丙戊酸对双峰驼成纤维细胞重编程影响

doi:10.3864/j.issn.0578-1752.2023.12.014

摘要/Abstract

摘要：

【目的】探讨提高双峰驼成纤维细胞重编程效率，减少因原癌基因引入造成致瘤风险。试验在成纤维细胞重编程过程中添加丙戊酸（valproic acid，VPA），探索小分子物质对双峰驼成纤维细胞重编程的影响，为双峰驼发病机制研究提供参考依据。【方法】采用3月龄双峰驼胎儿成纤维细胞，结合经典诱导组合OSKM（Oct4、Sox2、Klf4和c-Myc及治疗）和EGFP等5种逆转录病毒对双峰驼成纤维细胞进行重编程（OSKM组），并在第二次病毒感染后添加VPA处理7 d（OSKM+VPA组）收集细胞。利用PCR技术对所得细胞进行内、外源基因检测以证实逆转录病毒对双峰驼成纤维细胞的修饰作用，根据RNA-seq数据从VPA影响较为明显的基因中随机挑选8个基因，检验其在添加VPA前后的变化趋势是否与RNA-seq数据趋势一致，以验证RNA-seq数据的准确性。通过GO分析对转录组样本基因进行分类，并使用KEGG通路富集分析和超几何验证分析明确目的基因显著富集通路。对收集到的细胞进行总RNA提取并结合RNA-seq技术和实时荧光定量PCR（Real time-quantitative interpretation，RT-qPCR）技术检测VPA对双峰驼成纤维细胞重编程的影响。【结果】使用PCR技术检测内、外源基因在不同分组的表达，结果显示nSox2、Sox2、Oct4、Klf4、c-Myc在OSKM和OSKM+VPA组中均有表达，且OSKM+VPA组表达量高于OSKM组，而其在BCEFs组不表达。随机挑选8个基因检测，结果表明与细胞周期信号通路有关的TP53、CCNB1、CDC20这3个基因在添加VPA后表达量下调；与癌症表型特征相关的S100A4、CKS2、VIM、MMP9表达下调；PI3k-Akt信号通路中VEGFC表达上调；此表达趋势与组学数据趋势一致。结果显示在添加VPA后，增殖基因Mki67和PCNA表达下调，而凋亡基因CASP7表达上调。对转录组数据进行KEGG和超几何验证分析，根据分析结果筛选出959个差异表达基因，富集在276个信号通路中，其中Q值小于0.05的信号通路有八条：类固醇生物合成、细胞周期、PPAR信号通路、孕酮介导的卵母细胞成熟、脂肪酸代谢、ECM-受体相互作用、细胞黏附分子和胆固醇代谢。经筛选获得与细胞周期、脂肪酸代谢、细胞黏附分子和胆固醇代谢等相关的26个差异表达基因，并随机选取其中四个基因进行检测，结果表明：VPA可使双峰驼成纤维细胞黏附分子信号通路中L1CAM、CNTN1、NFASC三个基因表达上调，细胞间互作增强，同时上调脂肪酸信号通路中CD36基因的表达。【结论】VPA可将细胞阻滞在分裂期之前，以减少重编程过程中分化几率。同时，VPA对双峰驼成纤维细胞重编程过程中多条信号通路均具有影响，调节信号通路中相关基因表达趋势，有效提高细胞重编程效率，在双峰驼成纤维细胞重编程中发挥重要作用。

关键词: 诱导多能干细胞, 双峰驼, 丙戊酸, 细胞重编程

Abstract:

