Resetting the Epigenomebeyond Pluripotency in the GermlineKatsuhiko Hayashi1and M.Azim Surani1,*1Wellcome Trust Cancer Research UK Gurdon Institute,The Henry Wellcome Building of Cancer and Developmental Biology,University of Cambridge,Tennis Court Road,Cambridge CB21QN,UK*Correspondence:a.surani@DOI10.1016/j.stem.2009.05.007Germ cells undergo comprehensive epigenetic reprogramming toward acquiringfitness for pluripotency and totipotency.Notably,the full extent of the epigenetic reprogramming experienced by germ cells remains unmatched by attempts to experimentally restore pluripotency in somatic cells.We propose that the defects present in experimentally generated cells are corrected upon differentiation into the germ cell lineage,as has been observed in cases of germline transmission.Unraveling the mechanisms responsible for germ cell-specific epigenetic reprogramming will likely have important implications for both basic and clinical stem cell research.Epigenetic reprogramming of somatic cells,for example,by nuclear transplantation into an oocyte,frequently leads to defects in the resulting conceptus and cells(Hochedlinger and Jaenisch,2002;Tamashiro et al.,2003).Many of these defects are eliminated upon transmission through the germline,suggest-ing that they are epigenetic in nature and reversible(Shimozawa et al.,2002;Tamashiro et al.,2002).Cells derived via recent advances in reprogramming,including experimentally induced pluripotent stem cells(iPSCs),require critical evaluation of their properties with respect to the events in the germline,given that these cells may lack all of the attributes of an authentic pluripo-tent state.Here,we discuss our growing knowledge of the mammalian germ cell lineage and the implications of thesefind-ings to the experimental manipulation of epigenetic states.The Foundation of the Germ Cell LineagePrimordial germ cells(PGCs)originate during development from postimplantation epiblast cells,which,in turn,arise from the pluripotent primitive ectoderm cells of the inner-cell mass (PEct/ICM)of blastocysts(McLaren and Lawson,2005).Devel-opment of the postimplantation epiblast is accompanied by epigenetic modifications that are generally irreversible,including X inactivation when this chromosome switches from early to late replication(Takagi et al.,1982);this alteration is perhaps a hall-mark of genome-wide irreversible epigenetic changes and may involve DNA methylation.Other changes,including histone modifications and DNA methylation,also ensue during differen-tiation of the epiblast and appear to be required in that their absence in the wake of mutation to several key epigenetic regu-lators results in early embryonic lethality(Surani et al.,2007). Thus,PGCs originate from epiblast cells that have initiated the process of differentiation toward somatic cell lineages(Ohinata et al.,2005),as reflected in their transcriptional profile. Whereas the majority of epiblast cells continue to develop toward diverse somatic fates,this trend is arrested in a few epiblast cells destined to form the PGCs(Figure1).Blimp1/ Prdm1,a transcriptional repressor and the key germ cell deter-minant in mammals,initiates the reversion of differentiating epiblast cells by repressing the somatic program and initiating the germ cell program at embryonic day(E) 6.25(Ohinata et al.,2005).Additional changes follow,including re-expression of pluripotency genes such as Nanog and Sox2(Yabuta et al., 2006;Yamaguchi et al.,2005)in nascent PGCs,but not in the other differentiating epiblast cells.These adjustments suggest a trend toward reversion to an earlier ICM-like epigenetic state (Figure1),although nascent PGCs still possess some character-istics of epiblast cells,such as an inactive X chromosome(Chuva de Sousa Lopes et al.,2008;de Napoles et al.,2007;Sugimoto and Abe,2007).This epigenetic memory of the cell’s initial trajec-tory toward a somatic fate is erased progressively in PGC precursors.Epigenetic Reprogramming in the Emerging Germ Cells Epigenetic reprogramming events commence immediately after PGC specification at E7.25,which is marked by the detection of Stella/Dppa3(Saitou et al.,2002;Sato et al.,2002).These epige-netic changes are accompanied by downregulation of genes impli-cated in DNA methylation and changes in histone modifications (Yabuta et al.,2006).Notably,genes,including Glp and Dnmt3b, are downregulated.As a result,a global loss of histone H3lysine 9(H3K9me2)methylation is observed in PGCs between E7.5and E8.5,whereas the Ezh2-dependent H3K27me3modification is accentuated(Hajkova et al.,2008;Seki et al.,2005).Although the specific loci remodeled by these global histone modifications in PGCs remain to be elucidated,the overall effect in PGCs is to shift them toward the ICM/ESC-like epigenetic state.Indeed,with these changes,PGCs acquire the potential to dedifferentiate into plurip-otent embryonic germ(EG)cells that are virtually identical to ESCs (see below).There are no published reports on the derivation of EG cells from PGCs prior to E8.5,which may indicate the significance of resetting the epigenome between E7.5and E8.5in nascent PGCs(Figure1).Recent evidence demonstrates that Prdm14, another PR domain-containing transcriptional regulator that is de-tected shortly after Blimp1/Prdm1in PGC precursors,plays a pivotal role in regulating epigenetic changes in nascent PGCs. Gene disruption of