CTSD对黔北麻羊卵泡颗粒细胞的调控机制及功能分析

doi:10.11843/j.issn.0366-6964.2021.05.014

摘要/Abstract

摘要： 旨在分析组织蛋白酶D（cathepsin D， CTSD）对黔北麻羊卵泡颗粒细胞的调控机制并初步探究其对产羔性状的影响机理。本研究以36周龄、健康、多胎黔北麻羊母羊（n=5）为研究对象，屠宰后采集卵巢组织分离培养卵泡颗粒细胞，构建真核表达载体pEGFP-N3-CTSD，将其导入细胞后在转录与翻译水平验证真核表达效率；通过CCK-8法检测在不同时间段内重组质粒对颗粒细胞增殖的影响；通过流式细胞仪检测重组质粒对颗粒细胞凋亡及周期的影响；随后使用RT-qPCR法在细胞水平检测重组质粒对细胞凋亡相关基因Bcl-2、Bax、Caspase-3，细胞周期相关因子Cyclin A1、Cyclin D2、Cyclin E mRNA表达水平的影响，最后，以多胎性状候选基因BMPR-IB、FSHR、INHA为功能基因，在转录与翻译水平上验证重组质粒对其mRNA与蛋白表达的影响。双酶切及测序结果证实，黔北麻羊CTSD基因真核表达载体pEGFP-N3-CTSD构建成功，且在转录与翻译水平极显著上调CTSD在颗粒细胞中的表达（P<0.01）；细胞增殖检测结果表明，颗粒细胞中上调CTSD后能够抑制细胞的增殖，其中在12、24、48、72 h对细胞增殖的抑制效率达到极显著（P<0.01）；细胞凋亡检测结果表明，CTSD的过表达能够极显著促进颗粒细胞的凋亡（P<0.01），且能够显著下调细胞抗凋亡基因Bcl-2的表达（P<0.05），极显著上调细胞促凋亡相关基因Bax、Caspase-3的表达（P<0.01）；此外，细胞周期检测发现，pEGFP-N3-CTSD在转染后能够极显著上调G0/G1期与G2/M期的细胞数量（P<0.01），极显著下调S期细胞数量（P<0.01），同时能够极显著提高细胞周期相关因子Cyclin A1的表达（P<0.01），极显著降低Cyclin D2的表达（P<0.01）。RT-qPCR及Western blot检测结果表明，在颗粒细胞中上调CTSD后，能够极显著的下调多胎性状候选基因与蛋白BMPR-IB、FSHR、INHA在转录和翻译水平中的表达（P<0.01）。本研究发现，CTSD的高表达能抑制细胞的增殖，促进细胞凋亡，改变颗粒细胞的周期进程；还能改变细胞凋亡相关因子Bcl-2、Bax、Caspase-3与细胞周期相关因子Cyclin A1、Cyclin D2、Cyclin E的表达；同时也能够极显著降低颗粒细胞中多胎性状候选基因与蛋白BMPR-IB、FSHR、INHA的表达量（P<0.01）。这提示，CTSD可能通过改变细胞水平相关因子的表达量来调控颗粒细胞的生物学行为以及影响山羊多胎性状候选基因的表达进而间接的成为影响山羊产羔性状的重要因子，本研究为进一步探明CTSD对山羊产羔性状调控机理及对颗粒细胞的影响机制奠定基础。