【Objective】 To improve the efficiency of the reprogramming process of Bactrian camel fibroblasts and to reduce the risk of tumorigenesis caused by the introduction of proto-oncogenes. In this experiment, valproic acid (VPA) was added to the fibroblast reprogramming process to explore the effect of small molecules on the reprogramming of Bactrian camel fibroblasts. 【Method】 Given this, March-aged Bactrian camel fetal fibroblasts were used as test materials, combined with classic induction combination OSKM (Oct4, Sox2, Klf4, and c-Myc) and EGFP five retrovirus reprogramming of Bactrian camel fibroblasts (OSKM group), and the cells by adding VPA treatment for 7 days after the second viral infection (OSKM+VPA group) were collected. Endogenous and exogenous genes were examined by using PCR to confirm the modification effect of retrovirus on Bactrian camel fibroblasts. Eight genes were randomly selected from those more significantly affected by VPA. According to RNA-seq data, whether their trends before and after VPA addition were consistent with the trends of RNA-seq data was checked to verify the accuracy of RNA-seq data. The transcriptome sample genes were classified by GO analysis and significant enrichment pathways for target genes were clarified by using KEGG pathway enrichment analysis and hypergeometric validation analysis. Total RNA was extracted from the collected cells, and then, combined with RNA-seq and Real time-quantitative interpretation (RT-qPCR) techniques to detect the effect of VPA on the reprogramming of Bactrian camel fibroblasts. 【Result】 It was detected by using PCR that the expression of endogenous and exogenous genes in different groups. The results showed that nSox2, Sox2, Oct4, Klf4, and c-Myc genes were expressed in both OSKM and OSKM+VPA groups, and the expression in OSKM+VPA group was higher than that in the OSKM group, while they were not expressed in BCEFs group. Eight genes were randomly selected for testing, and the results showed that: three genes of TP53, CCNB1, and CCD20 were down-regulated in expression after the addition of VPA, which were related to the cell cycle signaling pathway. S100A4, CKS2, VIM, and MMP9 genes signaling were down-regulated in expression, which was related to the phenotypic characteristics of cancer; VEGFC gene expression was up-regulated in the PI3k-Akt signaling pathway. This expression trend was consistent with the trend of the histological data. The results showed that the expression of proliferation genes Mki67 and PCNA were down-regulated, while the expression of apoptosis gene CASP7 was up-regulated after the addition of VPA. KEGG and hypergeometric validation analyses of the transcriptome data were performed, and 959 differentially expressed genes were screened according to the analysis results, which were enriched in 276 signaling pathways, including eight signaling pathways with Q values less than 0.05: steroid biosynthesis, cell cycle, PPAR signaling pathway, progesterone-mediated oocyte maturation, fatty acid metabolism, ECM-receptor interactions, cell adhesion molecules, and cholesterol metabolism. The 26 differentially expressed genes related to cell cycle, fatty acid metabolism, cell adhesion molecule, and cholesterol metabolism were screened, and four of which were randomly selected for testing, showing that VPA upregulated the expression of L1CAM, CNTN1 and NFASC genes in the Bactrian camel fibroblast adhesion molecule signalling pathway and enhanced intercellular interactions. It was also upregulated that the expression of CD36 gene in the fatty acid signaling pathway. 【Conclusion】 The results showed that the VPA blocked cell before the split phase to reduce risk differentiation during the process of reprogramming. Meanwhile, VPA affected several signaling pathways in the reprogramming process of Bactrian camel fibroblasts, and regulated the expression trend of related genes in the signaling pathways, which effectively improved the reprogramming efficiency of the cells and played an important role in the reprogramming of Bactrian camel fibroblasts.

Key words: induced pluripotent stem cells, Bactrian camel, valproic acid, cell reprogramming

张启冉, 李宗帅, 马甜, 李怡娜, 赵兴绪, 张勇. 丙戊酸对双峰驼成纤维细胞重编程影响[J]. 中国农业科学, 2023, 56(12): 2407-2420.

ZHANG QiRan, LI ZongShuai, MA Tian, LI YiNa, ZHAO XingXu, ZHANG Yong. Effect of Valproic Acid on Reprogramming of Bactrian Camel Fibroblasts[J]. Scientia Agricultura Sinica, 2023, 56(12): 2407-2420.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.12.014