Prdm14hampers both the loss of H3K9me2 and the enhancement of K3K27me3.Consequently,PGC develop-ment and the derivation of EG cells are impaired in the Prdm14 Cell Stem Cell4,June5,2009ª2009Elsevier Inc.493mutant embryos (Yamaji et al.,2008).Thus,Blimp1/Prdm1and Prdm14appear to work in tandem to repress the somatic program and initiate the germ cell-specific epigenetic program.Coupled with the global epigenetic modifications that are observed in PGCs,some key pluripotency genes are also re-expressed.Included in this list are Nanog and Sox2(Yabuta et al.,2006;Yamaguchi et al.,2005),which are normally repressed in postimplantation epiblast cells and are the very factors involved in the reversion of somatic cells to pluripotent stem cells (Silva et al.,2006;Takahashi and Yamanaka,2006).A recent study also reveals that the binding of Nanog,Oct4,and Sox2to a regulatory element of Xist ,a noncoding RNA,may help to induce reactivation of the X chromosome (Navarro et al.,2008).However,reactivation of the X chromosome occurs in a pro-tracted manner in PGCs compared to PEct;X reactivation commences at around E7.0and is not completed until E12.5in PGCs,but it occurs within a day in PEct (Mak et al.,2004;Okamoto et al.,2004).This difference may be because Xist repression in PEct involves histone modifications,but the late replication of the X chromosome in epiblast cells (Takagi et al.,1982)may be coupled with DNA methylation,which is initially inherited by nascent PGCs.The Relationship between Germ Cells and Pluripotent Stem CellsAs described above,reprogramming in early germ cells results in PGCs from E8.5–E11.5embryos being in a permissive state with respect to their potential to give rise to pluripotent EG cells.EG cells are virtually identical to ESCs,except for the loss of DNA methylation from imprinted gene loci in EG cells (Shovlin et al.,2008;Tada et al.,2001).Both ESCs and EG cells have two active X chromosomes in female cells,can contribute to chimeras and the germline,and have transcriptomes that are very similar (Shar-ova et al.,2007).Despite their similarities,EG cells and ESCs have distinctive origins from PGC and PEct,respectively.PGCs are the founders of a unipotent lineage that generates sperm and eggs only,whereas PEct give rise to all of the somaticfetalFigure 1.Stepwise Differentiation and Reprogramming during Mouse DevelopmentA totipotent zygote develops into a blastocyst,followed by differentiation into ICM/PEct and trophectoderm (TE).ICM/PEct are pluripotent cells but no longer totipotent.After implantation,ICM/PEct differentiates into the epiblast,coupled with random X inactivation,a hallmark of the epigenetic state of this population.During gastru-lation,epiblast cells give rise to both germ cells and somatic cells.Whereas somatic cells undergo further differentiation,PGCs revert to a state that resembles the ICM/PEct population,with the exception that they are unipotent.Further epige-netic reprogramming events take place in germ cells,including genome-wide DNA demethylation and remodeling of retrotransposon-linked genes.tissues,as well as to germ cells (Fig-ure 1).To restrict their cell fate,PGCs exhibit lineage-specific gene expression,including Blimp1and Nanos3(Ohinata et al.,2005;Tsuda et al.,2003).Of partic-ular note for the maintenance of early unipotent germ cell lineage is the presence of a Blimp1-Prmt5repressive complex;Prmt5is a histone H2A/H4symmetrical arginine 3demethylase (H2A/H4R3me2s)(Ancelin et al.,2006).During EG cell derivation,Blimp1is rapidly downregulated (Durcova-Hills et al.,2008),which likely reverses restriction on the germ cell lineage,while Prmt5assumes another role in promoting pluripotency.The Blipm1-Prmt5complex translocates to the cytoplasm at E12.5(Ancelin et al.,2006),precisely when the ability to generate EG cells from PGCs ceases.Thus,the Blimp1-Prmt5complex may safeguard unipotency of early germ cells,but it may also have a role in epigenetic reprogramming itself.These hypotheses are testable predictions that will be interesting to tackle in the future.It is known that human ESCs that resemble pluripotent epiblast stem cells (EpiSCs)(Brons et al.,2007;Tesar et al.,2007)can generate cells with characteristics resembling PGCs (Clark et al.,2004).It is possible that the significance of epigenetic reprogramming in PGCs may become evident by investigating EpiSCs,which are derived from E5.5–E6.5postimplantation embryos.EpiSCs differ significantly from ESC/EG cells in their overall transcription profile and in their epigenetic state (Brons et al.,2007;Hayashi et al.,2008;Tesar et al.,2007),even though this population also exhibits expression of key pluripotency-specific genes (Figure 2A).However,EpiSCs have an inactive X chromosome and possibly hypermethylation of CpG sequences of some pluripotency genes,such as stella and Rex1/Zfp42.EpiSCs can neither contribute to adult chimeras,which precludes their contribution to PGCs in vivo (Brons et al.,2007),nor be easily converted to ESCs (Guo et al.,2009).If PGCs can be derived from EpiSCs in vitro and if these PGCs undergo appropriate epigenetic reprogramming,they may,in turn,be induced to give rise to EG cells.These results would demonstrate that reprogramming in PGCs is a route through which the EpiSC epigenome can be re-modeled and perhaps reverted to the ICM/ESC-like pluripotent state (Figure 2A).Thus,the epigenetic barrier that