关键词: CTSD, 黔北麻羊, 颗粒细胞, 调控机制, 产羔性状

Abstract: The aim of this study was to analyze the regulatory mechanism of cathepsin D (CTSD) on the follicular granulosa cells of Qianbei Ma goat and to explore its effect mechanism on litter size traits.In this study, 36-week-old, healthy, polytocous female Qianbei Ma goat (n=5) were used as the research object, follicular granulosa cells from ovarian tissues collected after slaughter were isolated and cultured. The eukaryotic expression vector pEGFP-N3-CTSD was constructed, and the eukaryotic expression efficiency at the transcription and translation levels after introducing it into cells was examined; CCK-8 assay was used to detect the effect of recombinant plasmid on the proliferation of granulosa cells in different time periods. The effects of recombinant plasmid on the apoptosis and cycle of granulosa cells were detected by flow cytometry; Subsequently, RT-qPCR was used to detect the effect of recombinant plasmids on the expression levels of apoptosis-related genes Bcl-2, Bax, Caspase-3, cell cycle-related factors Cyclin A1, Cyclin D2, and Cyclin E mRNA at the cellular level. Finally, the prolificacy traits candidate genes BMPR-IB, FSHR, INHA were used as functional genes to verify the effect of recombinant plasmids on their mRNA and protein expression at the transcription and translation levels. The results of double enzyme digestion and sequencing confirmed that the eukaryotic expression vector pEGFP-N3-CTSD with CTSD gene of Qianbei Ma goat was successfully constructed, and the expression of CTSD in granulosa cells could be extremely significantly increased at the transcription and translation levels (P<0.01); The cell proliferation test results showed that the up-regulation of CTSD in granulosa cells could inhibit cell proliferation, and the inhibition efficiency on cell proliferation was extremely significant at 12, 24, 48 and 72 h (P<0.01); The results of apoptosis detection showed that the overexpression of CTSD could significantly promote the apoptosis of granulosa cells(P<0.01) and significantly down-regulate the expression of anti-apoptosis gene Bcl-2 (P<0.05), significantly up-regulate the expression of apoptosis-related genes Bax and Caspase-3 (P<0.01). In addition, the results of cell cycle detection showed that the recombinant plasmid pEGFP-N3-CTSD could significantly up-regulate the number of cells at G0/G1 and G2/M phases (P<0.01), and significantly down-regulate the number of cells at S phase(P<0.01), significantly increase the expression of cell cycle related factors Cyclin A1(P<0.01), and significantly decrease the expression of Cyclin D2 (P<0.01). The results of RT-qPCR and Western blot detection showed that the up-regulation of CTSD in granulosa cells could significantly down-regulate the expression of prolificacy trait candidate genes and proteins BMPR-IB, FSHR and INHA at transcriptional and translational levels (P<0.01).In this study, it was found that the high expression of CTSD could inhibit cell proliferation, promote cell apoptosis, change the cycle process of granulosa cells, change the expression of apoptosis-related factors Bcl-2, Bax, Caspase-3 and cell cycle-related factors Cyclin A1, Cyclin D2, Cyclin E, and significantly reduce the expression of prolificacy trait candidate genes and proteins BMPR-IB, FSHR and INHA in granulosa cells(P<0.01). It is suggested that CTSD may regulate the biological behavior of granulosa cells and affect the expression of candidate genes of prolific traits in goats by changing the expression of related factors at the cell level, and then indirectly become an important factor affecting litter size traits in goats. This study laid a foundation for further exploring the regulation mechanism of CTSD on litter size traits and the mechanism of its effect on granulosa cells in goats.

Key words: CTSD, Qianbei Ma goat, granulosa cell, regulation mechanism, litter size traits

中图分类号:

S827.3

周志楠, 陈祥, 张艳, 杨沛方, 惠茂茂, 唐文, 洪磊. CTSD对黔北麻羊卵泡颗粒细胞的调控机制及功能分析[J]. 畜牧兽医学报, 2021, 52(5): 1278-1292.

ZHOU Zhinan, CHEN Xiang, ZHANG Yan, YANG Peifang, HUI Maomao, TANG Wen, HONG Lei. Regulatory Mechanism and Functional Analysis of CTSD on Follicular Granulosa Cells of Qianbei Ma Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1278-1292.

