水牛原代体细胞慢病毒感染体系的建立

doi:10.16431/j.cnki.1671-7236.2019.10.024

摘要/Abstract

摘要： 试验旨在建立水牛原代体细胞慢病毒感染体系。通过比较慢病毒（lentivirus）感染水牛颗粒细胞（buffalo granulosa cell，BuGC）、水牛乳腺上皮细胞（buffalo mammary epithelial cell，BuMEC）、水牛成纤维细胞（buffalo fibroblast cell，BuFC）的效率及荧光强度，筛选出3种水牛原代体细胞的最佳感染复数（MOI）。分离培养获得BuGC、BuMEC及BuFC。将慢病毒质粒pLVX-Puro-GFP和包装质粒pSPAX2、pMAD2.G脂质体转染HEK293T细胞，于转染后48和72 h收集病毒并通过稀释计数法测定慢病毒滴度。将慢病毒按不同的感染复数（100、200、400、600、800）感染BuGC、BuMEC和BuFC，感染72 h后于倒置荧光显微镜下观察拍照，统计感染效率及荧光强度。慢病毒感染3种水牛原代体细胞后，用嘌呤霉素筛选。结果显示，在MOI≤200时，慢病毒感染后细胞荧光强度BuMEC最强，而BuGC和BuFC间无显著差异；在MOI≥400时，慢病毒感染后细胞荧光强度为：BuMEC>BuGC>BuFC。在MOI=100时，慢病毒感染效率为：BuGC>BuMEC>BuFC；在MOI=200时，BuMEC和BuGC感染效率达到100%；在MOI=800时，BuFC感染效率达到100%。不同细胞类型对慢病毒的毒性耐受存在差异，在嘌呤霉素的筛选和维持下，获得了3种水牛原代体细胞绿色荧光蛋白（green fluorescent protein，GFP）过表达慢病毒稳转细胞株。慢病毒感染BuMEC、BuGC、BuFC的最佳MOI分别为200、400和800；3种水牛原代体细胞均能通过慢病毒获得过表达外源基因细胞株。本研究结果为水牛的乳腺组织发育分化、泌乳调控、生殖发育及转基因动物制备等提供了较好的细胞模型。

关键词: 水牛; 慢病毒; 颗粒细胞; 乳腺上皮细胞; 成纤维细胞

Abstract: This study was aimed to establish a system of buffalo primary somatic lentivirus infection.The efficiency and fluorescence intensity of buffalo granulosa cell (BuGC),buffalo mammary epithelial cell (BuMEC) and buffalo fibroblast cell (BuFC) infected by lentivirus were compared.The optimal multiplicity of infection (MOI) values of three buffalo primary somatic cells were selected.BuGC,BuMEC and BuFC were isolated and cultured.HEK293T cell was transfected with pLVX-Puro-GFP,pSPAX2 and pMAD2.G by liposome,and then the virus was collected at 48 and 72 h after transfection,and the titer of lentivirus was determined by dilution counting method.BuGC,BuMEC and BuFC were exposed to different multiplicity of infection (100,200,400,600 and 800) of the lentivirus.After 72 h,the cells were observed by the inverted fluorescence microscope,the transfection efficiency and fluorescence intensity were calculated.Three buffalo primary somatic cells were infected with lentivirus and screened with puromycin.The results showed that at MOI ≤ 200,the fluorescence intensity of cells after lentivirus infection of BuMEC was the strongest,and there was no significant difference between BuGC and BuFC;At MOI ≥ 400,the fluorescence intensity of cells after lentivirus infection was BuMEC>BuGC>BuFC.At MOI=100,the lentivirus infection efficiency was BuGC>BuMEC>BuFC;At MOI=200,the lentivirus infection efficiency of BuMEC and BuGC were 100%.At MOI=800,the lentivirus infection efficiency of BuFC was 100%.Different cell types had different tolerance to lentivirus.Under the selection and maintenance of puromycin,three buffalo primary somatic cell lines with overexpression of green fluorescent protein (GFP) in buffalo primary somatic cells were obtained.The optimal MOI of BuMEC,BuGC and BuFC infected by lentivirus were 200,400 and 800,respectively.All three buffalo primary somatic cells were able to establish the cell lines which overexpressed stably a foreign gene with lentivirus.This results provided good cell models for the study of buffalo breast tissue development and differentiation,lactation regulation,reproductive development and preparation of transgenic animals.