is created during development of the epiblast and EpiScs may be breached during PGC development.494Cell Stem Cell 4,June 5,2009ª2009Elsevier Inc.Genomic Reprogramming beyond PluripotencyThe most significant epigenetic reprogramming event in the germline is genome-wide DNA demethylation and extensive histone modifications that take place in gonadal PGCs (Hajkova et al.,2008).Though DNA demethylation does occur to some extent during preimplantation development (Howlett and Reik,1991;Monk et al.,1991),there is no process equivalent to the reprogramming observed in germ cells reported in cells of other lineages,and this process accounts for the complete erasure and resetting of the epigenome in the germline.This specialized reprogramming may account for the rare incidences of transge-nerational inheritance of epimutations (Whitelaw and Whitelaw,2008).The targeted modifications in germ cells also ensurethatEpiblastABFigure 2.Impact of PGC-MediateEpigenetic Reprogramming on Pluripotent Stem Cells(A)Mouse ESCs (top)derived from ICM/PEct exhibit an epigenetic status similar to mouse EG cells because PGC-mediate reprogramming has the potential to erase epigenetic memory of epiblast.It is proposed that derivation of PGCs from EpiSCs may similarly erase the epigenetic memory of the latter.Human ESCs (bottom)may lack the ICM/PEct reprogramming event observed in the mouse ICM/PEct,making hESCs more like the EpiESCs.However,epigenetic reprogramming in human germ cells renders them responsive to LIF-STAT3signaling and,therefore,similar to mouse ES/EG cells,which suggests equivalent resetting of the epigenome in the mouse and human germline.(B)Experimentally generated pluripotent stem cells may exhibit defects in their potential for terminal differentiation and,thus,may not be capable of development to term in tetraploid host blastocysts.These limitations may result from the presence of epigenetic defects retained during an incomplete reprogramming process.Epigenetic defects encountered in experimentally generated pluripotent cells may be corrected upon their transmission through the germline (top).imprinted genes and retrotransposons,such as LINE1,are remodeled appropri-ately (Hajkova et al.,2002).Notably,there is no strictly equivalent phenomenon in PEct/ICM,in ESCs,or in other experimen-tally derived pluripotent cells.The lack of a similar remodeling of the genome in the experimentally generated pluripotent cells may affect their functional properties (see below).Evidence suggests that remodeling of retrotransposon-associated and im-printed genes in germ cells also con-tributes to totipotency by restoring the potential for subsequent fetal and placental development.Genes associ-ated with retrotransposable elements include Peg10and Rtl1(also known as Peg11),which are essential for placental development (Ono et al.,2006;Sekita et al.,2008).Several human placentalgenes,such as Endothelin B receptor,Insl4,Leptin,Midline1,and Pleiotrophin,are also associated with retrotransposable elements (Rawn and Cross,2008).It is likely that genome-wide DNA demethylation exclusively in gonadal PGCs contributes to resetting these genes and others that are critical for develop-ment of the conceptus after fertilization.Remodeling of the epigenetic status of retrotransposable elements,however,may cause mutagenesis in the genome by active transposition.Recent studies elegantly demonstrate that small RNA pathways,such as piRNA (also known as gsRNA)and endogenous siRNA,play a crucial role in suppressing the expression of retrotransposons (Aravin et al.,2006;Girard et al.,2006;Watanabe et al.,2006,2008).Disruption of theseCell Stem Cell 4,June 5,2009ª2009Elsevier Inc.495genes,interestingly,has an impact on germ cell development, but not that of PEct or ESCs,suggesting that germ cells possess both the specific circumstance in which retrotransposons can be activated and specific mechanisms to suppress them,which imparts tolerance for these activities.Recent studies also reveal characteristic remodeling of the epigenetic status of Rhox genes,which were initially identified as a gene cluster of the reproductive homeobox on the X chro-mosome.Detailed analysis clearly revealed that expression of these genes commences exclusively in PGCs at E12.5PGCs (Daggag et al.,2008),when massive DNA demethylation occurs. Comparison of the epigenetic status of the Rhox gene cluster in PGCs and in fetal and placental tissues will clarify the signifi-cance of remodeling these genes in PGCs.Implications of Germline-Specific Reprogrammingin HumansApart from the germline,epigenetic reprogramming also takes place during the establishment of pluripotent cells in the ICM,as exemplified by reactivation of the inactive paternal X chromosome (Mak et al.,2004;Okamoto et al.,2004).Whether such an event occurs in the human ICM is unclear.Notably,expression of Xist, a noncoding RNA important for X inactivation,is apparently de-tected from both parental alleles in human embryos and not just from the paternal allele as in the mouse(Daniels et al.,1997; Ray et al.,1997).It is possible that there may not be an epigenetic reprogramming event in the human PEct/ICM consistent with that seen in the mouse ICM(Figure2A).If substantiated,this hypoth-esis may explain why mouse ESCs differ from human ESCs;for example,mouse female ESCs express two active X chromo-somes,whereas the vast majority of