糖酵解调控鸡体外PGCLC形成的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-1323

摘要/Abstract

摘要：

研究显示糖酵解过程主要参与细胞的重编程并维持细胞全能性,但其在生殖细胞分化过程中发挥的作用还所知甚少。旨在以骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导鸡胚胎干细胞(embryonic stem cells,ESC)分化原始生殖细胞(primordial germ cell,PGC)的体外模型为基础,初步探索糖酵解系统在PGC形成中的功能,为从代谢水平解析鸡PGC形成的分子机制奠定理论基础。收集BMP4诱导过程中分别添加糖酵解抑制剂维生素K3(VK3)和激活剂DASA58,通过qRT-PCR检测诱导过程中糖酵解相关基因磷酸甘油酸激酶1(phosphoglycerate kinase 1,Pgk1)、己糖激酶1(hexokinase 1,Hk1)、丙酮酸激酶M2(pyruvate kinase M2,Pkm2)、乳酸脱氢酶A(lactate dehydrogenase A,Ldha)、1型葡萄糖转运蛋白(glucose transporter type 1,Glut1)、磷酸果糖激酶(phosphofructokinase,platelet,Pfkp)和醛缩酶,果糖-双磷酸C(aldolase,fructose-bisphosphate C,Aldoc)的表达变化;细胞形态学观察不同处理下细胞形态的变化;流式细胞分析不同处理下诱导第6天时DDX4阳性细胞比例(PGC样细胞,PGCLC)。qRT-PCR结果显示,BMP4诱导鸡ESC分化为PGC样细胞的过程中,糖酵解相关基因都出现显著下调趋势,糖酵解途径被抑制;而添加VK3后,糖酵解途径极显著抑制,添加DASA58后,糖酵解途径则被显著激活。细胞形态学观察结果表明,与正常诱导过程相比,添加VK3后,第6天的类胚体显著增加;而添加DASA58后,类胚体则显著减少。流式细胞分析发现,添加VK3后,DDX4阳性细胞比例增加,添加DASA58后,DDX4阳性细胞比例减少。该研究结果显示抑制糖酵解过程能够促进体外鸡PGC-like的形成,即糖酵解过程在鸡PGCs形成过程中被抑制。

关键词: 糖酵解, 鸡, 胚胎干细胞, 原始生殖样细胞

Abstract:

Studies have shown that glycolysis is mainly involved in cell reprogramming and maintaining cell totipotency,but its role in germ cell differentiation is still poorly understood. This study aims to explore the function of glycolysis system in the formation of Primordial Germ Cell(PGC)based on the in vitro model of inducing PGC differentiation in chicken Embryonic Stem Cell(ESC)induced by Bone Morphogenetic Protein 4(BMP4),and to lay a theoretical foundation for analyzing the molecular mechanism of PGC formation in chickens from the metabolic level. The expression variations of glycolysis related genes of Pgk1(phosphoglycerate kinase 1),Hk1(hexokinase 1),Pkm2(pyruvate kinase M2),Ldha(lactate dehydrogenase A),Glut1(glucose transporter type 1),Pfkp(phosphofructokinase,platelet,)and Aldoc(aldolase,fructose-bisphosphate C)were detected by qRT-PCR during BMP4 induction with glycolysis inhibitor VK3 and activator DASA58. Cell morphology was observed under different treatments. Flow cytometry was used to analyze the percentage of DDX4 positive cells(PGC like cells,PGCLC)on the day 6 after induction. qRT-PCR results showed that during the differentiation of chicken ESC into PGC-like induced by BMP4,the glycolysis related genes were significantly down regulated,and the glycolysis pathway was inhibited;while the glycolysis pathway was significantly inhibited after adding VK3,and was significantly activated after adding DASA58. Morphological observation of cells showed that the number of embryoid bodies increased significantly after adding VK3 compared with the normal induction process,but decreased significantly after adding DASA58. Analysis by flow cytometry showed that the proportion of DDX4 positive cells increased after VK3 addition,and decreased after adding DASA58. In sum,the results indicate that inhibition of glycolysis may promote the formation of chicken PGC-like in vitro,that is,glycolysis is suppressed in the process of chicken PGCs formation.

