绵羊绒毛膜滋养层细胞与子宫内膜腔上皮细胞共培养体系的建立
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摘要
本研究旨在探索绵羊绒毛膜滋养层细胞(sheep trophoblast cells, STCs)和子宫内膜腔上皮细胞(endometrial luminal epithelial cells, ELECs)的共培养条件,为绵羊胎盘绒毛膜滋养层多核细胞的形成提供试验依据。在屠宰场采集妊娠45～60 d的健康绵羊子宫及胎盘组织,用酶消化法体外分离培养STCs和ELECs,并进行细胞免疫荧光鉴定,同时利用姬姆萨氏染色法确定STCs和ELECs最佳的共培养条件;然后转染空质粒pEGFP-C1到ELECs 48 h后,与STCs共培养,并利用激光共聚焦显微镜观察滋养层多核细胞的形态。结果显示:STCs CK-7与ELECs CK-18均呈阳性;当STCs与ELECs细胞数量比值为2∶1,共培养48 h,多核细胞数量最多,且与对照组相比差异显著(P<0.05);CK-7在STCs中表达呈红色荧光,且细胞中有pEGFP-C1标记的绿色荧光,胞核蓝染,3种荧光共表达呈现出了白光。综上表明,本研究建立了STCs和ELECs共培养条件,ELECs参与绵羊滋养层多核细胞的形成。
The study aims to explore the co-culture conditions of sheep trophoblast cells(STCs) and the endometrial luminal epithelial cells(ELECs), and provides experimental basis for chorionic trophoblastic coenocyte formation. Harvesting healthy sheep uterus and placental tissue from 45 to 60 days of gestation at the slaughter house, and the enzyme digestion method has been used to isolate and culture the STCs and ELECs in vitro, and they were identified by cellular immunofluorescence. Simultaneously, the optimal co-culture conditions for STCs and ELECs were determined by Giemsa staining. Then, the empty plasmid pEGFP-C1 was transfected into ELECs for 48 h, and then co-cultured with STCs. The morphology of chorionic trophoblastic coenocyte was observed by confocal laser scanning microscopy. The results showed that STCs CK-7 and ELECs CK-18 were positive.When the ratio of STCs to ELECs cells was 2∶1, the number of multinucleated cells reaches the maximum when the co-culture time was 48 h, and it was significantly different from the control group(P<0.05). The results of immunofluorescence showed that CK-7 expressed red fluorescence in STCs, and there were pEGFP-C1 labeled green fluorescence in the cells, and the nucleus was blue-stained, and the 3 fluorescent co-expressions showed white light. This study established the conditions for co-culture of STCs and ELECs, and showed that ELECs were involved in the formation of polynuclear cells in sheep trophoblasts.
The study aims to explore the co-culture conditions of sheep trophoblast cells(STCs) and the endometrial luminal epithelial cells(ELECs), and provides experimental basis for chorionic trophoblastic coenocyte formation. Harvesting healthy sheep uterus and placental tissue from 45 to 60 days of gestation at the slaughter house, and the enzyme digestion method has been used to isolate and culture the STCs and ELECs in vitro, and they were identified by cellular immunofluorescence. Simultaneously, the optimal co-culture conditions for STCs and ELECs were determined by Giemsa staining. Then, the empty plasmid pEGFP-C1 was transfected into ELECs for 48 h, and then co-cultured with STCs. The morphology of chorionic trophoblastic coenocyte was observed by confocal laser scanning microscopy. The results showed that STCs CK-7 and ELECs CK-18 were positive.When the ratio of STCs to ELECs cells was 2∶1, the number of multinucleated cells reaches the maximum when the co-culture time was 48 h, and it was significantly different from the control group(P<0.05). The results of immunofluorescence showed that CK-7 expressed red fluorescence in STCs, and there were pEGFP-C1 labeled green fluorescence in the cells, and the nucleus was blue-stained, and the 3 fluorescent co-expressions showed white light. This study established the conditions for co-culture of STCs and ELECs, and showed that ELECs were involved in the formation of polynuclear cells in sheep trophoblasts.
引文
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[2] LI Y,ZHENG R,WANG R.Involvement of nephrin in human placental trophoblast syncytialization[J].Reproduction,2015,149(4):339-346.
