Apoptosis of human primary ovarian granulose cells infected with lentivirus carrying bcl-2 gene

doi:10.3969/j.issn.2095-4344.2013.28.018

Abstract

Abstract:

BACKGROUND: Lentivirus can infect divided and undivided cells. It remains uncertain whether the lentivirus can successfully infect primary ovarian granulosa cells.

OBJECTIVE: To investigate infecting ratio and cell apoptosis of lentivirus carrying bcl-2 gene in primary human ovarian granulose cells cultured in vitro.

METHODS: The lentiviral vector carrying bcl-2 gene was constructed using molecular biology, and packaged into lentivirus with high titer. The resulting recombinant lentivirus carrying bcl-2 genes were then used to infect primary human ovarian granulosa cells in vitro at different multiplicity of infection, 10, 50, 100, 200, and 400. Infection efficiency and cell proliferation were observed at 24, 48, 72, and 96 hours following infection. Cell apoptosis was detected by flow cytometry, and bcl-2 gene transcription was assessed using reverse transcription PCR.

RESULTS AND CONCLUSION: Primary human ovarian granulosa cells adhered at 24 hours, and exhibited polygon- or fusiform-shape and colony-like growth. When multiplicity of infection was 100, cell appearance and growth remained unchanged, and infection efficiency was high, which reached the peak up to 72 hours. Moreover, the positive rate was up to 60% in granulosa cells. Lentivirus carrying bcl-2gene could increase expression of Bcl-2 protein and inhibit apoptosis of primary ovarian granulosa cells.

Key words: tissue construction, cytology experiment in tissue construction, bcl-2 gene, lentivirus, ovarian granulosa cells, cell apoptosis, gene transfection, vector construction, premature ovarian failure, enhanced green fluorescent protein gene, National Natural Science Foundation of China

CLC Number:

R318

Wang Xue-feng, Tan Feng, Chen Yan-ying, Liu Mu-biao, He Yuan-li. Apoptosis of human primary ovarian granulose cells infected with lentivirus carrying bcl-2 gene[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(28): 5209-5215.

Figures/Tables 5

References

[1]Maman E, Prokopis K, Levron J, et al. Does controlled ovarian stimulation prior to chemotherapy increase primordial follicle loss and diminish ovarian reserve? An animal study. Hum Reprod. 2009;24(1):206-210.

[2]Lie Fong S, Lugtenburg PJ, Schipper I, et al. Anti-müllerian hormone as a marker of ovarian function in women after chemotherapy and radiotherapy for haematological malignancies. Hum Reprod. 2008;23(3):674-678.

[3]Mathelin C, Brettes JP, Diemunsch P. Premature ovarian failure after chemotherapy for breast cancer.Bull Cancer. 2008;95(4):403-412.

[4]Sasagawa S, Shimizu Y, Nagaoka T, et al. Dienogest, a selective progestin, reduces plasma estradiol level through induction of apoptosis of granulosa cells in the ovarian dominant follicle without follicle-stimulating hormone suppression in monkeys.J Endocrinol Invest. 2008;31(7):636-641.

[5]Yuan P, Bartlam M, Lou Z,et al. Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site.Nature. 2009;458(7240):909-913.

[6]王雪峰,何援利. 大鼠B细胞淋巴瘤-2基因的克隆及其慢病毒表达载体的构建[J].解放军医学杂志,2009,34(1):51-54.

[7]郜虹媛,匡延平. IVF-ET中促性腺激素释放激素激动剂的超促排卵应用与剂量研究[J].生殖与避孕,2012,32(6):403-407.

[8]王国鹏,罗凌惠,谢静,等.慢病毒载体和腺病毒载体转染原代培养的螺旋神经节细胞的比较[J].临床耳鼻咽喉头颈外科杂志,2011, 25(4):172-175.

[9]陈彩云,刘亚京,牛忠英.慢病毒载体的研究进展及应用[J].口腔颌面修复学杂志,2012,13(2):117-120.

[10]Engelbert D, Schnerch D, Baumgarten A,et al.The ubiquitin ligase APC(Cdh1) is required to maintain genome integrity in primary human cells.Oncogene. 2008;27(7):907-917.

[11]Yew NS, Przybylska M, Ziegler RJ,et al. High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter. Mol Ther. 2001;4(1):75-82.

[12]Conaway RC, Brower CS, Conaway JW. Emerging roles of ubiquitin in transcription regulation. Science. 2002;296 (5571): 1254-1258.

[13]Stanley BJ, Ehrlich ES, Short L,et al. Structural insight into the human immunodeficiency virus Vif SOCS box and its role in human E3 ubiquitin ligase assembly. J Virol. 2008;82(17): 8656-8663.

[14]Böcker W, Rossmann O, Docheva D,et al. Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells.J Gene Med. 2007;9(7):585-595.