Key words: glycolysis, chicken, embryonic stem cells, primordial germ like cells

张晨, 左其生, 邹艺琛, 赵娟娟, 张亚妮, 李碧春. 糖酵解调控鸡体外PGCLC形成的功能研究[J]. 生物技术通报, 2021, 37(6): 163-170.

ZHANG Chen, ZUO Qi-sheng, ZOU Yi-chen, ZHAO Juan-juan, ZHANG Ya-ni, LI Bi-chun. Study on the Function of Glycolysis in Inducing Chicken PGCLC in vitro Formation[J]. Biotechnology Bulletin, 2021, 37(6): 163-170.

图/表 5

表1 qRT-PCR引物

Table 1 Primers used for qRT-PCR

引物名称 Primer name	5' 端引物 Primer sequence(5')	3' 端引物 Primer sequence(3')
Hk1	CCTCTTGGCTTCACATTC	TTCACAGTTTGGGTCTTCAT
Pkm2	GGCACCCACGAGTATCAT	CATTGTCCAGCGTCACTTT
Pfkp	GCCACAACAAACCTATAA-CA	ATCAAAGGCAGACGAACA
Ldha	TGGGCATCCATCCTCTGA	CCTGCTTGTGAACCTCCT
Glut1	TGTTTGGCTTGGACTTGAT	TCTTGAGGACGCTCTTGG
Hif1a	TTGACAAGGCATCCATTA	TCCTCAGAAAGCACCATA
Aldoc	CTGACGACGGCACTCCTTT	GACAGCCCATCCAGACCCT
Pgk1	AGGGCTGCATCACCATTA	CCACCTCCAGTGCTAACG

图1 PGC-like形成中糖酵解相关基因的表达

Fig. 1 Expression of glycolysis-related genes in the formation of PGC-like

图2 体外诱导过程中糖酵解抑制剂/激活剂对糖酵解相关基因的影响

Fig. 2 Effects of glycolysis inhibitors/activators on glycolysis-related genes during the induction in vitro

图3 体外诱导过程中糖酵解抑制剂/激活剂对类胚体形成的影响 A:细胞形态学观察;B:直方图显示类胚体数量

Fig.3 Effect of glycolysis inhibitor/activator on embryoid body formation during the induction in vitro A:Observation of cell morphology. B:The histogram shows the number of embryoid bodies

图4 体外诱导过程中糖酵解抑制剂/激活剂对PGC-like形成的影响 A:流式细胞分析显示阳性细胞比例;B:直方图显示阳性细胞比例

Fig. 4 Effect of glycolysis inhibitor/activator on PGC-like formation during the induction in vitro A:Proportion of positive cells with flow cytometry. B:Histogram showed the proportion of positive cells