https://www.chinaagrisci.com/CN/Y2023/V56/I12/2407

图/表 8

表1

图1

图2

表2

表3

图3

图4

图5

参考文献 36

[1]	TAKAHASHI K, YAMANAKA S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663-676. doi: 10.1016/j.cell.2006.07.024 pmid: 16904174
[2]	YU J, VODYANIK M A, SMUGA-OTTO K, ANTOSIEWICZ- BOURGET J, FRANE J L, TIAN S, NIE J, JONSDOTTIR G A, RUOTTI V, STEWART R, SLUKVIN I I, THOMSON J A. Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007, 318(5858): 1917-1920. doi: 10.1126/science.1151526 pmid: 18029452
[3]	EZASHI T, TELUGUA B P V. L., ALEXENKO A P., SACHDEV S, SINHA S, ROBERTS M. Derivation of induced pluripotent stem cells from pig somatic cells. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(27): 10993-10998.
[4]	HAN X, HAN J, DING F, CAO S, LIM S S, DAI Y, ZHANG R, ZHANG Y, LIM B, LI N. Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells. Cell Research, 2011, 21(10): 1509-1512. doi: 10.1038/cr.2011.125 pmid: 21826109
[5]	LIAO J, CUI C, CHEN S, REN J, CHEN J, GAO Y, LI H, JIA N, CHENG L, XIAO H, XIAO L. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell, 2009, 4(1): 11-15. doi: 10.1016/j.stem.2008.11.013 pmid: 19097959
[6]	NAGY K, SUNG H, ZHANG P, LAFLAMME S, VINCENT P, AGHA-MOHAMMADI S, WOLTJEN K, MONETTI C, MICHAEL I P, SMITH L C, NAGY A. Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Reviews and Reports, 2011, 7(3):693-702. doi: 10.1007/s12015-011-9239-5 pmid: 21347602
[7]	LIU J, BALEHOSUR D, MURRAY B, KELLY J M, SUMER H, VERMA P J. Generation and characterization of reprogrammed sheep induced pluripotent stem cells. Theriogenology, 2012, 77(2): 338-346. doi: 10.1016/j.theriogenology.2011.08.006 pmid: 21958637
[8]	LIU H, ZHU F, YONG J, ZHANG P, HOU P, LI H, JIANG W, CAI J, LIU M, CUI K, QU X, XIANG T, LU D, CHI X, GAO G, JI W, DING M, DENG H. Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell, 2008, 3(6): 587-590. doi: 10.1016/j.stem.2008.10.014 pmid: 19041774
[9]	BUEHR M, MEEK S, BLAIR K, YANG J, URE J, SILVA J, MCLAY R, HALL J, YING Q, SMITH A. Capture of authentic embryonic stem cells from rat blastocysts. Cell, 2008, 135(7): 1287-1298. doi: 10.1016/j.cell.2008.12.007 pmid: 19109897
[10]	LI P, TONG C, MEHRIAN-SHAI R, JIA L, WU N, YAN Y, MAXSON R E, SCHULZE E N, SONG H, HSIEH C, PERA M F, YING Q. Germline competent embryonic stem cells derived from rat blastocysts. Cell, 2008, 135(7): 1299-1310. doi: 10.1016/j.cell.2008.12.006 pmid: 19109898
[11]	WERNIG M, LENGNER C J, HANNA J, LODATO M A, STEINE E, FOREMAN R, STAERK J, MARKOULAKI S, JAENISCH R. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nature Biotechnol, 2008, 26(8): 916-924. doi: 10.1038/nbt1483
[12]	HUANGFU D, OSAFUNE K, MAEHR R, GUO W, EIJKELENBOOM A, CHEN S, MUHLESTEIN W, MELTON D A. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature Biotechnol, 2008, 26(11): 1269-1275. doi: 10.1038/nbt.1502
[13]	ESTEBAN M A, WANG T, QIN B, YANG J, QIN D, CAI J, LI W, WENG Z, CHEN J, NI S, CHEN K, LI Y, LIU X, XU J, ZHANG S, LI F, HE W, LABUDA K, SONG Y, PETERBAUER A, WOLBANK S, REDL H, ZHONG M, CAI D, ZENG L, PEI D. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 2010, 6(1): 71-79. doi: 10.1016/j.stem.2009.12.001 pmid: 20036631
[14]	HUANGFU D, MAEHR R, GUO W, EIJKELENBOOM A, SNITOW M, CHEN A E, MELTON D A. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature Biotechnol, 2008, 26(7): 795-797. doi: 10.1038/nbt1418
[15]	GURVICH N, TSYGANKOVA O M, MEINKOTH J L, KLEIN P S. Histone deacetylase is a target of valproic acid-mediated cellular differentiation. Cancer Research, 2004, 64(3): 1079-1086. doi: 10.1158/0008-5472.can-03-0799 pmid: 14871841
[16]	PHIEL C J, ZHANG F, HUANG E Y, GUENTHER M G, LAZAR M A, KLEIN P S. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. The Journal of Biological Chemistry, 2001, 276(39): 36734-36741. doi: 10.1074/jbc.M101287200
[17]	GIORGETTI A, MONTSERRAT N, AASEN T, GONZALEZ F, RODRíGUEZ-PIZà, VASSENA R, RAYA A, BOUé S, BARRERO M J, CORBELLA B A, TORRABADELLA M, VEIGA A, BELMONTE J C I. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell, 2009, 5(4): 353-357. doi: 10.1016/j.stem.2009.09.008 pmid: 19796614
[18]	CHEN S, ZHOU Y, CHEN Y, GU J. fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34(17): i884-i890. doi: 10.1093/bioinformatics/bty560
[19]	KIM D, LANGMEAD B, SALZBERG S L. HISAT: A fast spliced aligner with low memory requirements. Nature Methods, 2015, 12(4): 357-360. doi: 10.1038/nmeth.3317 pmid: 25751142