参考文献 38

[1]	孙振梅.黔北麻羊BMP4和BMPR-IB基因克隆表达及其多态性与繁殖性状的关联分析[D].贵阳:贵州大学,2017.SUN Z M.Cloning expression and polymorphisms analysis of BMP4 and BMPR-IB genes associated with reproductive traits in Qianbei Ma goat[D].Guiyang:Guizhou University,2017.(in Chinese)
[2]	翁吉梅.黔北麻羊FTO基因多态性及其表达与肉质性状的关联性研究[D].贵阳:贵州大学,2019.WENG J M.Association study for polymorphism and expression of FTO gene with meat quality traits in Qianbei brown goat[D].Guiyang:Guizhou University,2019.(in Chinese)
[3]	MAN S M,KANNEGANTI T D.Regulation of lysosomal dynamics and autophagy by CTSB/cathepsin B[J].Autophagy, 2016,12(12):2504-2505.
[4]	CHENG X W,HUANG Z,KUZUYA M,et al.Cysteine protease cathepsins in atherosclerosis-based vascular disease and its complications[J].Hypertension,2011,58(6):978-986.
[5]	PATEL S,HOMAEI A,EL-SEEDI H R,et al.Cathepsins:proteases that are vital for survival but can also be fatal[J].Biomed Pharmacother,2018,105:526-532.
[6]	曾广智,谭宁华,贾锐锐,等.组织蛋白酶及其抑制剂研究进展[J].云南植物研究,2005,27(4):337-354.ZENG G Z,TAN N H,JIA R R,et al.Cathepsins:structures,functions and inhibitors[J].Acta Botanica Yunnanica,2005, 27(4): 337-354.(in Chinese)
[7]	ROSSI A,DEVERAUX Q,TURK B,et al.Comprehensive search for cysteine cathepsins in the human genome[J].Biol Chem, 2004,385(5):363-372.
[8]	ZAIDI N,MAURER A,NIEKE S,et al.Cathepsin D:a cellular roadmap[J].Biochem Biophys Res Commun,2008,376(1):5-9.
[9]	周瑞.Cathepsin D缺陷小鼠的行为学及其机制的研究[D].杭州:浙江大学,2015.ZHOU R.Behavioral phenotype of Cathepsin D-deficient mice[D].Hangzhou:Zhejiang University,2015.(in Chinese)
[10]	RUSSO V,FONTANESI L,SCOTTI E,et al.Single nucleotide polymorphisms in several porcine cathepsin genes are associated with growth,carcass,and production traits in Italian large white pigs[J].J Anim Sci,2008,86(12):3300-3314.
[11]	SONG G,SPENCER T E,BAZER F W.Cathepsins in the ovine uterus:regulation by pregnancy,pro-gesterone,and interferon tau[J].Endocrinology,2005,146(11):4825-4833.
[12]	CAO W,LI Y,LI M,et al.Txn1,Ctsd and Cdk4 are key proteins of combination therapy with taurine,epigallocatechin gallate and genistein against liver fibrosis in rats[J].Biomed Pharmacother,2017,85:611-619.
[13]	GAO Y H,GAO Y T,HUANG B,et al.Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus)[J].Sci Rep,2020,10(1):823.
[14]	MANCHANDA M,DAS P,GAHLOT G P S,et al.Cathepsin L and B as potential markers for liver fibrosis:insights from patients and experimental models[J].Clin Transl Gastroenterol,2017,8(6):e99.
[15]	RIBECA C,BONFATTI V,CECCHINATO A,et al.Association of polymorphisms in calpain 1,(mu/I) large subunit, calpastatin, and cathepsin D genes with meat quality traits in double-muscled Piemontese cattle[J].Anim Genet,2013, 44(2): 193-196.
[16]	梅盈洁,李加琪,陈瑶生,等.猪CTSD全长cDNA的克隆和表达分析[J].畜牧兽医学报,2007,38(5):432-436.MEI Y J,LI J Q,CHEN Y S,et al.Cloning and expression of porcine Cathepsin D gene full-length cDNA[J].Acta Veterinaria et Zootechnica Sinica,2007,38(5):432-436.(in Chinese)
[17]	邓桂馨.牛溶酶体组织蛋白酶家族及其抑制基因的克隆、表达及遗传效应分析[D].北京:中国农业科学院,2011.DENG G X.Cloning,expression and genetics effect analysis of Cathepsin family genes and their inhibitor of cattle[D].Beijing: Chinese Academy of Agricultural Sciences,2011.(in Chinese)
[18]	王念璐.天府肉羊CTSD和CSTB基因克隆及其组织表达测定[D].成都:四川农业大学,2014.WANG N L.cDNA cloning and tissue expression of CTSD,CSTB in Tianfu goat[D].Chengdu:Sichuan Agricultural University, 2014.(in Chinese)
[19]	周志楠,陈祥,敖叶,等.黔北麻羊RARRES1基因的克隆表达及功能初步研究[J].畜牧兽医学报,2020,51(7):1548-1562.ZHOU Z N,CHEN X,AO Y,et al.Cloning and expression of RARRES1 gene in Qianbei Ma goat and preliminary study on its function[J].Acta Veterinaria et Zootechnica Sinica,2020,51(7):1548-1562.(in Chinese)