参考文献 35

[1]	Brinster RL, Harstad H . Energy metabolism in primordial germ cells of the mouse[J]. Experimental Cell Research, 1977, 109(1):111-117. doi: 10.1016/0014-4827(77)90050-7 URL
[2]	Koppenol WH, Bounds PL, Dang CV. Otto Warburg’s contributions to current concepts of cancer metabolism[J]. Nature Reviews Cancer, 2011, 11:325-337. doi: 10.1038/nrc3038 URL
[3]	Zheng J. Energy metabolism of cancer:Glycolysis versus oxidative phosphorylation(Review)[J]. Oncology Letters, 2012, 4(6):1151-1157. pmid: 23226794
[4]	Hayashi Y, Otsuka K, Ebina M, et al. Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells[J]. Proceedings of the National Academy of ences of the United States of America, 2017, 114(31):8289.
[5]	Kanatsu-Shinohara M, Tanaka T, Ogonuki N, et al. Myc/Mycn-mediated glycolysis enhances mouse spermatogonial stem cell self-renewal[J]. Genes & Development, 2016, 30(23):2637. doi: 10.1101/gad.287045.116 URL
[6]	Folmes CL, Nelson T, Martinez-Fernandez A, et al. Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming[J]. Cell Metabolism, 2011, 14(2):264-271. doi: 10.1016/j.cmet.2011.06.011 URL
[7]	冯科珂, 刘晶锦, 姚其正. VK₃及其类似物的抗肿瘤作用[J]. 国外医药:抗生素分册, 2009, 30(4):171-177.
	Feng KK, Liu JJ, Yao QZ. The antineoplastic effect of vitamin K₃ and its derivates [J]. World Notes on Antibiotics, 2009, 30(4):171-177.
[8]	Chen J, Jiang Z, Wang B, et al. Vitamin K₃ and K₅ are inhibitors of tumor pyruvate kinase M2[J]. Cancer Letter, 2012, 316(2):204-10. doi: 10.1016/j.canlet.2011.10.039 URL
[9]	Wu FY, Liao WC, Chang HM. Comparison of antitumor activity of vitamins K₁, K₂ and K₃ on human tumor cells by two(MTT and SRB)cell viability assays[J]. Life Science, 1993, 52(22):1797-804.
[10]	Hitomi M, Yokoyama F, Kita Y, et al. Antitumor effects of vitamins K₁, K₂ and K₃ on hepatocellular carcinoma in vitro and in vivo[J]. International Journal of Oncology, 2005, 26(3):713-720.
[11]	Giannoni E, Taddei ML, Morandi A, et al. Targeting stromal-induced pyruvate kinase M2 nuclear translocation impairs oxphos and prostate cancer metastatic spread[J]. Oncotarget, 2015, 6(27):24061-24074. pmid: 26183399
[12]	Palsson-McDermott EM, Curtis AM, Goel G, et al. Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages[J]. Cell Metabolism, 2015, 21(1):65-80. doi: 10.1016/j.cmet.2014.12.005 URL
[13]	施青青, 张振韬, 李鹏程, 等. BMP4诱导鸡胚胎干细胞向雄性生殖细胞分化的研究[J]. 畜牧兽医学报, 2013, 44(11):1749-1757.
	Shi QQ, Zhang ZT, Li PC, et al. Study on differentiation of chicken embryonic stem cells to male germ cells by BMP4[J]. Chinese Journal of Animal and Veterinary Sciences, 2013, 44(11), 1749-1757.
[14]	于丹, 王莹, 高原, 等. Akt/GSK-3β/Snail通路在TGF-β_1诱导A549/DDP细胞上皮间质转化中的作用[J]. 中国病理生理杂志, 2018(6):1124-1128.
	Yu D, Wang Y, Gao Y, et al. Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1[J]. Chinese Journal of Pathophysiology, 2018(6):1124-1128.
[15]	杨海燕, 孙敏, 田智泉, 等. 鸡X期胚盘细胞分散培养与整胚培养比较初探[J]. 中国家禽, 2010, 32(7):14-17.
	Yang HQ, Sui M, Tian ZQ, et al. Preliminary comparison of dispersed culture and whole embryo culture of chicken blastodermal cells on X period[J]. China Poultry, 2010, 32(7):14-17.
[16]	靳锴, 汪怡临, 左其生, 等. 睾丸注射Mx-NA双基因制备抗病转基因鸡的研究[J]. 中国家禽, 2014, 36(14):8-12.
	Jin K, Wang YL, Zuo QS, et al. Generation of disease-resistant transgenic chicken by testis-mediated injection method of combined Mx-NA gene[J]. China Poultry, 2014. 36(14):8-12.
[17]	Yeung SJ, Pan J, Lee MH. Roles of p53, MYC, and HIF-1 in regulating glycolysis:the seventh hallmark of cancer[J]. Cellular and Molecular Life Sciences, 2008, 65:3981-3999. doi: 10.1007/s00018-008-8224-x pmid: 18766298
[18]	Hsu PP, Sabatini DM. Cancer cell metabolism:Warburg and beyond[J]. Cell, 2008, 134:703-707. doi: 10.1016/j.cell.2008.08.021 URL