human female ESCs retain an inactive X chromosome(Dhara and Benvenisty,2004;Shen et al.,2007).In addition,hESCs more closely resemble mouse EpiSCs than ESCs,and both of the former require bFGF/Activin for their self-renewal,whereas mouse ESCs/EG cells require LIF/STAT3signaling to retain their pluripotent state(Niwa et al., 1998).Whether the observed differences in patterns of X inactiva-tion and the signaling requirements of mouse and human ESCs are functionally connected remains to be determined.However, it is possible that the ICM/PEct in human embryos may continue development toward a postimplantation epiblast-like stage during the derivation of hESC from blastocysts.Based on the available evidence,it does appear that the extensive epigenetic reprogramming observed in mouse germ cells may also occur in the human,given that extensive DNA demethylation would be required to reset the imprints and for X reactivation.That these modifications occur seems particularly likely because human EG cells,unlike hESC,are dependent on LIF/STAT signaling(Figure2A)(Shamblott et al.,1998).Sper-matogonia-derived human pluripotent stem cells are also similar to mouse ES/EG cells(Conrad et al.,2008).The nature of epige-netic reprogramming events in human germ cells could be exam-ined in PGCs derived from hESC,and parallel experiments may also be possible with mouse EpiSCs in vitro.Perspective on Experimentally Induced PluripotencyA classical approach to restore totipotency/pluripotency in somatic nuclei is by transplantation into an oocyte(SCNT) (Campbell et al.,1996;Wakayama et al.,1998).The transferred somatic nuclei are exposed to reprogramming factors in the oocyte,and as a result,they may acquire totipotency.Further-more,during subsequent development of such reconstituted embryos to the blastocyst stage,donor nuclei may also undergo reprogramming in the ICM.In spite of this two-step reprogram-ming of somatic nuclei in early embryos,the resulting concep-tuses often show both fetal and placental abnormalities, suggesting that neither the oocyte nor the ICM has the compre-hensive potential to reset the epigenetic state of the somatic nucleus(Bao et al.,2005;Bortvin et al.,2003).Many of these defects are,however,corrected upon transmission through the germline(Shimozawa et al.,2002;Tamashiro et al.,2002), demonstrating the comprehensive nature of epigenetic reprog-ramming upon passage through the germ cell lineage (Figure2B).Though some of the epigenetic defects could be erased during epigenetic reprogramming in the ICM,rather than in germ cells,any defects present in the trophectoderm and other extraembryonic lineages would remain uncorrected. The experimentally generated human pluripotent stem cells (hESCs)and iPSC may show even greater defects compared to the mouse because,as discussed above,it is unclear whether human cells experience an equivalent reprogramming event as observed in the ICM of the mouse.In any case,it seems that the extensive epigenetic reprogramming and resetting of the epi-genome observed in the germline,including genome-wide DNA demethylation as well as wide-ranging histone modifications,do not occur in the oocyte or in the ICM.Germline Reprogramming and the iPSCThe most important recent advance toward restoring pluripo-tency in somatic cells comes from Yamanaka’s work using transcription factors,including Oct4,Sox2,Klf4,and c-Myc,to convert somatic cells into iPSCs that appear overtly equivalent to pluripotent ESCs(Takahashi and Yamanaka,2006).Consider-able attention has been paid to the low frequency of the deriva-tion of iPSCs,the protracted nature of the process,and some of the key properties of these cells(Hochedlinger and Plath,2009). From our perspective,however,the most important remaining question is how closely do iPSCs truly resemble ESCs derived from normal blastocysts.To generate iPSCs,the pluripotency-specific transcriptional factors introduced into somatic cells probably help to establish a new genetic network that evidently approximates the authentic pluripotency network,although it cannot be excluded that subtle yet important differences may be present.However,the genera-tion of iPSCs from somatic cells does not appear to require systematic reversal of the entire developmental program that originally resulted in all of the diverse somatic cells with distinct phenotypes.Differentiation of somatic cells requires robust silencing of genes and regulatory elements that are not required in specific cell types.It is possible that epigenetic modifications associated with such silent genes that are robustly repressed in specific differentiated cells by DNA methylation may be difficult to reverse,as is the case with methylation of imprinted genes, which could make some key regulatory elements inaccessible to tissue-specific binding factors(Xu et al.,2007).This hypothesis is plausible,given that pluripotency is the property being selected for in these experiments rather than the ability of the newly estab-lished iPSC to undergo terminal differentiation to a specific496Cell Stem Cell4,June5,2009ª2009Elsevier Inc.lineage.Thus,traces of residual epigenetic memory could exist in iPSCs,in that some genes that are silenced in specific cell types may remain silenced,and the residual memory marks may only be erased upon transmission through the germline.Therefore,it is essential to carry out comprehensive epigenetic