[20]	敖叶,陈祥,周志楠,等.SMAD1基因对黔北麻羊卵巢颗粒细胞的影响及组织表达分析[J].畜牧兽医学报,2020, 51(7): 1607-1618.AO Y,CHEN X,ZHOU Z N,et al.Effect of SMAD1 gene on ovarian granulosa cells and its tissue expression analysis in Qianbei Ma goats[J].Acta Veterinaria et Zootechnica Sinica,2020,51(7):1607-1618.(in Chinese)
[21]	ZI X D,LU J Y,ZHOU H,et al.Omparative analysis of ovarian transcriptomes between prolific and non‐prolific goat breeds via high‐throughput sequencing[J].Reprod Domest Anim,2018,53(2):344-351.
[22]	SHIMIZU T,MURAYAMA C,SUDO N,et al.Involvement of insulin and growth hormone (GH) during follicular development in the bovine ovary[J].Anim Reprod Sci,2008,106(1-2):143-152.
[23]	QUINN R L,SHUTTLEWORTH G,HUNTER M G.Immunohistochemical localization of the bone morphogenetic protein receptors in the porcine ovary[J].J Anat,2004,205(1):15-23.
[24]	MAHDAVI M,NANEKARANI S,HOSSEINI S D.Mutation in BMPR-IB gene is associated with litter size in Iranian Kalehkoohi sheep[J].Anim Reprod Sci,2014,147(3-4):93-98.
[25]	KEMPISTY B,JACKOWSKA M,WOŹNA M,et al.Expression and cellular distribution of INHA and INHB before and after in vitro cultivation of porcine oocytes isolated from follicles of different size[J].J BioMed Biotechnol,2011,2012:742829.
[26]	YOON S H,CHOI Y M,HONG M A,et al.Inhibin α gene promoter polymorphisms in Korean women with idiopathic premature ovarian failure[J].Hum Reprod,2012,27(6):1870-1873.
[27]	CUI Z F,LIU L B,ZHAO X L,et al.Analysis of expression and single nucleotide polymorphisms of INHA gene associated with reproductive traits in chickens[J].Biomed Res Int,2019,2019:8572837.
[28]	SUN D,BAI M Z,JIANG Y Y,et al.Roles of follicle stimulating hormone and its receptor in human metabolic diseases and cancer[J].Am J Transl Res,2020,12(7):3116-3132.
[29]	LIU P,JI Y T,YUEN T,et al.Blocking FSH induces thermogenic adipose tissue and reduces body fat[J].Nature,2017, 546(7656):107-112.
[30]	HAN P,XIN H Y,PENG J Y,et al.Identification and expression of X-linked inhibitor of apoptosis protein during follicular development in goat ovary[J].Theriogenology,2017,98:30-35.
[31]	FENG X,LI F Z,WANG F,et al.Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep[J].Biosci Rep,2018,38(2):BSR20171350.
[32]	DEISS L P,GALINKA H,BERISSI H,et al.Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha[J].Embo J,1996,15(15):3861-3870.
[33]	ABOELENAIN M,KAWAHARA M,BALBOULA A Z,et al.Status of autophagy,lysosome activity and apoptosis during corpus luteum regression in cattle[J].J Reprod Dev,2015,61(3):229-236.
[34]	MINAROWSKA A,MINAROWSKI L,KARWO-WSKA A,et al.Regulatory role of cathepsin D in apoptosis[J].Folia Histochem Cytobiol,2007,45(3):159-163.
[35]	曹晓涵.Bcl-2相关的凋亡基因在绵羊繁殖器官中的表达特征及Bad基因在绵羊发情周期中的作用[D].北京:中国农业科学院,2018.CAO X H.Expression of the Bcl-2-associated of cell death genes in the sheep reproductive organs and the functional analysis of Bad gene during the reproductive cycle[D].Beijing:Chinese Academy of Agricultural Sciences,2018.(in Chinese)
[36]	XU X G,ZHANG H L,ZHANG Q,et al.Porcine epidemic diarrhea virus N protein prolongs S-phase cell cycle,induces endoplasmic reticulum stress,and up-regulates interleukin-8 expression[J].Vet Microbiol,2013,164(3-4):212-221.
[37]	熊永洁.ATF6在小鼠胚胎植入期子宫和卵巢颗粒细胞凋亡过程中的作用研究[D].杨凌:西北农林科技大学,2017.XIONG Y J.Study on function of ATF6 in mouse periimplantation uterus and granulosa cell apoptosis[D].Yangling:Northwest A&F University,2017.(in Chinese)
[38]	陈任真.Cx43与AKAP95竞争性结合cyclinE调控细胞G1-S期转变[D].厦门:厦门大学,2019.CHEN R Z.Cx43 and AKAP95 regulate cell G1-S transition by competitively binding to cyclinE[D].Xiamen:Xiamen University, 2019.(in Chinese)