[19]	Graziano F, Ruzzo A, Giacomini E, Ricciardi T, et al. Glycolysis gene expression analysis and selective metabolic advantage in the clinical progression of colorectal cancer[J]. Pharmacogenomics, 2017, 17(3):258-264.
[20]	Kakinuma Y, Miyauchi T, Suzuki T, et al. Enhancement of glycolysis in cardiomyocytes elevates endothelin-1 expression through the transcriptional factor hypoxia-inducible factor-1 alpha[J]. Clinical Science, 2002, 103(Suppl 48):210S-214S. doi: 10.1042/CS103S210S URL
[21]	Zhou W, Choi M, Margineantu D, et al. HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition[J]. EMBO Journal, 2014, 31(9):2103-2116. doi: 10.1038/emboj.2012.71 URL
[22]	Taper HS, de Gerlache J, Lans M, et al. Non-toxic potentiation of cancer chemotherapy by combined C and K3 vitamin pre-treatment[J]. International Journal of Cancer, 1987, 40:575-579. pmid: 3666992
[23]	Taper HS, Keyeux A, Roberfroid M. Potentiation of radiotherapy by nontoxic pretreatment with combined vitamins C and K3 in mice bearing solid transplantable tumor[J]. Anticancer Research, 1996, 16:499-503.
[24]	Taper HS, Jamison JM, Gilloteaux J, et al. Inhibition of the development of metastases by dietary vitamin C:K3 combination[J]. Life Science, 2004, 75:955-967.
[25]	De Loecker W, Janssens J, Bonte J, et al. Effects of sodium ascorbate(vitamin C)and 2-methyl-1, 4-naphtho-quinone(vitamin K3)treatment on human tumor cell growth in vitro, II:synergism with combined chemotherapy action[J]. Anticancer Research, 1993, 13:103-106.
[26]	Sakagami H, Satoh K, Hakeda Y, et al. Apopto-sis-inducing activity of vitamin C and vitamin K[J]. Cellular and Molecular Biology, 2000, 46:129-143. pmid: 10726979
[27]	Jamison JM, Gilloteaux J, Taper HS, et al. Evaluation of the in vitro and in vivo antitumor activities of vitamin C and K-3 combinations against human prostate cancer[J]. Journal of Nutrition, 2001, 131:158S-160S. doi: 10.1093/jn/131.1.158S URL
[28]	Buc Calderon P, Cadrobbi J, Marques C, et al. Potential therapeutic application of the association of vitamins C and K3 in cancer treatment[J]. Current Medicinal Chemistry, 2002, 9:2271-2285. doi: 10.2174/0929867023368674 URL
[29]	Verrax J, Stockis J, Tison A, et al. Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice[J]. Biochem Pharmacol, 2006, 72:671-680. doi: 10.1016/j.bcp.2006.05.025 URL
[30]	Verrax J, Vanbever S, Stockis J, et al. Role of glycolysis inhibition and poly(-ADP-ribose)polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemia cells[J]. International Journal of Cancer, 2007, 120:1192-1197. doi: 10.1002/(ISSN)1097-0215 URL
[31]	Kim YH, Heo JS, Han HJ. High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways[J]. Journal of Cellular Physiology, 2006, 209:94-102. doi: 10.1002/(ISSN)1097-4652 URL
[32]	Crespo FL, Sobrado VR, Gomez L, et al. Mitochondrial reactive oxygen species mediate cardiomyocyte formation from embryonic stem cells in high glucose[J]. Stem Cells, 2010, 28:1132-1142.
[33]	Khoo MLM, McQuade LR, Smith MSR, et al. Growth and differentiation of embryoid bodies derived from human embryonic stem cells:Effect of glucose and basic fibroblast growth factor[J]. Biology of Reproduction, 2005, 73:1147-1156. doi: 10.1095/biolreprod.104.036673 URL
[34]	Chae HD, Lee MR, Broxmeyer HE. 5-Aminoimidazole-4-carboxyamide ribonucleoside induces G₁/S arrest and Nanog downregulation via p53 and enhances erythroid differentiation[J]. Stem Cells, 2012, 30(2):140-149. doi: 10.1002/stem.778 URL
[35]	Hayashi Y, Otsuka K, Ebina M, et al. Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells[J]. Proceedings of the National Academy of ences of the United States of America, 2017, 114(31):8289.