山羊FST慢病毒载体构建及其对卵巢卵泡颗粒细胞增殖的影响

doi:10.11843/j.issn.0366-6964.2019.09.017

Abstract

Abstract: This experiment was conducted to study the effect of follistatin (FST) on the proliferation of goat ovarian follicular granulosa cells. Primary ovarian follicular granulosa cells were isolated and cultured from goat ovarian tissue. The goat FST was cloned by RT-PCR, shRNA fragment was designed, and lentiviral overexpression vector and interference vector were constructed. The effect of overexpression and interference were detected by qPCR technique, and the proliferation effect of the cells was detected by CCK-8 assay. Goat ovarian follicular granulosa cells were identified by immunofluorescence of follicle stimulating hormone receptor (FSHR), and the positive rate was 95%. After the lentivirus infected granulosa cells, the expression of fluorescent protein accounted for 80%. The effect of overexpression of FST and interference with lentiviral vector were both significant (P<0.05) by qPCR. Overexpression of FST significantly (P<0.05) inhibited the proliferation of granulosa cells, and interfered with FST significantly (P<0.05) promoted granulosa cell proliferation. The ovarian follicular granulosa cells were isolated; the FST overexpression and interference vectors were successfully constructed. The infection results indicated that FST could inhibit the proliferation of goat ovarian follicular granulosa cells. It provides basic data for further study of the role of FST in the regulation of mammalian follicular development and its mechanism.

Key words: follistatin gene, ovarian follicular granulosa cells, lentivirus vector, shRNA, overexpression vector, cell proliferation

CLC Number:

LING Yinghui, ZHU Lu, WU Hao, CHEN Qing, QUAN Qing, LIU Yong, LI Wenyong, ZHANG Yunhai. Construction of Goat FST Lentivirus Vectors and Its Effect on the Proliferation of Ovarian Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(9): 1888-1896.

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References

[1] GOSDEN R G, TELFER E. Numbers of follicles and oocytes in mammalian ovaries and their allometric relationships[J]. J Zool, 1987, 211(1):169-175.
[2] REGAN S L P, KNIGHT P G, YOVICH J L, et al. Granulosa cell apoptosis in the ovarian follicle-a changing view[J]. Front Endocrinol (Lausanne), 2018, 9:61.
[3] UENO N, LING N, YING S Y, et al. Isolation and partial characterization of follistatin:a single-chain Mr 35000 monomeric protein that inhibits the release of follicle-stimulating hormone[J]. Proc Natl Acad Sci U S A, 1987, 84(23):8282-8286.
[4] FUNG R S K, BAI J, YUEN K W Y, et al. Activin/follistatin system in grass carp pituitary cells:-regulation by local release of growth hormone and luteinizing hormone and its functional role in growth hormone synthesis and secretion[J]. PLoS One, 2017, 12(6):e0179789.
[5] KÖNINGER A, SCHMIDT B, DAMASKE D, et al. Follistatin during pregnancy and its potential role as an ovarian suppressing agent[J]. Eur J Obstet Gynecol Reprod Biol, 2017, 212:150-154.
[6] MUTTUKRISHNA S, TANNETTA D, GROOME N, et al. Activin and follistatin in female reproduction[J]. Mol Cell Endocrinol, 2004, 225(1-2):45-56.
[7] COCKRELL A S, KAFRI T. Gene delivery by lentivirus vectors[J]. Mol Biotechnol, 2007, 36(3):184-204.
[8] LAWSON S K, DOBRIKOVA E Y, SHVEYGERT M, et al. p38α mitogen-activated protein kinase depletion and repression of signal transduction to translation machinery by miR-124 and -128 in neurons[J]. Mol Cell Biol, 2013, 33(1):127-135.
[9] 徐敏,许厚强,杨洋,等. FGF10基因真核表达载体的构建及其在从江香猪不同组织中的表达[J]. 农业生物技术学报, 2019, 27(3):471-480.
XU M, XU H Q, YANG Y, et al. Construction of eukaryotic expression vector of FGF10 gene and its expression in different tissues of Congjiang Xiang Pig (Sus scrofa)[J]. Journal of Agricultural Biotechnology, 2019, 27(3):471-480. (in Chinese)
[10] 吴锦艳, 田宏,蒙学莲,等. 慢病毒介导稳定表达增强小反刍兽疫病毒复制的山羊Vero/SLAM细胞系的建立[J]. 畜牧兽医学报, 2019, 50(1):218-226.
WU J Y, TIAN H, MENG X L, et al. Establishment of Vero/SLAM cell lines with stable expression SLAM to enhanced PPRV replication using the lentiviral expression system[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(1):218-226. (in Chinese)
[11] ZHU K, LI S, LIU J S, et al. Role of RAB5A in FSHR-mediated signal transduction in human granulosa cells[J]. Reproduction, 2018, 155(6):505-514.
[12] XU D J, HE H S, JIANG X H, et al. SIRT2 plays a novel role on progesterone, estradiol and testosterone synthesis via PPARs/LXRα pathways in bovine ovarian granular cells[J]. J Steroid Biochem Mol Biol, 2019, 185:27-38.
[13] MÁTRAI J, CHUAH M K, VANDENDRIESSCHE T. Recent advances in lentiviral vector development and applications[J]. Mol Ther, 2010, 18(3):477-490.
[14] HENRIKSEN J R, BUECHNER J, L LØKKE C, et al. Inhibition of gene function in mammalian cells using short-hairpin RNA (shRNA)[M]//NIELSEN H. RNA:Methods and Protocols. New York:Humana Press, 2011:189-204.
[15] 王曼,孙长花,李东,等. 鸡FGF8基因RNA干扰载体的构建及其对PGCs形成的影响[J]. 农业生物技术学报, 2018, 26(9):1457-1466.
WANG M, SUN C H, LI D, et al. Construction of RNA interference vector of chicken (Gallus domesticus) FGF8 gene and its effect on the formation of PGCs[J]. Journal of Agricultural Biotechnology, 2018, 26(9):1457-1466. (in Chinese)
[16] RICHARDS J A S. From follicular development and ovulation to ovarian cancers:an unexpected journey[J]. Vitam Horm, 2018, 107:453-472.
[17] PATEL K. Follistatin[J]. Int J Biochem Cell Biol, 1998, 30(10):1087-1093.
[18] MEEHAN T, SCHLATT S, O'BRYAN M K, et al. Regulation of germ cell and sertoli cell development by activin, follistatin, and FSH[J]. Dev Biol, 2000, 220(2):225-237.
[19] KIMURA F, BONOMI L M, SCHNEYER A L. Follistatin regulates germ cell nest breakdown and primordial follicle formation[J]. Endocrinology, 2011, 152(2):697-706.
[20] SILVA J R V, THARASANIT T, TAVERNE M A M, et al. The activin-follistatin system and in vitro early follicle development in goats[J]. J Endocrinol, 2006, 189(1):113-125.
[21] NAKAMURA T, TAKIO K, ETO Y, et al. Activin-binding protein from rat ovary is follistatin[J]. Science, 1990, 247(4944):836-838.
[22] XU R H, SAMPSELL-BARRON T L, GU F, et al. NANOG is a direct target of TGFβ/activin-mediated SMAD signaling in human ESCs[J]. Cell Stem Cell, 2008, 3(2):196-206.
[23] YU C, ZHANG Y L, FAN H Y. Selective Smad4 knockout in ovarian preovulatory follicles results in multiple defects in ovulation[J]. Mol Endocrinol, 2013, 27(6):966-978.

Construction of Goat FST Lentivirus Vectors and Its Effect on the Proliferation of Ovarian Follicular Granulosa Cells

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