[3] 周跃华,陆琴.miR-29a在胎盘滋养细胞中的功能[J].中华保健医学杂志,2018,20(2):113-116.ZHOU Y H,LU Q.The function of miR-29a in placental trophoblasts[J].Chinese Journal of Health Care and Medicine,2018,20(2):113-116. (in Chinese)
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[6] 刘丽珍,刘淑英.妊娠早期蒙古绵羊子宫内膜enJSRV及IFN-τ基因表达水平的分析[J].畜牧兽医学报,2012,43(6):979-984.LIU L Z,LIU S Y.Analysis of the expression of enJSRV and IFN-τ in endometrium of Mongolian sheep in early pregnancy[J].Acta Veterinaria et Zootechnica Sinica,2012,43(6):979-984. (in Chinese)
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[8] LEROUX C,GIRARD N,COTTIN V,et al.Jaagsiekte sheep retrovirus (JSRV):from virus to lung cancer in sheep[J].Vet Res,2007,38(2):211-228.
[9] FU J J,Lü X Y,LIN H Y,et al.Ubiquitin ligase cullin 7 induces epithelial-mesenchymal transition in human choriocarcinoma cells[J].J Biol Chem,2010,285(14):10870-10879.
[10] SPENCER T E,PALMARINI M.Application of next generation sequencing in mammalian embryogenomics:lessons learned from endogenous betaretroviruses of sheep[J].Anim Reprod Sci,2012,134(1-2):95-103.
[11] DUNLAP K A,PALMARINI M,ADELSON D L,et al.Sheep endogenous betaretroviruses (enJSRVs) and the hyaluronidase 2 (HYAL2) receptor in the ovine uterus and conceptus[J].Biol Reprod,2005,73(2):271-279.
[12] WEIER J F,WEIER H U G,JUNG C J,et al.Human cytotrophoblasts acquire aneuploidies as they differentiate to an invasive phenotype[J].Dev Biol,2005,279(2):420-432.
[13] NAKAYA Y,MIYAZAWA T.The roles of syncytin-like proteins in ruminant placentation[J].Viruses,2015,7(6):2928-2942.
[14] 刘猛,樊凤娇,石璞洁,等.不同浓度牛乳铁蛋白对成骨细胞与破骨细胞共培养的影响[J].食品研究与开发,2017,38(24):1-6.LIU M,FAN F J,SHI P J,et al.Effects of different concentrations of bovine lactoferrin on osteoblast/osteoclast co-cultures[J].Food Research and Development,2017,38(24):1-6. (in Chinese)
[15] 马欣,邢书涵,刘云海,等.绵羊子宫内膜上皮细胞的纯化培养与鉴定[J].中国农学通报,2015,31(11):63-67.MA X,XING S H,LIU Y H,et al.Cultivation,purification and characterization of ovine endometrial epithelial cells[J].Chinese Agricultural Science Bulletin,2015,31(11):63-67. (in Chinese)
[16] 刘慧,刘淑英,刘丽珍,等.绵羊胎盘滋养层细胞的体外培养与鉴定[J].中国兽医科学,2012,42(3): 309-313.LIU H,LIU S Y,LIU L Z,et al.Culture and identification of sheep trophoblast cells in vitro[J].Chinese Veterinary Science,2012,42(3):309-313. (in Chinese)
[17] BUKUR J,JASINSKI S,SELIGER B.The role of classical and non-classical HLA class I antigens in human tumors[J].Semin Cancer Biol,2012,22(4):350-358.
[18] 吴晓莉,齐景伟,刘淑英,等.Real-Time PCR和组织原位杂交法检测enJSRV在绵羊胎儿免疫器官及肺脏中的表达[J].畜牧兽医学报,2010,41(8):1018-1023.WU X L,QI J W,LIU S Y,et al.Expression of enJSRV in immune organs and lung of the fetus by in situ hybridization and real-time PCR[J].Acta Veterinaria et Zootechnica Sinica,2010,41(8):1018-1023. (in Chinese)
[19] 张宇飞,石晶,刘淑英.enJSRV囊膜蛋白及其受体的瞬时表达与绵羊绒毛膜滋养层细胞融合的诱导研究[J].畜牧兽医学报,2015,46(11):1924-1933.ZHANG Y F,SHI J,LIU S Y.Study on the transient expression of enJSRV envelope protein and its receptor and the induction of trophoblast cell fusion in sheep[J].Acta Veterinaria et Zootechnica Sinica,2015,46(11):1924-1933. (in Chinese)
[20] 廖乃顺,陈文列,黄云梅,等.成骨细胞与破骨细胞共培养及其应用研究进展[J].中国骨伤,2013,26(4):349-353.LIAO N S,CHEN W L,HUANG Y M,et al.Research propress of co-culture system of osteoblast with osteoclast and its applications[J].China Journal of Orthopaedics and Traumatology,2013,26(4):349-353. (in Chinese)