analysis of iPSCs,for example,by using genome-wide methylation analysis by methylDip(Farthing et al.,2008),in carefully controlled exper-iments to rigorously address the hypothesis that iPSCs retain tissue restriction patterns from their parental cells.The most stringent test to establish whether cells,such as iPSCs,are indeed pluripotent is to demonstrate that full-term embryos can be derived exclusively from the putative pluripotent cells.To do so,the candidate donor cells are introduced into ‘‘tetraploid’’host blastocysts,in which the donor cells do not contribute to placental development.Though it remains possible that iPSC do possess this potential,no live young have yet been reported from iPSCs in any such experiments,even though they have in some instances reached an advanced stage of E14of gestation(Meissner et al.,2007;Woltjen et al.,2009).Indeed, one could predict that iPSCs might display relatively greater defects at later stages during terminal differentiation of somatic lineages,with the notable exception of germ cells and gametes, given that all epigenetic modifications are erased and reset in this lineage(Figure2B).Very careful systematic epigenetic anal-ysis,together with a comprehensive examination of differentia-tion and precise distribution of iPSCs into all of the tissues in chimeric adults,could reveal their true developmental potential. Following this line of reasoning,one might also expect that the nature of the residual epigenetic memory would depend on the original somatic cells used in these experiments,notwith-standing the stochastic nature of the process.It is crucial also to confirm rigorously whether iPSC-derived differentiated cells are equivalent to normal terminally differentiated cells.The proposed use of terminally differentiated iPSCs in cell therapy and disease models requires them to be phenotypically and physiologically identical to the in vivo terminally differentiated cells.We predict that any residual epigenetic memory in iPSCs would be erased as the epigenome is reset in the germline (Figure2B).Notably,transdifferentiation of closely related cell types would be predicted to be less affected by the residual epigenetic memory because these cells would share many of their key epigenetic properties(Zhou et al.,2008).It is known from several studies that mouse ESCs exhibit functional and epigenetic heterogeneity(Enver et al.,2009;Graf and Stadtfeld,2008;Hayashi et al.,2008).It is possible that such heterogeneity may be prevalent in experimentally generated pluripotent cells,which may be accentuated in human iPSCs,as they lack the ICM-like reprogramming event that we have described here.Under ideal conditions,following reprogramming, each cell should exhibit an equivalent epigenetic state and iden-tical potential for pluripotency,which may not be the case.For example,during establishment of iPSCs,only a minority of trans-duced cells are converted to pluripotency and selected under the applied culture conditions,and it is unknown whether all of these cells acquire identical properties.Indeed,even the process of re-deriving‘‘secondary’’iPSCs using conditional reprogramming vectors remains inefficient(Jaenisch and Young,2008;Maherali and Hochedlinger,2008).It is possible that the protracted nature of this procedure could result in daughter cells with diverse epige-netic states.This possibility can also be evaluated by rigorous clonal analysis of iPSCs for their phenotypic and epigenetic prop-erties.Resetting of the epigenome in the germline may not only be extensive,but it also benefits from rigorous in vivo selection,as seen during spermatogenesis(Ueno et al.,2009).ConclusionsBased on the existing evidence,we suggest that the most comprehensive process of epigenetic reprogramming that ensures authentic pluripotency occurs upon passage through the germline.The wide-ranging erasure of epigenetic modifica-tions,including DNA demethylation,ensures removal of most, if not all,of the extraneous epigenetic information.This conver-sion apparently does not occur in the experimentally restored pluripotent state.In the mouse,the form of reprogramming that takes place in the ICM provides a possibility for approaching the authentic pluripotent state,but even so,the result is often variable,owing to the stochastic nature of the process(Bortvin et al.,2003).hESCs may be more compromised in terms of their functional pluripotency,as these populations evidently lack the ICM-specific reprogramming step.This distinction sets hESCs apart from mESCs and may account for the relatively heteroge-neous nature of hESCs,as has been observed with mEpiSCs. 