酸刺激对人体细胞特性影响的研究
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摘要
目的:探索体外培养的体细胞经过低pH处理后的特性和分化特征,为感音神经性聋提供新的治疗策略。方法:选取人成熟体细胞作为目标细胞,选择几种pH值不同溶液分别处理细胞,对细胞的形态进行观察,从碱性磷酸酶(AKP)活性、免疫组织化学染色、分子生物学等方面对细胞的特性进行鉴定,选出最适应pH值。对诱导后细胞体外进行神经干定向诱导,再将基膜以胆红素处理,建立感音神经性聋体外模型,验证诱导后细胞的体外分化能力以及治疗功能;验证方法为形态学、AKP活性、免疫组织化学、分子生物学等手段。结果:实验组细胞生长明显优于对照组;实验组AKP活性高于对照组;Nanog和Oct4的表达在2组均为阳性;将处理后的细胞转移到神经干细胞培养液中,Nestin表达阳性。结论:低pH处理后的人体细胞显示出与诱导多能干细胞相似的特性,但其功能和治疗效果有待进一步研究。
Objective:The purpose of the present study was to explore the characteristics and differentiation of somatic cells in vitro undergoing a low pH treatment,so as to provide new therapeutic strategies for treating sensorineural hearing loss.Method:The human mature somatic cells were selected as the target cells,and the cells were treated with different pH values to observe the cell morphology.The cell characteristics were identified from alkaline phosphatase(AKP)activity,immunohistochemical staining and molecular biology,and the most suitable pH value was selected.In addition,a mouse model of the cochlear lesion was constructed using bilirubin.Subsequently,the characteristics and therapeutic effect of somatic cells undergoing low pH treatment were examined by morphology,AKP activity,immunofluorescence assay and Q-PCR.Result:The cell growth of the experimental group was significantly better than those in the control group.The activity of AKP in the experimental group was higher than that in the control group.The expression of Nanog and Oct4 was both positive in the two groups.When the cells were changed to neurobasol medium,the marker of Nestin was positive.Conclusion:The human somatic cells undergoing a low pH treatment showed the similar characteristics as those of induced pluripotent stem(iPS)cells;although the functions and therapeutic effect of these altered human somatic cells need to be further studied.
Objective:The purpose of the present study was to explore the characteristics and differentiation of somatic cells in vitro undergoing a low pH treatment,so as to provide new therapeutic strategies for treating sensorineural hearing loss.Method:The human mature somatic cells were selected as the target cells,and the cells were treated with different pH values to observe the cell morphology.The cell characteristics were identified from alkaline phosphatase(AKP)activity,immunohistochemical staining and molecular biology,and the most suitable pH value was selected.In addition,a mouse model of the cochlear lesion was constructed using bilirubin.Subsequently,the characteristics and therapeutic effect of somatic cells undergoing low pH treatment were examined by morphology,AKP activity,immunofluorescence assay and Q-PCR.Result:The cell growth of the experimental group was significantly better than those in the control group.The activity of AKP in the experimental group was higher than that in the control group.The expression of Nanog and Oct4 was both positive in the two groups.When the cells were changed to neurobasol medium,the marker of Nestin was positive.Conclusion:The human somatic cells undergoing a low pH treatment showed the similar characteristics as those of induced pluripotent stem(iPS)cells;although the functions and therapeutic effect of these altered human somatic cells need to be further studied.
引文
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[16]ANG Y S,TSAI S Y,LEE D F,et al.Wdr5mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network[J].Cell,2011,145:183-197.
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[20]ADAMSON C L,REID M A,MO Z L,et al.Firing features and potassium channel content of murine spiral ganglion neurons vary with cochlear location[J].JComp Neurol,2002,447:331-350.
[21]CHEN W,JONGKAMONWIWAT N,ABBAS L,et al.Restoration of auditory evoked responses by human ES-cell-derived otic progenitors[J].Nature,2012,490:278-282.
[2]YU J Y,VODYANIK M A,SMUGA-OTTO K,et al.Induced pluripotent stem cell lines derived from human somatic cells[J].Science,2007,318:1917-1920.
[3]TISCORNIA G,VIVAS E L,BELMONTE J C.Diseases in a dish:modeling human genetic disorders using induced pluripotent cells[J].Nature Med,2011,17:1570-1576.
[4]TAKAHASHI J.Stem cells and regenerative medicine for neural repair[J].Curr Opin Biotechnol,2018,52:102-108.
[5]TAKAHASHI K,YAMANAKA S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,2006,126:663-676.
[6]THORPE T A.History of plant tissue culture[J].Mol Biotechnol,2007,37:169-180.
[7]BERG J S,GOODELL M A.An argument against a role for Oct4in somatic stem cells[J].Cell Stem Cell,2007,1:359-360.
[8]D’IPPOLITO G,DIABIRA S,HOWARD G A,et al.Marrow-isolated adult multilineage inducible(MI-AMI)cells,a unique population of postnatal young and old human cells with extensive expansion and differentiation potential[J].J Cell Sci,2004,117:2971-2981.
[9]KUCIA M,RECA R,CAMPBELL F R,et al.A population of very small embryonic-like(VSEL)CXCR41SSEA-11Oct-41stem cells identified in adult bone marrow[J].Leukemia,2006,20:857-869.
[10]KURODA Y,KITADA M,WAKAO S,et al.Unique multipotent cells in adult human mesenchymal cell populations[J].Proc Natl Acad Sci U S A,2010,107:8639-8643.
[11]LENGNER C J,WELSTEAD G G,JAENISCH R.The pluripotency regulator Oct4:a role in somatic stem cells[J]?Cell Cycle,2008,7:725-728.
[12]OBOKATA H,KOJIMA K,WESTERMAN K,et al.The potential of stem cells in adult tissues representative of the three germ layers[J].Tissue Eng,2011,17:607-615.
[13]XU Q Q,GUO W W,WANG X,et al.Acid stimulation-induced semi-pluripotent characteristics in human somatic cells[J].Acta Otolaryngol,2019,8:1-7.
[14]WRAY J,KALKAN T,SMITH A G.The ground state of pluripotency[J].Biochem Soc Trans,2010,38:1027-1032.
[15]YING Q L,WRAY J,NICHOLS J,et al.The ground state of embryonic stem cell self-renewal[J].Nature,2008,453:519-23.
[16]ANG Y S,TSAI S Y,LEE D F,et al.Wdr5mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network[J].Cell,2011,145:183-197.
[17]BRONS I G,SMITHERS L E,TROTTER M W,et al.Derivation of pluripotent epiblast stem cells from mammalian embryos[J].Nature,2007,448:191-195.
[18]NIWA H.How is pluripotency determined and maintained[J]?Development,2007,134:635-646.
[19]BYRNES W M.Ernest Everett Just,Johannes Holtfreter,and the origin of certain concepts in embryo morpHogenesis[J].Mol Reprod Dev,2009,76:912-921.
[20]ADAMSON C L,REID M A,MO Z L,et al.Firing features and potassium channel content of murine spiral ganglion neurons vary with cochlear location[J].JComp Neurol,2002,447:331-350.
[21]CHEN W,JONGKAMONWIWAT N,ABBAS L,et al.Restoration of auditory evoked responses by human ES-cell-derived otic progenitors[J].Nature,2012,490:278-282.