Key words: buffalo; lentivirus; granulosa cell; mammary epithelial cell; fibroblast

中图分类号:

S852.65⁺3

段安琴, 陆杏蓉, 马小娅, 梁莎莎, 庞春英, 梁贤威, 邓廷贤. 水牛原代体细胞慢病毒感染体系的建立[J]. 中国畜牧兽医, 2019, 46(10): 3023-3031.

DUAN Anqin, LU Xingrong, MA Xiaoya, LIANG Shasha, PANG Chunying, LIANG Xianwei, DENG Tingxian. Establishment of Buffalo Primary Somatic Lentivirus Infection System[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(10): 3023-3031.

参考文献

[1] 简保权,秦学敏,龚芳,等.世界水牛奶业发展现状和典型模式分析[J].世界农业,2015,3:115-118. JIAN B Q,QIN X M,GONG F,et al.Analysis on the status and typical models of world water buffalo milk industry[J].Word Agriculture,2015,3:115-118.(in Chinese)
[2] YANG B,LIANG X,QIN J,et al.Brief introduction to the development of Chinese dairy buffalo industry[J].Buffalo Bulletin,2013,32(1):111-120.
[3] 毛颖佳,郑源强,石艳春,等.慢病毒载体及其应用的研究进展[J].中国生物制品学杂志,2009,22(2):196-200. MAO Y J,ZHENG Y Q,SHI Y C,et al.Progress in study on sentiviral sectors and their application[J].Chinese Journal of Biologicals,2009,22(2):196-200.(in Chinese)
[4] FENG G,SHI D,YANG S,et al.Co-culture embedded in cumulus clumps promotes maturation of denuded oocytes and reconstructs gap junctions between oocytes and cumulus cells[J]. Zygote,2013,21(3):231-237.
[5] KAUR G,ALI S A,PACHAURI S,et al.Buffalo leukemia inhibitory factor induces differentiation and dome-like secondary structures in COS-1 cells[J].Cytogenetic and Genome Research,2017,151(3):119-130.
[6] KUMAR D,SHARMA P,VIJAYALAKSHMY K,et al.Generation of Venus fluorochrome expressing transgenic handmade cloned buffalo embryos using Sleeping Beauty transposon[J].Tissue & Cell,2018,51:49-55.
[7] PANDA M,ASMITA P,KUMAR S,et al.Effect of exogenous progesterone on cumulus characteristics of buffalo oocytes by allowing passage of more number of sperm through cumulus but not essentially fertilization[J].Asian Pacific Journal of Reproduction,2018,7(2):79-86.
[8] SHANDILYA U K,SHARMA A,SODHI M,et al.Matrix-based three-dimensional culture of buffalo mammary epithelial cells showed higher induction of genes related to milk protein and fatty acid metabolism[J].Cell Biology International,2016,40(2):232-238.
[9] YADAV P,KUMAR P,MUKESH M,et al.Kinetics of lipogenic genes expression in milk purified mammary epithelial cells (MEC) across lactation and their correlation with milk and fat yield in buffalo[J].Research in Veterinary Science,2015,99:129-136.
[10] 段安琴,庞春英,朱鹏,等.腺病毒载体转染水牛不同原代体细胞的效果比较[J].中国畜牧兽医,2016,43(10):2661-2665. DUAN A Q,PANG C Y,ZHU P,et al.Comparison of adenovirus vector mediated gene transfer into different buffalo cells[J].China Animal Husbandry ＆ Veterinary Medicine,2016,43(10):2661-2665.(in Chinese)
[11] 段安琴,庞春英,朱鹏,等.水牛乳汁中乳腺上皮细胞的分离培养及鉴定[J].中国畜牧兽医,2017,44(11):3243-3249. DUAN A Q,PANG C Y,ZHU P,et al.Isolation,culture and identification of buffalo mammary epithelial cells from milk[J]. China Animal Husbandry ＆ Veterinary Medicine,2017,44(11):3243-3249.(in Chinese)
[12] 李松,丁方荣,戴蕴平,等.不同转染方法转染牛体细胞效率的比较[J].农业生物技术学报,2011,19(6):1027-1033. LI S,DING F R,DAI Y P,et al.Comparison of different methods for transfection efficiency of bovine somatic cells[J]. Journal of Agricultural Biotechnology,2011,19(6):1027-1033.(in Chinese)
[13] 孟凡荣,陈琛,万海粟,等.慢病毒载体及其研究进展[J].中国肺癌杂志,2014,17(12):870-876. MENG F R,CHEN C,WAN H S,et al.Advances of lentiviral vectors[J].Chinese Journal of Lung Cancer,2014,17(12):870-876.(in Chinese)