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doi:10.3969/j.issn.1000⁃484X.2020.07.003帕金森病模型细胞中p53㊁增殖细胞核抗原表达与细胞增殖和凋亡①李 季 郭继东 张晓杰 杨 宏 李尊严 宋天琦 孙 微 冯 伟 李大伟(北华大学第一临床医院,吉林132011) 中图分类号 R74 文献标志码 A 文章编号 1000⁃484X (2020)07⁃0780⁃05①本文为吉林省卫生技术创新项目(No.2016J078)和吉林省教育厅 十三五”科学技术(JJKH20170047K)资助项目㊂作者简介:李 季,男,硕士,主治医师,主要从事神经系统变性病及神经免疫方面的研究,E⁃mail:99831817@㊂通讯作者及指导教师:郭继东,男,硕士,主任医师,硕士生导师,主要从事神经系统变性病及神经免疫方面的研究㊂[摘 要] 目的:探讨帕金森病(PD)模型细胞中p53㊁增殖细胞核抗原(PCNA)表达与细胞增殖㊁凋亡相关性及病理变化特征㊂方法:以不同浓度的1⁃甲基⁃4⁃苯基⁃吡啶离子(MPP +)诱导PC12细胞损伤,建立PD 模型,PC12细胞分为空白组和MPP +不同浓度组(0.5mmol /L㊁1mmol /L㊁2mmol /L)共4组,采用MMT 方法检测每组细胞生存率;DCFH⁃DA 检测活性氧水平(ROS);吖啶橙和溴化乙锭(AO /EB)凋亡染色及酶联免疫吸附法(ELISA)测定单链DNA;Western blot 检测各组p53和PCNA 表达㊂结果:随MPP +浓度增加,与对照组相比,PC12细胞生存率及该细胞凋亡呈剂量依赖性降低(P <0.05或P <0.01),ROS 生成逐渐增加与剂量正相关(P <0.05或P <0.01);PCNA 蛋白表达逐渐减少,其上游信号p53蛋白表达逐渐升高㊂结论:MPP +诱导PC12细胞凋亡可能机制之一是通过p53介导PCNA 降解实现的㊂[关键词] 帕金森病;MPP +;PC12细胞;增殖细胞核抗原;p53Expressions of p53and proliferative cell nucleoantigen with neuronal proliferation and apoptosis in a cell model of Parkinson′s diseaseLI Ji ,GUO Ji⁃Dong ,ZHANG Xiao⁃Jie ,YANG Hong ,LI Zun⁃Yan ,SONG Tian⁃Qi ,SUN Wei ,FENG Wei ,LI Da⁃Wei .The First Hospital of Beihua University ,Jilin 132011,China[Abstract ] Objective :To investigate the pathological characteristics and correlation of expressions of p53and proliferating cellnuclear antigen (PCNA)with neuronal proliferation and apoptosis in a cell model of Parkinson′s disease.Methods :The cell model ofParkinson′s disease was created by rat pheochromocytoma (PC12)cells with different concentrations of MPP +(0,0.5,1,2mmol /L).The MTT assay was performed to detect the cells viability,reactive osygen species (ROS)was detected by DCFH⁃DA assay.AO /EB staining and ELISA assay were done to confirm cells apoptosis and ssDNA respectively.Western blot was performed to detect the expressions of p53and PCNA.Results :The measurements revealed a decrease in cell viability and a increase in apoptotic neurons and DNA fragmentation (P <0.05or P <0.01)following the exposure of PC12cells to MPP +in a dose⁃dependent manner,and exposure to MPP +induced a significant increase in ROS production (P <0.05or P <0.01)in PC12cells.The results also revealed that PCNAprotein expression was downregulated in PC12cells treated with MPP +,whereas p53expression was upregulated.Conclusion :One of the possible mechanisms of MPP +⁃induced PC12apoptosis is achieved by p53⁃mediated PCNA degradation.[Key words ] Parkinson′s disease;MPP +;PC12cell;Proliferating cell nuclear antigen;p53 帕金森病(Parkinson′s disease,PD)是神经系统常见变性疾病,其主要病理变化为中脑黑质多巴胺能神经元逐渐变性凋亡,进而递质失衡而出现运动障碍为主的临床症状[1]㊂国内外文献证明外源性及内源性神经毒素诱导多巴胺能神经细胞变性凋亡在PD 发病机制中发挥重要作用,而氧化应激在PD病理过程中起重要作用[2,3]㊂1⁃甲基⁃4⁃苯基⁃1,2,3,6⁃四氢吡啶(MPTP)是诱发PD 的常见环境毒素㊂MPTP 通过血脑屏障后代谢生成毒性1⁃甲基⁃4⁃苯基吡啶离子(MPP +),进入线粒体抑制呼吸链进而生成大量活性氧(reactive osygen species,ROS),最终导致多巴胺能神经细胞变性凋亡[4]㊂氧化应激导致多巴胺能神经细胞变性凋亡的分子机制至今未完全阐明,但DNA 氧化损伤在PD 病理过程中具有重要作用[5]㊂目前,体外培养黑质多巴胺能神经元非常困难,因此可行的方案是以能大量增殖的类神经细胞作为对象来探讨MPP+细胞毒作用机制㊂PC12细胞系是一种分化程度较低的肿瘤细胞,从大鼠肾上腺嗜铬细胞瘤分离得到,在形态㊁生理和生化功能等方面相似于正常神经细胞,且具有中等水平的多巴胺β⁃羟化酶活性㊂诱导PC12细胞损伤模型,模拟体内自由基诱导细胞凋亡的过程,可作为多种神经性疾病的体外细胞模型[6]㊂本研究建立了MPP+诱导的PC12细胞PD模型,探讨p53㊁PCNA表达与细胞增殖㊁凋亡及病理变化特征,探索多巴胺能神经元在氧化应激病理条件下变性凋亡的分子机制,为PD治疗提供新靶点及策略㊂1 材料与方法1.1 材料1.1.1 主要试剂 DMEM高糖培养液及胎牛血清购于美国Gibco公司;胰蛋白酶购于瑞士LONZA公司; MPP+㊁2′,7′⁃二氯荧光黄双乙酸盐(DCFH⁃DA)㊁二甲基亚砜(DMSO)购于美国Sigma公司;小鼠抗大鼠PCNA单克隆抗体和p53抗体购于美国BD公司;辣根过氧化物酶包被抗小鼠二抗购于美国Pierce公司;单链DNA试剂盒购于美国Chemicon Int公司㊂1.1.2 细胞株 PC12细胞(大鼠肾上腺嗜铬细胞瘤细胞)购于中科院上海细胞库㊂1.1.3 主要仪器 CO2培养箱购于美国Cellstar公司;超净工作台购于北京亚泰科隆公司;2S⁃1型紫外线消毒车购于上海跃进医疗器械厂;液氮生物容器购于上海跃进医疗器械厂;电热恒温水浴箱购于上海跃进医疗器械厂;4℃冰箱购于中国西门子公司;荧光倒置显微镜购于日本欧林帕斯公司;550型酶标仪购于美国Bio⁃Rad公司;流式细胞仪购于美国BD公司, FACSCantoⅡ㊂1.2 方法1.2.1 PC12细胞复苏与传代 从液氮罐中取出PC12细胞冻存管,常规消毒㊂无菌条件下稍松动瓶盖后拧紧,封口膜封闭,快速置于37℃水浴箱中摇动融化㊂细胞解冻后取出冻存管,超净台中常规消毒后取出细胞悬液,经离心处理后弃上清,重悬细胞并接种于25cm2培养瓶中,放置于37℃㊁5%CO2㊁90%湿度培养箱中进行培养㊂倒置显微镜观察细胞生长状态,细胞增殖达80%左右汇合进行传代㊂弃培养液并用PBS清洗细胞,然后用0.25%胰蛋白酶消化贴壁细胞㊁细胞计数㊂将细胞悬液在1000r/min㊁18℃的条件下离心5min㊂根据计数结果调整细胞密度为1×105个/ml接种传代培养㊂传至3代细胞状态良好时选取对数生长期的细胞进行试验㊂1.2.2 MPTP诱发PC12损伤模型的建立 参考文献[6]建立MPTP诱发PC12损伤模型㊂取对数生长期PC12细胞,调整至1×105个/ml,吸取0.2ml 接种于96孔板中,放入37℃㊁5%CO2培养箱中孵育24h,弃去培养液,加入浓度为0(空白)㊁0.5㊁1㊁2mmol/L的MPP+的DMEM培养基200μl/孔,37℃㊁5%CO2培养箱中继续孵育24h,每孔加入10μl MTT(5mg/ml),37℃㊁5%CO2培养箱培养4h,弃掉孔内液体,每孔加入150μl DMSO并轻轻振荡以充分溶解甲臜颗粒,在酶标仪570nm处测量各孔的吸光度(OD)值,观察MPP+对PC12细胞的毒性损伤作用㊂1.2.3 细胞内ROS检测 取对数生长期PC12细胞,按损伤模型进行分组,加入无胎牛血清稀释的DCFH⁃DA(终浓度20μmol/L),37℃继续培养30min㊂收集各组细胞,流式细胞仪530nm波长处检测ROS㊂1.2.4 细胞凋亡检测 DNA氧化应激损伤使用AO/EB凋亡染色进行检测㊂PC12细胞处理完成后加入荧光染料AO/EB,在荧光显微镜下观察细胞核形态㊂细胞核完整且被染成绿色的为存活细胞,核浓缩的为早期凋亡细胞,浓缩和碎裂且染成橘红色的为晚期凋亡细胞㊂同时,采用酶联免疫单链DNA 试剂盒检测各组细胞DNA损伤,加入抗单链DNA 单克隆抗体和过氧化物酶标记的二抗,在酶标仪405nm处测各孔吸光值㊂1.2.5 Western blot检测p53和PCNA表达 各组细胞经相应处理培养结束后,收集细胞并裂解提取蛋白,测定浓度㊂参考文献[6]方法,进行各组蛋白电泳分离㊁转膜㊁洗膜㊂β⁃actin作为内参㊂小鼠抗大鼠PCNA单克隆抗体孵育过夜,经TBST洗涤,加入二抗室温孵育2h显影㊂1.3 统计学处理 每组实验重复3次,相同条件设4个平行,结果以x±s表示㊂统计分析采用单因素方差分析或t检验,P<0.05表示差异具有统计学意义㊂2 结果2.1 不同浓度MPP+对细胞存活率影响 采用不同浓度MPP+(0㊁0.5㊁1㊁2mmol/L)处理PC12细胞,反应48h后,使用MTT法检测细胞活力,结果显示细胞活力随MPP+浓度增加而相应减少,呈剂量依赖性,与对照组比较,差异具有统计学意义(P<0.05, P<0.01),见图1㊂2.2 细胞ROS测定 MPP+诱导PC12细胞产生ROS,可以通过荧光染料DCFH⁃DA由流式细胞仪测定㊂DCFH⁃DA 可通过细胞膜并在细胞内酯酶作用下生成DCFH㊂在细胞内ROS 作用下,DCFH 生成具有较强荧光性的DCF㊂结果显示随着MPP +浓度增强,荧光强度逐渐增强,与对照组比较,差异具有统计学意义(P <0.01),见图2㊂2.3 A0/EB 凋亡染色检测细胞凋亡 单链DNA 测定进一步证实随着MPP +浓度增加,PC12细胞变图1 不同浓度MPP +对PC12细胞存活率影响Fig.1 Effects of different concerntration of MPP +onviability of PC12cellsNote:Compared with control,*.P <0.05,**.P <0.01.图2 不同浓度MPP +对PC12细胞产生ROS 的影响Fig.2 Effects of different concentration of MPP +onROS productionNote:Compared with control,**.P <0.01.图3 A0/EB 染色观察不同浓度MPP +对PC12细胞凋亡影响(×200)Fig.3 Effects of MPP +on apoptosis of PC12cells (×200)Note:Compared with control,*.P <0.05,**.P <0.01.性凋亡亦随之增加,与对照组比较,差异具有统计学意义(P <0.05,P <0.01),见图3㊁4㊂2.4 MPP +对PCNA 和p53蛋白表达的影响 采用Western blot 检测了PD 细胞模型中PCNA 和p53蛋白的表达变化㊂结果表明当使用1mmol /L MPP +处理PC12细胞后,在12㊁24㊁48h MTT 测得的细胞活力分别为87%㊁77%㊁61%,而PCNA蛋白表达水平图4 不同浓度MPP +对PC12变性凋亡DNA 变化的影响Fig.4 Effects of MPP +on DNA fragments of apoptosis ofPC12cell linesNote:Compared with control,*.P <0.05,**.P <0.01.图5 MPP +对细胞活力和PCNA 表达影响Fig.5 Effects of MPP +on viability and expression ofPCNA of PC12cellsNote:Compared with control,*.P <0.05,**.P <0.01.图6 不同浓度MPP +对p53和PCNA 蛋白表达影响Fig.6 Effects of different concentration of MPP +on expr⁃ession of p53and PCNANote:Compared with control,*.P <0.05,**.P <0.01.