[20]	LOVE M I, HUBER W, ANDERS S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 2014, 15(12): 550-571. doi: 10.1186/s13059-014-0550-8
[21]	SUN J, LI W, SUN Y, YU D, WEN X, WANG H, CUI J, WANG G, HOFFMAN A R, HU J. A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies. Nucleic Acids Research, 2014, 42(15): 9588-9601. doi: 10.1093/nar/gku549 pmid: 25092925
[22]	苗向阳, 陈晓瑛. 诱导性多能干细胞的研究及应用. 中国农业科学, 2012, 45(2): 369-375. doi: 10.3864/j.issn.0578-1752.2012.02.020. doi: 10.3864/j.issn.0578-1752.2012.02.020
	MIAO X Y, CHEN X Y. Study and application of induced pluripotent stem cells. Scientia Agricultura Sinica, 2012, 45(2): 369-375. doi: 10.3864/j.issn.0578-1752.2012.02.020. (in Chinese) doi: 10.3864/j.issn.0578-1752.2012.02.020
[23]	ZHAI Y, CHEN X, YU D, LI T, CUI J, WANG G, HU J, LI W. Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming- induced senescence stress. Experimental Cell Research, 2015, 337(1): 61-67. doi: 10.1016/j.yexcr.2015.06.003
[24]	魏如雪, 郝海生, 赵学明, 杜卫华, 朱化彬. 抑制体细胞衰老促进诱导性多潜能干细胞(iPSC)生成的研究进展. 中国农业科学, 2015, 48(16): 3258-3265. doi: 10.3864/j.issn.0578-1752.2015.16.015. doi: 10.3864/j.issn.0578-1752.2015.16.015
	WEI R X, HAO H S, ZHAO X M, DU W H, ZHU H B. Studies of improved efficiency of induced pluripotent stem cell generation by restraining somatic cell senescence. Scientia Agricultura Sinica. 2015, 48(16): 3258-3265. doi: 10.3864/j.issn.0578-1752.2015.16.015. (in Chinese) doi: 10.3864/j.issn.0578-1752.2015.16.015
[25]	YANG Y B, PANDURANGAN M, HWANG I. Targeted suppression of mu-calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle. Cell Biology International, 2012, 36(9):843-849. doi: 10.1042/CBI20120050
[26]	LAKHANI S A, MASUD A, KUIDA K, JR G A P, BOOTH C J, MEHAL W Z, INAYAT I, FLAVELL R. Caspases 3 and 7: Key mediators of mitochondrial events of apoptosis. Science, 2006, 311(5762): 847-851. doi: 10.1126/science.1115035 pmid: 16469926
[27]	WERNIG M, MEISSNER A, FOREMAN R, BRAMBRINK T, KU M, HOCHEDLINGER K, BERNSTEIN B E, JAENISCH R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 2007, 448(7151): 318-324. doi: 10.1038/nature05944
[28]	TANOSAKI S, TOHYAMA S, FUJITA J, SOMEYA S, HISHIKI T, MATSUURA T, NAKANISH H, OHTO-NAKANISH T, AKIYAMA T, MORITA Y, KISHINO Y, OKADA M, TANI H, SOMA Y, NAKAJIMA K, KANAZAWA H, SUGIMOTO M, KO M S H, SUEMATSU M, FUKUDA K. Fatty acid synthesis is indispensable for survival of human pluripotent stem cells. iScience, 2020, 23(9): 101535-101568. doi: 10.1016/j.isci.2020.101535
[29]	ABUMRAD N A, EL-MAGHRABI M R, AMRI E Z, LOPEZ E, GRIMALDI P A. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. Homology with human CD36. The Journal of Biologic Chemistry, 1993, 268(24): 17665-17668. doi: 10.1016/S0021-9258(17)46753-6
[30]	LI L, BENNETT S A L, WANG L. Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells. Cell Adhesion Migration, 2012, 6(1): 59-70. doi: 10.4161/cam.19583
[31]	SAMANTA D, ALMO S C. Nectin family of cell-adhesion molecules: structural and molecular aspects of function and specificity. Cellular and Molecular Life Sciences, 2015, 72(4): 645-658. doi: 10.1007/s00018-014-1763-4 pmid: 25326769
[32]	PROKHOROVA T A, RIGBOLT K T G, JOHANSEN P T, HENNINGSEN J, KRATCHMAROVA I, KASSEM M, BLAGOEV B. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells. Molecular Cellular Proteomics, 2009, 8(5): 959-970. doi: 10.1074/mcp.M800287-MCP200
[33]	LI L, WANG S, JEZIERSKI A, MOALIM-NOUR L, MOHIB K, PARKS R J, RETTA S F, WANG L. A unique interplay between Rap1 and E-cadherin in the endocytic pathway regulates self-renewal of human embryonic stem cells. Stem Cells, 2010, 28(2): 247-257. doi: 10.1002/stem.289 pmid: 20039365
[34]	ROWLAND T J, MILLER L M, BLASCHKE A J, DOSS E L, BONHAM A J, HIKITA S, JOHNSON L V, CLEGG D O. Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin. Stem Cells and Development, 2010, 19(8): 1231-1240. doi: 10.1089/scd.2009.0328 pmid: 19811096
[35]	CHEN T, YUAN D, WEI B, JIANG J, KANG J, LING K, GU Y, LI J, XIAO L, PEI G. E-cadherin-mediated cell-cell contact is critical for induced pluripotent stem cell generation. Stem Cells, 2010, 28(8): 1315-1325. doi: 10.1002/stem.456 pmid: 20521328
[36]	SON Y S, SEONG R H, RYU C J, CHO Y S, BAE K, CHUNG S J, LEE B, MIN J, HONG H J. Brief report: L1 cell adhesion molecule, a novel surface molecule of human embryonic stem cells, is essential for self-renewal and pluripotency. Stem Cells, 2011, 29(12): 2094-2099.