[15]Wang YY, Yang Y, Chen Q,et al.Simultaneous knockdown of p18INK4C, p27Kip1 and MAD1 via RNA interference results in the expansion of long-term culture-initiating cells of murine bone marrow cells in vitro.Acta Biochim Biophys Sin (Shanghai). 2008;40(8):711-720.

[16]杨萍,严金川,刘培晶.siRNA反向转染法提高原代悬浮细胞转染效率的应用[J].江苏大学学报:医学版,2010,20(3):267-269.

[17]李华玲,周晓霞.阳离子脂质体转染人类骨骼肌原代细胞的初步研究[J].生物学杂志,2006,23(6):8-10.

[18]张余琴,林晓琳,贾俊双,等. 红色荧光蛋白和荧光素酶双报告基因转基因小鼠的建立[J].中国癌症杂志,2012,22(5):321-328.

[19]王露露,刘勤,周晓杨,等.慢病毒介导HLA-G转基因小鼠的建立[J]. 第三军医大学学报,2012,34(14):1392-1396.

[20]Monniaux D. Oocyte apoptosis and evolution of ovarian reserve. Gynecol Obstet Fertil. 2002;30(10):822-826.

[21]Wang X, Wang Y, Kim HP, et al. Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation.J Biol Chem. 2007;282(3):1718-1726.

[22]Rho GJ, Kim DS. Influence of in vitro oxygen concentrations on preimplantation embryo development, gene expression and production of Hanwoo calves following embryo transfer. Mol Reprod Dev. 2007;74(4):486-496.

[23]Gupta MK, Uhm SJ, Han DW, et al. Embryo quality and production efficiency of porcine parthenotes is improved by phytohemagglutinin. Mol Reprod Dev. 2007;74(4):435-444..

[24]孙懿,胡志德,黄元兰,等. IL-22抑制ox-LDL诱导的CRL-1730细胞凋亡并上调其Bcl-2表达[J].第二军医大学学报,2011,32(3): 233-237.

[25]朱华,胡燕,帅茨霞,等. survivin、bcl-2在宫颈癌和癌前病变组织中的表达及与人乳头瘤病毒16/18感染的相关性[J]. 中华医学杂志,2010,90(7):469-473.

[26]Wang X, Wang Y, Kim HP,et al. Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation.J Biol Chem. 2007;282(3):1718-1726.

[27]Orike N, Middleton G, Borthwick E,et al. Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons.J Cell Biol. 2001;154(5):995-1005.

[28]Vairo G, Innes KM, Adams JM. Bcl-2 has a cell cycle inhibitory function separable from its enhancement of cell survival. Oncogene. 1996;13(7):1511-1519.

[29]Baghdassarian N, Bertrand Y, Ffrench P,et al. Role of BCL-2 and cell cycle regulatory proteins for corticosensitivity assessment in childhood acute lymphoblastic leukaemia. Br J Haematol. 2000;109(1):109-116.

[30]Zhou W, Liu J, Liao L,et al. Effect of bisphenol A on steroid hormone production in rat ovarian theca-interstitial and granulosa cells.Mol Cell Endocrinol. 2008;283(1-2):12-18.

[31]Aerts JM, De Clercq JB, Andries S,et al. Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing.Cryobiology. 2008;57(2):163-169.

[32]Aerts JM, Bols PE. Ovarian follicular dynamics: a review with emphasis on the bovine species. Part I: Folliculogenesis and pre-antral follicle development. Reprod Domest Anim. 2010; 45(1):171-179.

[33]Santos HB, Thomé RG, Arantes FP,et al. Ovarian follicular atresia is mediated by heterophagy, autophagy, and apoptosis in Prochilodus argenteus and Leporinus taeniatus (Teleostei: Characiformes).Theriogenology. 2008;70(9): 1449-1460.

[34]Chitnis SS, Navlakhe RM, Shinde GC,et al. Granulosa cell apoptosis induced by a novel FSH binding inhibitory peptide from human ovarian follicular fluid. J Histochem Cytochem. 2008;56(11):961-968.

[35]Yu YS, Sui HS, Han ZB,et al. Apoptosis in granulosa cells during follicular atresia: relationship with steroids and insulin-like growth factors.Cell Res. 2004;14(4):341-346.

[36]Tilly JL, Tilly KI, Kenton ML,et al. Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels. Endocrinology. 1995;136(1):232-241.

[37]Choi D, Hwang S, Lee E,et al. Expression of mitochondria-dependent apoptosis genes (p53, Bax, and Bcl-2) in rat granulosa cells during follicular development.J Soc Gynecol Investig. 2004;11(5):311-317.

[38]Yoshida Y, Hosokawa K, Dantes A,et al. Theophylline and cisplatin synergize in down regulation of BCL-2 induction of apoptosis in human granulosa cells transformed by a mutated p53 (p53 val135) and Ha-ras oncogene.Int J Oncol. 2000; 17(2):227-235.

[39]姜勋平,周东蕊,杨利国,等.鸡Bcl-2基因的克隆及其对卵泡颗粒细胞凋亡的影响[J].中国兽医学报,2002,22(1):87-89.