亦逐渐降低㊂随着MPP+浓度增加,p53蛋白表达水平逐渐增加PCNA蛋白表达水平逐渐降低,见图5㊁6㊂3摇讨论PD临床主要特征为肌肉强直㊁运动减少㊁震颤及姿势步态异常,主要由中脑黑质多巴胺能神经元变性凋亡导致,其机制至今未明[7]㊂但更多证据表明,氧化应激促发了一系列凋亡信号,参与了PD多巴胺能神经元变性凋亡过程[8]㊂多巴胺能神经元因富含铁和脂质以及多巴胺自身代谢的原因,更易受氧化攻击[9,10]㊂PD尸检和动物实验均证实DNA 氧化损伤更易出现在中脑黑质多巴胺能神经元,也证实了DNA氧化损伤是PD的重要病理机制[7]㊂DNA对细胞死亡和存活具有决定作用,因常遭受体内外有害物质攻击,因此DNA修复对于保持完整性极为重要㊂本研究在PD细胞模型中显示随MPP+浓度增加,ROS生成增多,呈剂量依赖性,多巴胺能神经元凋亡随之增加,进一步证实氧化应激与细胞凋亡关系密切,以及氧化应激参与PD病理过程,与国外报道一致[6,10]㊂PCNA作为多功能蛋白,在染色体重组及DNA 修复和细胞周期调控中具有重要作用[8]㊂PCNA无内在酶活性,可通过调节多种蛋白如周期蛋白依赖性激酶㊁周期蛋白依赖性激酶抑制因子p21等发挥其调节功能㊂PCNA在DNA氧化损伤修复中亦有重要作用[9]㊂实验证实随MPP+浓度和剂量增加, PCNA蛋白表达呈剂量依赖性减少,支持此蛋白参与了MPP+对多巴胺能神经元毒性损伤过程㊂另一方面,PCNA对维持DNA稳定性,抵御各种致病因素包括氧化应激对DNA的损伤具有重要作用,表达较少加重DNA在病理条件下损伤[10]㊂酶联免疫单链DNA测定同样证实了随着MPP+浓度增加,DNA 损伤逐渐加重㊂提示PCNA表达降低与DNA氧化损伤程度具有相关性㊂转录因子p53调节一系列靶基因,参与细胞生物过程,包括细胞周期㊁DNA修复㊁细胞凋亡和细胞应激反应[11]㊂在神经组织,p53介导细胞凋亡主要通过DNA损伤途径实现㊂氧化应激激活转录因子p53,引起DNA在氧化应激状态下损伤,其损伤机制在PD中未见报道㊂p53是PCNA上游信号蛋白,与特定序列结合后,调节此蛋白表达㊂高浓度野生型p53抑制PCNA启动子,减少PCNA蛋白生成[12]㊂在PD动物模型中已证实p53高表达与多巴胺能神经元变性凋亡密切相关,p53抑制因子可有效阻止PD模型中多巴胺能神经元在氧化应激状态下损伤[13]㊂本实验在PD细胞模型中,MPP+增加ROS 生成和p53表达,证实氧化应激激活p53使其高表达,及p53参与PD病理多巴胺能神经元变性凋亡过程,与文献报道一致[14,15]㊂本实验同样证实了随着MPP+增加PCNA表达减少,且与p53表达量呈负相关;随着PCNA表达减少,DNA氧化损伤逐渐加重,多巴胺神经元凋亡逐渐增加㊂这些实验提示在PD模型中,PCNA介导细胞凋亡可能是PD多巴胺能神经元变性凋亡分子机制之一,且ROS/p53/PCNA信号途径参与这一过程㊂通过PC12细胞建立的PD模型病理变化特征,我们推测MPP+介导多巴胺能神经元氧化损伤可能机制之一是通过p53/PCNA信号途径实现的㊂氧化应激激活p53,减少下游信号蛋白PCNA生成,加重DNA氧化损伤,导致多巴胺能神经元变性凋亡㊂这一分子机制需进一步证实,可为PD治疗提供新分子靶点和策略㊂参考文献:[1] Subramaniam SR,Chesselet MF.Mitochondrial dysfunction andoxidative stress in Parkinson′s disease[J].Prog Neurobiol,2013, 106⁃107:17⁃32.[2] Park J,Lim CS,Seo H,et al.Pain perception in acute model miceof Parkinson′s disease induced by1⁃methyl⁃4⁃phenyl⁃1,2,3,6⁃tet⁃rahydropyridine(MPTP)[J].Mol Pain,2015,11:28. 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分子生物学词汇(P1)分子生物学词汇(Pl)分子生物学词汇(P1)p element p因子［果蝇的可动遗传因子，会造成杂种不育，可用作外源基因的载体］p nucleotide p核苷酸［见于免疫球蛋白及t细胞受体等基因，为重排中根据模板信息所插入］pacemaker起搏点，起搏器pacemaker enzyme 定步酶pachynema粗线期packaging 包装packaging ratio包装率［一条染色单体基本纤维的全长与dna 双螺旋的全长之比，反映dna分子的凝聚状态］packed填充的packed cellvolume收集细胞体积［用以表示培养物的相对增长率］paddle blender浆式捣碎器［利用往复运动的浆叶捣碎密封塑料袋中的材料］paedogenetic parthenogenesis 幼体孤雌生殖paired sib method同胞对照法pairing酉己对palindrome回文序列，回文结构palindromic sequence 回文序列palisade tissue 栅栏组织palytoxin岩沙海葵毒素pancreas 胰腺pancreastatin胰抑制素［可抑制胰岛素分泌］pancreatic 胰的pancreatin胰酶制剂pancreozymin促伊妹儿素panmixis随机交配panning 淘选panning technique淘选技术［如通过亲和层析纯化细胞］panose潘糖panoxadiol 人参二醇panoxatriol人参三醇pantetheine泛酰巯基乙胺pantothenate泛酸；泛酸盐、酯、根pantropic virus泛嗜性病毒papain木瓜蛋白酶papaverine 罂粟碱paper raft nursing technique纸桶保育技术［可用于培养单个植物细胞］papilla ［植物］乳突毛；［动物］乳头papillomavirus乳头瘤病毒papovavirus孚L多空病毒parabasal body 副基粒paracasein副酪蛋白，衍酪蛋白paracentric inversion 臂内倒位parachromatin 副染色质paracodon副密码子［trna上被氨酰trna合成酶识别的碱基，与trna识别氨基酸有关］paracrine旁分泌paracrystal 次晶paracrystalline state 次晶态paradoxical sleep 异相睡眠paraffin imbedding 石蜡包埋法parafilm ［商］石蜡膜，石蜡封口膜［american can公司的商标］ paraformaldehyde低聚甲醛，仲甲醛，多聚甲醛parafuscin草履虫融膜蛋白paraglobulin副球蛋白paralbumin副蛋白，副清蛋白parallel平行的paralogous gene共生同源基因，平行进化同源基因paramagnetic 顺磁的paramecin草履虫素，草履虫蛋白paramecium 草履虫paramucin异粘液素paramucosin仲唾液蛋白paramutation 副突变paramylum原生动物糖paramyosin 副肌球蛋白paramyxovirus 副粘病毒paranemic joint平行汇接［由双链dna分子在重组区解旋而形成］paranemic spiral平行螺旋，反向双股螺旋pararosaniline碱性副品红parasexuality 准性生殖parasite寄生虫parasitism 寄生parasporal crystal 伴胞晶体parathion对硫磷parathyrin甲状旁腺素parathyroid gland 甲状旁腺paratope ［抗原］互补位parenchyma ［植物］薄壁组织；［动物］实质parental亲本的，亲代的parental imprinting亲本印记［配子发生过程中亲本基因的选择性差异表达］parental type亲本型［如用于描述子代性状］parental virus 亲代病毒parity宇称parkinson disease 帕金森病paromomycin巴龙霉素parotin腮腺素pars amorpha ［核仁］无定形区pars fibrosa ［核仁］纤维区pars granulosa ［核仁］颗粒区parsnip yellow fleck virus 欧防风黄点病毒parthenocarpy 单性结实parthenogamy孤雌核配parthenogenesis孤雌生殖［雌体产生不需受精即可发育的卵子］；孤雌发育［卵子不经受精进行发育］parthenogenetic embryo 单性胚，孤雌胚parthenogonidium孤雌生殖细胞parthenomixis孤雌两核融合partial molar偏摩尔的particle gun基因枪，粒子枪partitivirus 分病毒parvalbumin小白蛋白，小清蛋白［如见于鲤鱼］parvovirus细小病毒passage佟细胞］传代passage type过渡形式pasteur effect巴斯德效应，巴氏效应［有氧氧化抑制酵解］pasteur pipet巴氏吸管，巴斯德吸管pasteurella巴氏菌属，巴斯德菌属pasteurization巴氏消毒法patch clamp膜片箝，膜片钳patch clamping technique膜片箝术，膜片钳术［可用于监测膜通道活性］patching膜片形成paternity test 亲权认定pathogen病原体pathovar致病变型patroclinal ingeritance 偏父遗传patrogenesis孤雄生殖pattern ［特征序列］模式patulin展青霉素pauli exclusion principle 泡利不相容原理pauperization 杂交弱势paxillin桩蛋白［见于粘着斑，被栓在膜上］pea enation mosaic virus 豌豆耳突花叶病毒pectamycin密旋霉素pectin果胶pectinase果胶酶pedigree 系谱pediocin片球菌素pellicle菌膜；（菌）醛"细胞］表膜penetrance夕卜显率penicillinase 青霉素酶penicillium 青霉属penicillium chrysogenum virus 产毒青霉病毒pentagastrin五肽胃泌素pentamer五聚体penton五邻体［见于腺病毒］pentosan戊聚糖pentose 戊糖pentyl戊基peplomer包膜突起peplos包膜pepscan肽扫描（技术）pepsin胃蛋白酶pepsinogen胃蛋白酶原pepsitensin胃酶解血管紧张肽pepstatin胃（蛋白）酶抑制剂，抑胃酶肽peptidase 肽酶peptide nucleic acid肽核酸［一类dna类似物，以氨基酸取代糖磷酸主链］peptide screening肽筛选［常指利用合成肽进行表位作图的方法］peptidergic fiber 肽能纤维peptidoglycan 肽聚糖peptidyl 肽基peptization 胶溶peptone 月东pepzyme肽性酶［人工合成的小分子肽催化剂］percoll ［商］珀可［pharmacia公司商标，是聚乙烯吡咯酮包被的二氧化硅壳粒的无菌胶体悬液，可以形成1.3g/ml以下的各种密度梯度］perforin穿孔素perfringocin产气荚膜竣菌素perfusion 灌流peri effect近位效应peri position 近位perianth花被；［苔藓］蒴萼periblem皮层原［见于植物］pericardial cavity 心包腔pericardium 心包膜pericentric inversion 臂间倒位periclinal 平周的pericycle中柱鞘periderm周被［见于植物］peridium ［粘菌］抱囊被；包被perikaryon 核周体periodic protein周期性蛋白［含有周期性重复序列］periosteum骨夕卜膜peripheral外周的，周边的peripherin外周蛋白［一种中间丝蛋白，最初发现于神经元］periplasm （外）周质periplast周质体perisperm夕卜胚孚Lperistalsis 蠕动peristaltic pump 蠕动泵perithecium 子囊壳peritoneum 腹膜perlecan基底膜（蛋白）聚糖permeability 通透性permeabilization透化（作用）［使通透性增加］permeabilizing 透化（处理）permease通透酶permissive