[21] 秦燕勤,陈玉龙,李建生.细胞共培养方法的研究进展[J].中华危重病急救医学,2016,28(8):765-768.QIN Y Q,CHEN Y L,LI J S.Research progress of cell co-culture method[J].Chinese Critical Care Medicine,2016,28(8):765-768. (in Chinese)
[22] 查雨锋,傅晓钟,张顺,等.大鼠脑微血管内皮细胞与周细胞、星形胶质细胞共培养建立体外血脑屏障模型[J].中国药理学通报,2015,31(5):730-735.ZHA Y F,FU X Z,ZHANG S,et al.Establishment of an in vitro blood-brain barrier model by co-culturing rat brain microvascular endothelial cells,pericytes and astrocytes[J].Chinese Pharmacological Bulletin,2015,31(5):730-735. (in Chinese)
[23] 韩翠萍,刘吉勇,高蕾,等.人脐血间充质干细胞向类肝细胞分化:人肝细胞共培养诱导法的可行性?[J].中国组织工程研究与临床康复,2010,14(1):48-52.HAN C P,LIU J Y,GAO L,et al.Human umbilical cord blood mesenchymal stem cells differentiate into hepatocypte-like cells:Feasibility of co-culture with human hepatocyte line LO2 in vitro[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2010,14(1):48-52. (in Chinese)
[24] 高大宽,章翔,费舟,等.脂肪干细胞诱导神经干细胞分化的体外实验研究[J].中华神经外科疾病研究杂志,2010,9(2):120-123.GAO D K,ZHANG X,FEI Z,et al.Study of differentiation of neural stem cells induced by adipose tissue derived stromal cells in vitro[J].Chinese Journal of Neurosurgical Disease Research,2010,9(2):120-123. (in Chinese)
[25] WANG R,DANG Y L,ZHENG R,et al.Live cell imaging of in vitro human trophoblast syncytialization[J].Biol Reprod,2014,90(6):117.
[2] LI Y,ZHENG R,WANG R.Involvement of nephrin in human placental trophoblast syncytialization[J].Reproduction,2015,149(4):339-346.
[3] 周跃华,陆琴.miR-29a在胎盘滋养细胞中的功能[J].中华保健医学杂志,2018,20(2):113-116.ZHOU Y H,LU Q.The function of miR-29a in placental trophoblasts[J].Chinese Journal of Health Care and Medicine,2018,20(2):113-116. (in Chinese)
[4] KAUFMANN P.Functional anatomy of the non-primate placenta[J].Placenta,1981,1(S1):13-28.
[5] 葛利江,田文儒,崔琳,等.细胞因子对小尾寒羊胎盘成熟的影响[J].中国兽医杂志,2004,40(1):7-10.GE L J,TIAN W R,CUI L,et al.Effect of some cytokines on placenta maturation in the small fat tail sheep[J].Chinese Journal of Veterinary Medicine,2004,40(1):7-10. (in Chinese)
[6] 刘丽珍,刘淑英.妊娠早期蒙古绵羊子宫内膜enJSRV及IFN-τ基因表达水平的分析[J].畜牧兽医学报,2012,43(6):979-984.LIU L Z,LIU S Y.Analysis of the expression of enJSRV and IFN-τ in endometrium of Mongolian sheep in early pregnancy[J].Acta Veterinaria et Zootechnica Sinica,2012,43(6):979-984. (in Chinese)
[7] GUILBERT L,ROBERTSON S A,WEGMANN T G.The trophoblast as an integral component of a macrophage-cytokine network[J].Immunol Cell Biol,1993,71(1):49-57.
[8] LEROUX C,GIRARD N,COTTIN V,et al.Jaagsiekte sheep retrovirus (JSRV):from virus to lung cancer in sheep[J].Vet Res,2007,38(2):211-228.
[9] FU J J,Lü X Y,LIN H Y,et al.Ubiquitin ligase cullin 7 induces epithelial-mesenchymal transition in human choriocarcinoma cells[J].J Biol Chem,2010,285(14):10870-10879.
[10] SPENCER T E,PALMARINI M.Application of next generation sequencing in mammalian embryogenomics:lessons learned from endogenous betaretroviruses of sheep[J].Anim Reprod Sci,2012,134(1-2):95-103.
[11] DUNLAP K A,PALMARINI M,ADELSON D L,et al.Sheep endogenous betaretroviruses (enJSRVs) and the hyaluronidase 2 (HYAL2) receptor in the ovine uterus and conceptus[J].Biol Reprod,2005,73(2):271-279.
[12] WEIER J F,WEIER H U G,JUNG C J,et al.Human cytotrophoblasts acquire aneuploidies as they differentiate to an invasive phenotype[J].Dev Biol,2005,279(2):420-432.
[13] NAKAYA Y,MIYAZAWA T.The roles of syncytin-like proteins in ruminant placentation[J].Viruses,2015,7(6):2928-2942.
