利用单细胞融合技术高效获得融合细胞的方法
|
摘要
试验以小鼠胚胎干细胞(mESC)作为细胞融合对象,用0.25%胰酶将mESC消化成单细胞,利用细胞凝集素构建一对一的单细胞对,在含有50%聚乙二醇的ES培养液中培养1 min,随后将细胞继续培养7 d,获得既表达红色荧光也表达绿色荧光的克隆即为融合成功。结果显示,单细胞融合法的克隆效率(46.25%)显著高于传统融合的效率(0.001 7%)。进一步检测融合细胞的生物学特性发现,虽然细胞形态与mESC具有很大的差异,但依然呈AP阳性,RNA水平上表达Oct4、Sox2和Nanog,具有向三胚层分化的能力等。结果表明,单细胞融合法是一种高效构建融合细胞的新途径。
Cell fusion is an important means to study cell pluripotency,fate determination and gene regulation.However,the efficiency of cell fusion methods currently reported is generally around 1%.In order to improve the efficiency of cell fusion,it is more effective to provide research materials for related research.This study proposed a single cell fusion method.Mouse embryonic stem cells(mESCs) were used as a cell fusion material,mESCs were digested with 0.25% trypsin into single cells,one to one pair was constructed with phytohemagglutinin,cultured in 50% polyethylene glycol for 1 min,then the cells were cultured for 7 days,got clones expressing both red and green fluorescence.The fusion efficiency of the single cell fusion method(46.25%) was significantly higher than that of the conventional fusion(0.001 7%).Throught further examination of the biological characteristics there was a great difference between the fusion cells,and mouse ES cell morphology,with AP positive;RNA expression of OCT4,SOX2 and Nanog and the ability to differentiate to the three germ layers and so on.The results indicate that the single cell fusion method is a new way to get fusion cells.
Cell fusion is an important means to study cell pluripotency,fate determination and gene regulation.However,the efficiency of cell fusion methods currently reported is generally around 1%.In order to improve the efficiency of cell fusion,it is more effective to provide research materials for related research.This study proposed a single cell fusion method.Mouse embryonic stem cells(mESCs) were used as a cell fusion material,mESCs were digested with 0.25% trypsin into single cells,one to one pair was constructed with phytohemagglutinin,cultured in 50% polyethylene glycol for 1 min,then the cells were cultured for 7 days,got clones expressing both red and green fluorescence.The fusion efficiency of the single cell fusion method(46.25%) was significantly higher than that of the conventional fusion(0.001 7%).Throught further examination of the biological characteristics there was a great difference between the fusion cells,and mouse ES cell morphology,with AP positive;RNA expression of OCT4,SOX2 and Nanog and the ability to differentiate to the three germ layers and so on.The results indicate that the single cell fusion method is a new way to get fusion cells.
引文
[1] LEE S,PEDEN K,DIMITROV D S,et al.Enhancement of human immunodeficiency virus type 1 envelope-mediated fusion by a CD4-gp120 complex-specific monoclonal antibody[J].J Virol,1997,71(8):6037-6043.
[2] ZANELLA I,VERARDI R,NEGRINI R,et al.New heteromyeloma cell lines for the production of human monoclonal antibodies[J].J Immunol Methods,1992,156(2):205-215.
[3] EHRENSTEIN M R,LEAKER B,ISENBERG D,et al.Production of human monoclonal antibodies to myeloperoxidase[J].Immunology,1992,77(4):617-620.
[4] KUIDA K,TANAKA T,KITAMURA F,et al.Reconstitution of the intermediate-affinity interleukin-2 receptor by cell fusion[J].Int Immunol,1992,4(10):1123-1128.
[5] LI X,CUI X L,WANG J Q,et al.Generation and application of mouse-rat allodiploid embryonic stem cells[J].Cell,2016,164(1/2):279-292.
[6] HARRIS H,MILLER O J,KLEIN G,et al.Suppression of malignancy by cell fusion[J].Nature,1969,223(5204):363-368.
[7] TEISSIE J,KNUTSON V P,TSONG T Y,et al.Electric pulse-induced fusion of 3T3 cells in monolayer culture[J].Science,1982,216(4545):537-538.