[14] BU W,XIN L,TONEFF M,et al.Lentivirus vectors for stably introducing genes into mammary epithelial cells in vivo[J].Journal of Mammary Gland Biology and Neoplasia,2009,14(4):401-404.
[15] QIAN W,WANG Y,LI R F,et al.Prolonged integration site selection of a lentiviral vector in the genome of human keratinocytes[J]. Medical Science Monitor,2017,23:1116-1122.
[16] 姚永曦,吴睿帆,汪以真,等.腺病毒与慢病毒感染猪原代细胞的比较研究[J].中国畜牧杂志,2018,54(4):83-88. YAO Y X,WU R F,WANG Y Z,et al.Comparative study on adenovirus and lentivirus infection of porcine primary cells[J].Chinese Journal of Animal Science,2018,54(4):83-88.(in Chinese)
[17] 黄乙涓,黄强,吕欧.高剂量pSin慢病毒载体介导的转染对诱导性多能干细胞影响的研究[J].中华细胞与干细胞杂志(电子版),2017,7(3):125-130. HUANG Y J,HUANG Q,LYU O.Effect of high dose pSin lentiviral vector-mediated transfection on pluripotent stem cells[J].Chinese Journal of Cell and Stem Cell (Electronic Edition),2017,7(3):125-130.(in Chinese)
[18] 肖渝,李彦林,王坤,等.三种携带GFP慢病毒转染兔骨髓间充质干细胞的效果[J].中国组织工程研究,2017,21(29):4642-4647. XIAO Y,LI Y L,WANG K,et al.Transfection efficiency of three different lentiviruses with green fluorescent protein gene to transfect rabbit bone marrow mesenchymal stem cells[J].Chinese Journal of Tissue Engineering Research,2017,21(29):4642-4647.(in Chinese)
[19] 夏静,夏蒙蒙,牛长敏,等.慢病毒介导的miR194-5p过表达精原细胞株的建立[J].中国男科学杂志,2018,32(2):10-15. XIA J,XIA M M,NIU C M,et al.Establishment of spermatogonium with miR194-5p overexpression by lentivirus mediated overexpression technology[J].Chinese Journal of Andrology,2018,32(2):10-15.(in Chinese)
[20] 欧阳寒梅,罗丹,王欣,等.高滴度慢病毒制备及其感染人原代T细胞的条件优化[J].成都医学院学报,2019,14(1):16-20. OUYANG H M,LUO D,WANG X,et al.Preparation of high-titer lentivirus and optimization of the infection conditions for human primary T cells[J].Journal of Chengdu Medical College,2019,14(1):16-20.(in Chinese)
[21] CAO F,XIE X,GOLLA T,et al.Comparison of gene-transfer efficiency in human embryonic stem cells[J].Molecular Imaging and Biology,2010,12(1):15-24.
[22] WINIARSKA M,NOWIS D,FIRCZUK M,et al.Selection of an optimal promoter for gene transfer in normal B cells[J].Molecular Medicine Reports,2017,16(3):3041-3048.
[23] KAUSHIK R,SINGH K P,KUMARI A,et al.Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line[J].Molecular Biology Reports,2014,41(9):5891-5902.
[24] GHANEIALVA H,LOTFI A S,ARJMAND S,et al.Comparison of transduction efficiency among various cell types by a lentivector containing CMV promoter[J].Comparative Clinical Pathology,2019,28(4):1077-1085.
[25] 林兰,徐之良.慢病毒介导不同启动子嵌合抗原受体CD19在T细胞中的表达[J].医学研究杂志,2017,46(6):92-98. LIN L,XU Z L.Comparision of the efficiency of different promoters driving the expression of CD19——Specifc chimeric antigen receptor (CAR)-redirected T lymphocytes mediated by lentiviral vector[J].Journal of Medical Research,2017,46(6):92-98.(in Chinese)
[26] WANG N,RAJASEKARAN N,HOU T,et al.Comparison of transduction efficiency among various lentiviruses containing GFP reporter in bone marrow hematopoietic stem cell transplantation[J]. Experimental Hematology,2013,41(11):934-943.
[27] FRECHA C,LEVY C,COSSET F L,et al.Advances in the field of lentivector-based transduction of T and B lymphocytes for gene therapy[J]. Molecular Therapy:The Journal of the American Society of Gene Therapy,2010,18(10):1748-1757.
[28] HORNICK A L,LI N,OAKLAND M,et al.Human,pig,and mouse interferon-induced transmembrane proteins partially restrict pseudotyped lentiviral vectors[J].Human Gene Therapy,2016,27(5):354-362.