[14] 刘猛,樊凤娇,石璞洁,等.不同浓度牛乳铁蛋白对成骨细胞与破骨细胞共培养的影响[J].食品研究与开发,2017,38(24):1-6.LIU M,FAN F J,SHI P J,et al.Effects of different concentrations of bovine lactoferrin on osteoblast/osteoclast co-cultures[J].Food Research and Development,2017,38(24):1-6. (in Chinese)
[15] 马欣,邢书涵,刘云海,等.绵羊子宫内膜上皮细胞的纯化培养与鉴定[J].中国农学通报,2015,31(11):63-67.MA X,XING S H,LIU Y H,et al.Cultivation,purification and characterization of ovine endometrial epithelial cells[J].Chinese Agricultural Science Bulletin,2015,31(11):63-67. (in Chinese)
[16] 刘慧,刘淑英,刘丽珍,等.绵羊胎盘滋养层细胞的体外培养与鉴定[J].中国兽医科学,2012,42(3): 309-313.LIU H,LIU S Y,LIU L Z,et al.Culture and identification of sheep trophoblast cells in vitro[J].Chinese Veterinary Science,2012,42(3):309-313. (in Chinese)
[17] BUKUR J,JASINSKI S,SELIGER B.The role of classical and non-classical HLA class I antigens in human tumors[J].Semin Cancer Biol,2012,22(4):350-358.
[18] 吴晓莉,齐景伟,刘淑英,等.Real-Time PCR和组织原位杂交法检测enJSRV在绵羊胎儿免疫器官及肺脏中的表达[J].畜牧兽医学报,2010,41(8):1018-1023.WU X L,QI J W,LIU S Y,et al.Expression of enJSRV in immune organs and lung of the fetus by in situ hybridization and real-time PCR[J].Acta Veterinaria et Zootechnica Sinica,2010,41(8):1018-1023. (in Chinese)
[19] 张宇飞,石晶,刘淑英.enJSRV囊膜蛋白及其受体的瞬时表达与绵羊绒毛膜滋养层细胞融合的诱导研究[J].畜牧兽医学报,2015,46(11):1924-1933.ZHANG Y F,SHI J,LIU S Y.Study on the transient expression of enJSRV envelope protein and its receptor and the induction of trophoblast cell fusion in sheep[J].Acta Veterinaria et Zootechnica Sinica,2015,46(11):1924-1933. (in Chinese)
[20] 廖乃顺,陈文列,黄云梅,等.成骨细胞与破骨细胞共培养及其应用研究进展[J].中国骨伤,2013,26(4):349-353.LIAO N S,CHEN W L,HUANG Y M,et al.Research propress of co-culture system of osteoblast with osteoclast and its applications[J].China Journal of Orthopaedics and Traumatology,2013,26(4):349-353. (in Chinese)
[21] 秦燕勤,陈玉龙,李建生.细胞共培养方法的研究进展[J].中华危重病急救医学,2016,28(8):765-768.QIN Y Q,CHEN Y L,LI J S.Research progress of cell co-culture method[J].Chinese Critical Care Medicine,2016,28(8):765-768. (in Chinese)
[22] 查雨锋,傅晓钟,张顺,等.大鼠脑微血管内皮细胞与周细胞、星形胶质细胞共培养建立体外血脑屏障模型[J].中国药理学通报,2015,31(5):730-735.ZHA Y F,FU X Z,ZHANG S,et al.Establishment of an in vitro blood-brain barrier model by co-culturing rat brain microvascular endothelial cells,pericytes and astrocytes[J].Chinese Pharmacological Bulletin,2015,31(5):730-735. (in Chinese)
[23] 韩翠萍,刘吉勇,高蕾,等.人脐血间充质干细胞向类肝细胞分化:人肝细胞共培养诱导法的可行性?[J].中国组织工程研究与临床康复,2010,14(1):48-52.HAN C P,LIU J Y,GAO L,et al.Human umbilical cord blood mesenchymal stem cells differentiate into hepatocypte-like cells:Feasibility of co-culture with human hepatocyte line LO2 in vitro[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2010,14(1):48-52. (in Chinese)
[24] 高大宽,章翔,费舟,等.脂肪干细胞诱导神经干细胞分化的体外实验研究[J].中华神经外科疾病研究杂志,2010,9(2):120-123.GAO D K,ZHANG X,FEI Z,et al.Study of differentiation of neural stem cells induced by adipose tissue derived stromal cells in vitro[J].Chinese Journal of Neurosurgical Disease Research,2010,9(2):120-123. (in Chinese)
[25] WANG R,DANG Y L,ZHENG R,et al.Live cell imaging of in vitro human trophoblast syncytialization[J].Biol Reprod,2014,90(6):117.