[8] RAO P N,JOHNSON R T.Mammalian cell fusion:studies on the regulation of DNA synthesis and mitosis[J].Nature,1970,225(5228):159-164.
[9] COWAN C A,ATIENZA J,MELTON D A,et al.Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells[J].Science,2005,309(5739):1369-1373.
[10] YU J,VODYANIK M A,HE P,et al.Human embryonic stem cells reprogram myeloid precursors following cell-cell fusion[J].Stem Cells,2006,24(1):168-176.
[11] HASEGAWA K,ZHANG P,WEI Z,et al.Comparison of reprogramming efficiency between transduction of reprogramming factors,cell-cell fusion,and cytoplast fusion[J].Stem Cells,2010,28(8):1338-1348.
[12] SUMER H,NICHOLLS C,LIU J,et al.Comparison of reprogramming ability of mouse ES and iPS cells measured by somatic cell fusion[J].Int J Dev Biol,2010,54(11/12):1723-1728.
[13] MATVEEVA N M,PRISTYAZHNYUK I E,TEMIROVA S A,et al.Unequal segregation of parental chromosomes in embryonic stem cell hybrids[J].Mol Reprod Dev,2005,71(3):305-314.
[14] JAMI J,FAILLY C,RITZ E.Lack of expression of differentiation in mouse teratoma-fibroblast somatic cell hybrids[J].Exp Cell Res,1973,76(1):191-199.
[15] SUMER H,NICHOLLS C,PINTO A R,et al.Chromosomal and telomeric reprogramming following ES-somatic cell fusion[J].Chromosoma,2010,119(2):167-176.
[2] ZANELLA I,VERARDI R,NEGRINI R,et al.New heteromyeloma cell lines for the production of human monoclonal antibodies[J].J Immunol Methods,1992,156(2):205-215.
[3] EHRENSTEIN M R,LEAKER B,ISENBERG D,et al.Production of human monoclonal antibodies to myeloperoxidase[J].Immunology,1992,77(4):617-620.
[4] KUIDA K,TANAKA T,KITAMURA F,et al.Reconstitution of the intermediate-affinity interleukin-2 receptor by cell fusion[J].Int Immunol,1992,4(10):1123-1128.
[5] LI X,CUI X L,WANG J Q,et al.Generation and application of mouse-rat allodiploid embryonic stem cells[J].Cell,2016,164(1/2):279-292.
[6] HARRIS H,MILLER O J,KLEIN G,et al.Suppression of malignancy by cell fusion[J].Nature,1969,223(5204):363-368.
[7] TEISSIE J,KNUTSON V P,TSONG T Y,et al.Electric pulse-induced fusion of 3T3 cells in monolayer culture[J].Science,1982,216(4545):537-538.
[8] RAO P N,JOHNSON R T.Mammalian cell fusion:studies on the regulation of DNA synthesis and mitosis[J].Nature,1970,225(5228):159-164.
[9] COWAN C A,ATIENZA J,MELTON D A,et al.Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells[J].Science,2005,309(5739):1369-1373.
[10] YU J,VODYANIK M A,HE P,et al.Human embryonic stem cells reprogram myeloid precursors following cell-cell fusion[J].Stem Cells,2006,24(1):168-176.
[11] HASEGAWA K,ZHANG P,WEI Z,et al.Comparison of reprogramming efficiency between transduction of reprogramming factors,cell-cell fusion,and cytoplast fusion[J].Stem Cells,2010,28(8):1338-1348.
[12] SUMER H,NICHOLLS C,LIU J,et al.Comparison of reprogramming ability of mouse ES and iPS cells measured by somatic cell fusion[J].Int J Dev Biol,2010,54(11/12):1723-1728.
[13] MATVEEVA N M,PRISTYAZHNYUK I E,TEMIROVA S A,et al.Unequal segregation of parental chromosomes in embryonic stem cell hybrids[J].Mol Reprod Dev,2005,71(3):305-314.
[14] JAMI J,FAILLY C,RITZ E.Lack of expression of differentiation in mouse teratoma-fibroblast somatic cell hybrids[J].Exp Cell Res,1973,76(1):191-199.
[15] SUMER H,NICHOLLS C,PINTO A R,et al.Chromosomal and telomeric reprogramming following ES-somatic cell fusion[J].Chromosoma,2010,119(2):167-176.