intravascular ultrasound and histology[J].J Biomed Biotechnol,2012,2012:121867.
[17]	JIMÉNEZ-GARCÍA A,BALONGO-GARCÍA R,ALCONERO F F,et al.Intestinal wall damage in simple ileus in rabbits:immune-modulator role of somatostatin[J].Hepatogastroenterology,2004,51(58):1030-1036.
[18]	DESANDO G,CAVALLO C,SARTONI F,et al.Intra-articular delivery of adipose derived stromal cells attenuates osteoarthritis progression in an experi-mental rabbit model[J].Arthritis Res Ther,2013,15(1):R22.
[19]	KANG S J,GROSSNIKLAUS H E.Rabbit model of retinoblastoma[J].J Biomed Biotechnol,2011, 2011:394730.
[20]	WOODRUFF-PAK D S,AGELAN A,DEL VALLE L.A rabbit model of Alzheimer's disease:valid at neuropathological, cognitive,and therapeutic levels[J].J Alzheimers Dis,2007,11(3):371-383.
[21]	LEICHTHAMMER F,BAUNACK E,BREM G.Behavior of living primordial germ cells of livestock in vitro[J]. Theriogenology,1990,33(6):1221-1230.
[22]	LEE C K,SCALES N,NEWTON G,et al.Isolation and initial characterization of primordial germ cell (PGC)-derived cells from goat,rabbit and rats[J].Theriogenology,1998,49(1):388.
[23]	李义书,王新庄,冯勋伟,等.不同饲养层对兔原始生殖细胞传代培养的影响[J].西北农业学报,2008, 17(3):37-41.LI Y S,WANG X Z,FENG X W,et al.Effect of different feeder layers on subculture of rabbit pri-mordial germ cells[J]. Acta Agriculturae Boreali-occidentalis Sinica,2008,17(3):37-41. (in Chinese)
[24]	王亚丹,虎啸,丁雪粉,等.饲养层种类及密度对体外培养兔原始生殖细胞的影响[J].中国细胞生物学学报,2018,40(8):1384-1391.WANG Y D,HU X,DING X F,et al.Effects of feeder layer type and density on culturing rabbit (Leporidae) primordial germ cells in vitro[J].Chinese Journal of Cell Biology,2018,40(8):1384-1391. (in Chinese)
[25]	黄奔,李童,石德顺,等.不同影响因素对水牛原始生殖细胞分离培养的影响[J].中国兽医学报,2008, 28(5):592-595.HUANG B,LI T,SHI D S,et al.Influence of different factor on the isolation and culture of the buffalo PGCs[J]. Chinese Journal of Veterinary Science,2008,28(5):592-595. (in Chinese)
[26]	MITALIPOVA M,BEYHAN Z,FIRST N L.Pluripotency of bovine embryonic cell line derived from precompacting embryos[J].Cloning,2001,3(2):59-67.
[27]	WANG L,DUAN E K,SUNG L Y,et al.Generation and characterization of pluripotent stem cells from cloned bovine embryos[J].Biol Reprod,2005,73(1):149-155.
[28]	郭志林,王新庄,张涌,等.从原始生殖细胞分离昆明小鼠EG细胞及其传代培养[J].农业生物技术学报, 2006, 14(5):688-691.GUO Z L,WANG X Z,ZHANG Y,et al.Isolation of Kunming mice embryonic germ cells derived from primordial germ cells and their passage culture[J].Journal of Agricultural Biotechnology,2006,14(5):688-691. (in Chinese)
[29]	陈永珍,张苏,朱旻,等.人胚胎生殖细胞的传代培养[J].江苏医药,2007,33(4):330-332.CHEN Y Z,ZHANG S,ZHU M,et al.Passage culture of human embryonic germ cells[J].Jiangsu Medical Journal, 2007,33(4):330-332. (in Chi-nese)
[30]	刘善荣,刘厚奇,汤淑萍,等.人胚胎生殖系细胞分离与体外培养的初步研究[J].第二军医大学学报, 2003,24(9):941-943.LIU S R,LIU H Q,TANG S P,et al.Isolation and cultivation of human embryonic germ cells[J].Academic Journal of Second Military Medical University,2003,24(9):941-943. (in Chinese)
[31]	KOSHIMIZU U,TAGA T,WATANABE M,et al.Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells[J]. Development,1996,122(4):1235-1242.
[32]	MOLYNEAUX K A,SCHAIBLE K,WYLIE C.GP130,the shared receptor for the LIF/IL6 cytokine family in the mouse,is not required for early germ cell differentiation,but is required cell-autonomously in oocytes for ovulation[J].Development,2003,130(18):4287-4294.
[33]	SATOH R,BANDO H,SAKAI N,et al.Function of leukaemia inhibitory factor in spermatogenesis of a teleost fish,the medaka Oryzias latipes[J].Zygote,2019,27(6):423-431.
[34]	SZEBENYI G,FALLON J F.Fibroblast growth factors as multifunctional signaling factors[J].Int Rev Cytol, 1999,185:45-106.
[35]	ZHANG Y,MA J,LI H,et al.bFGF signaling-mediated reprogramming of porcine primordial germ cells[J]. Cell Tissue Res,2016,364(2):429-441.
[36]	WANG C Z,DENG Y F,CHEN F,et al.Basic fibroblast growth factor is critical to reprogramming buffalo (Bubalus bubalis) primordial germ cells into embryonic germ stem cell-like cells[J].Theriogenology, 2017, 91:112-120.
[37]	RESNICK J L,BIXLER L S,CHENG L Z,et al.Long-term proliferation of mouse primordial germ cells in culture[J].Nature,1992,359(6395):550-551.
[38]	GU S C,FENG X H.TGF-β signaling in cancer[J].Acta Biochim Biophys Sin (Shanghai),2018,50(10):941-949.
[39]	MASSAGUÉ J.TGFβ signalling in context[J].Nat Rev Mol Cell Biol,2012,13(10):616-630.
[40]	DAVID C J,MASSAGUÉ J.Contextual determinants of TGFβ action in development,immunity and cancer[J]. Nat Rev Mol Cell Biol,2018,19(7):419-435.
[41]	LIU C,PENG G D,JING N H.TGF-β signaling pathway in early mouse development and embryonic stem cells[J].Acta Biochim Biophys Sin (Shanghai),2018,50(1):68-73.

Study on the Culture of Rabbit (Oryctolagus cuniculus) Primordial Germ Cells in Vitro

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