样本 Sample	总计 Total	未映射 Unmapped (%)	单映射 Unique_mapped (%)	多映射 Multiple_mapped (%)	总映射 Total_mapped (%)
CK-1	61182534	5873073 (9.60%)	53914224 (88.12%)	1395237 (2.28%)	55309461 (90.40%)
CK-2	65291602	6449818 (9.88%)	57337110 (87.82%)	1504674 (2.30%)	58841784 (90.12%)
CK-3	65980288	6383674 (9.68%)	58071438 (88.01%)	1525176 (2.31%)	59596614 (90.32%)
T-1	69181666	6651458 (9.61%)	60990701 (88.16%)	1539507 (2.23%)	62530208 (90.39%)
T-2	67916304	6710952 (9.88%)	59640972 (87.82%)	1564380 (2.30%)	61205352 (90.12%)
T-3	67343960	6228926 (9.25%)	59624392 (88.54%)	1490642 (2.21%)	61115034 (90.75%)

样本 Sample	参考基因 Refer genes	序列参考基因 Sequenced refer genes (%)	新基因 Novel genes	新基因序列 Sequenced novel genes (%)	总基因 Total genes	序列总基因 Sequenced total genes (%)
all	19172	15643 (81.59%)	576	576 (100.00%)	19748	16219 (82.13%)
CK-1	19172	14377 (74.99%)	576	563 (97.74%)	19748	14940 (75.65%)
CK-2	19172	14332 (74.75%)	576	562 (97.57%)	19748	14894 (75.42%)
CK-3	19172	14399 (75.10%)	576	561 (97.40%)	19748	14960 (75.75%)
T-1	19172	14367 (74.94%)	576	556 (96.53%)	19748	14923 (75.57%)
T-2	19172	14427 (75.25%)	576	564 (97.92%)	19748	14991 (75.91%)
T-3	19172	14347 (74.83%)	576	556 (96.53%)	19748	14903 (75.47%)