action允许作用［如特指激素间一种协同作用］permselective membrane选择透性膜，选择（性）通透膜permselectivity选择通透性permutation变换，置换；排列peroxidase过氧化物酶persitol鳄梨糖醇perturbation 微扰pertussis toxin百日咳毒素pervaporation 全蒸发perxisome过氧化物酶体pesticin鼠疫菌素pestivirus瘟病毒属petri dish培养皿petroselinic acid岩芹酸，6-十八（碳）烯酸pfu dna polymerase pfu dna 聚合酶［来自pyrococcus furiosus的耐热dna聚合酶（stratagene公司专利产品）兼具5'-3-dna聚合活性及3'-5'外切校正活性］phaeophyll 叶褐素phaeophyta 褐藻门phaeophytin 褐藻素10phaeoplast 叶褐体phage antibody噬菌体抗体［噬菌体蛋白与免疫球蛋白的融合体，表达于噬菌体表面］phage display噬菌体展示［将抗体或肽表位展示于噬菌体表面］phage typing噬菌体分型［利用噬菌体进行细菌分型］phagecyte吞噬细胞phagemid噬菌粒，噬粒phagetype噬菌体型phagevar噬菌体变型phagocytosis 吞噬（作用）phagosome吞噬体phallacidin类鬼笔（毒）环肽phallin鬼笔溶血（毒）环肽，白鬼笔（毒）环肽phallisin类鬼笔素phalloidin鬼笔（毒）环肽phalloin鬼笔素phallotoxin鬼笔毒素，鬼笔毒蕈肽［类名，包括鬼笔素，鬼笔环肽等］pharynx 咽phaseolin云扁豆蛋白phaseoline 菜豆碱11phaseolotoxin菜豆丁香假单胞杆菌毒素，菜豆菌毒素phasmid噬菌粒，噬粒phastgel ［商］快速凝胶［phastsystem所用现成凝胶］phastsystem ［商］快速凝胶电泳系统［pharmacia公司生产的快速聚丙烯酰胺凝胶电泳］phenanthrene 菲phenanthroline 菲咯啉phenazine 吩嗪phenobarbital 苯巴比妥phenocopy拟表型，表型模拟phenogenetics发育遗传学phenotype 表型phenotypic 表型的phentolamine酚妥拉明phenylacetamidase 苯乙酰胺酶phenylacetamide 苯乙酰胺phenylacetic acid 苯乙酸phenylalanini 苯丙氨酸phenylarsenic oxide 氧化苯胂phenylethanol 苯基乙醇phenylethanolamine 苯基乙醇胺phenylethylamine 苯乙胺12 phenylisothiocyanate 异硫氰酸苯酯phenylketonuria苯丙酮尿症phenylthiocarbamide 苯确脲phenylthiohydantoin 乙内酰苯硫脲pheophyll叶褐素pheophytin褐藻素，脱镁叶绿素pheromone信息素，外激素pheron脱辅（基）酶phlobaphene 鞣红phloem韧皮部phloroglucinol藤黄酚，间苯三酚phorbol佛波醇phosgene 光气phosphagen 磷酸原phosphatase 磷酸酶phosphatidase 磷脂酶phosphatide 磷脂phosphatidylcholine 磷脂酰胆碱phosphatidylethanolamine 磷脂酰乙醇胺phosphatidylglycerol 磷脂酰甘油phosphatidylserine磷脂酰丝氨酸13phosphine 膦phosphoarginine磷酸精氨酸phosphocellulose 磷酸纤维素phosphocreatine 磷酸肌酸phosphodiester 磷酸二酯phosphodiesterase 磷酸二酯酶phosphoenolpyruvate烯醇丙酮酸膦酸phosphoeptide 磷酸肽phosphoester 磷酸酯phosphofructokinase 果糖磷酸激酶phosphoglucoisomerase葡糖磷酸异构酶phosphoglucomutase葡糖磷酸变位酶phosphogluconate shunt葡糖酸膦酸支路phosphogluconolactone磷酸葡糖酸内酯phosphoglycerate 磷酸甘油酸phosphoglyceride 磷酸甘油phosphohistidine 磷酸组氨酸phosphokinase 磷酸激酶phospholamban 受磷蛋白phospholipase 磷脂酶phospholipid 磷脂14phospholipoprotein 磷酸脂蛋白phosphonate 膦酸酯phosphonoacetic acid膦酰乙酸，膦羧乙酸phosphonomycin 磷霉素phosphoramidite 亚磷酰胺phosphoramidon膦酰二肽[一种来自微生物的蛋白酶抑制剂，即n-（a-鼠李吡喃糖基膦酰胺-ser-trp] phosphorescence 磷光phosphoribosyl磷酸核糖的phosphorimeter 磷光计phosphorodithioate 二硫代磷酸酯phosphorolysis 磷酸解phosphorotein 磷蛋白phosphorothioate 硫代磷酸（酯）phosphorylase 磷酸化酶phosphorylation 磷酸化phosphoserine磷酸丝氨酸phosphothreonine 磷酸苏氨酸phosphotransferase 磷酸转移酶phosphotriester 磷酸三酯phosphotyrosine磷酸酪氨酸phosphtidylinositol 磷脂酰肌醇15phosvitin卵黄高磷蛋白photoabsorption 光吸收photoacoustic 光声的photoactivation 光活化photoactive光活性的，光敏的photoaffinity 光亲和的photoallergy光变态反应photoautotroph光（能）自养生物photoautotrophic 光能自养的photoautotrophy 光（能）自养photoautoxidation 光（能）自动氧化photobacteria （发）光细菌photobiology光生物学photobiont共生光合生物photobiotin ［商］光生物素［澳大利亚bresa公司的商标通过带电荷的连接臂将具有光化学反应活性的芳基叠氮基团连接于生物素］photobleaching 光漂白photocatalysis 光催化（作用）photocatalyst 光催化剂photochemical 光化学的photochemistry 光化学photochromism光致变色（性）16 photoconductive 光导的photodecomposition 光（分）解（作用）photodegradable 光降解的photodegradation 光降解（作用）photodensitometer 光密度计photodensitometry 光密度分析（法）photodigoxigenin ［商］光（敏）地高辛配体，光（敏）洋地黄毒苷photodiode 光（电）二极管photodissociation 光解离photoelectric 光电的photoelectrocatalysis 光电催化photoelectron 光电子photogene nucleic acid detection system ［商］光化学核酸检测系统［lifetechnologies 公司（brl）商标］photohemolysis 光致溶血photoheterotroph光（能）异养生物photoheterotrophic 光（能）异养菌photoheterotrophy 光（能）异养photoinduction 光诱导photoisomerization 光异构化17 photolithotrophic光（能）无机营养的photolithotrophy光（能）无机营养photoluminescence 光致发光photolyase 光解酶photolysis光解（作用）photomedicine 光医学photomicrogr叩hy显微摄影（术），显微照相（术）photomovement 光运动photomultiplier光电倍增管photon光子photonasty 感光性photoorganotroph光能有机营养生物photoorganotrophic 光能有机营养的photoorganotrophy光能有机营养photooxidation 光氧化（作用）photoperiodism光周期现象，光周期性photoperoid 光周期photophase光照阶段photophosphorylation 光合磷酸化photopolymerization 光（致）聚合（作用）photopotential 光电位photopsin光视蛋白18photoreaction 光反应photoreactivation 光复活photoreactive光敏的，光反应性的photorearrangement 光重排photoreception感光，光感受（作用）photoreceptor 光感受器photoreceptor transduction 感光传导photoredox reaction光致氧化还原（反应）photoreduction 光还原（反应）photorespiration 光呼吸（作用）photosensitive 光敏的photosensitivity 光敏感性photosensitization 光敏化（作用）photosensitizer 光敏剂photosensory 感光的photostage光照阶段photosynthate 光合产物photosynthesis 光合作用photosynthetic 光合的photosystem 光系统phototaxis趋光性［（细胞）受光源方向或强度的影响进行定向运动］19phototroph光养生物phototrophic 光养的phototrophy 光（营）养phototropic 向光的phototropism向光性［受光源方向或强度影响的（细胞）定向生长］photoxidation 光氧化phragmoplast 成膜体phrenosin羟脑苷脂phycobilin藻胆（色）素phycobiliprotein藻胆（色素）蛋白phycobilisome藻胆体，藻胆蛋白体phycobiont 共生藻phycochrome 藻色素phycocyanin藻蓝蛋白，藻青蛋白phycocyanobilin 藻蓝素phycodnavirus 藻dna 病毒phycoerythrin 藻红蛋白phycoerythrobilin 藻红（胆）素phycomycetes藻状菌纲phycophaein 藻褐素phycophage 噬藻体20 phycovirus 藻病毒phycoxanthine 藻黄素phylaxin抵抗素phyllocaerulin叶泡雨蛙肽phyllocaline 成叶素phyllolitorin叶泡雨滨蛙肽phyllospheric microganism 叶际微生物phylogenesis系统发育phylogenetic系统发育的，系统的phylogeny系统发育physalaemin 泡蛙肽physical selection物理选择［根据突变体的特有性状进行选择］physostigmine 毒扁豆碱phytanic acid 植烷酸phytic acid 植酸phytoalexin植物抗毒素phytochelatin植物螯合肽phytochemistry 植物化学phytochrome （植物）光敏（色）素phytocide除草剂phytocidin植物杀菌素phytoecdysteroid植物蜕皮类固醇，植物蜕皮甾体21phytoferritin植物铁蛋白phytohemagglutinin 植物凝集素phytohormone植物激素phytol叶绿醇，植醇phytoplankton 浮游植物phytosphingosine植物鞘氨醇，4-羟二氢鞘氨醇phytotoxin毒植物素［微生物产生的对植物有毒害作用的一种物质］phytotron人工气候室phytylmenaquinone叶绿甲基萘醍，维生素k1picking挑取（菌落、噬斑、蚀斑等）picolinic acid吡啶甲酸［色氨酸代谢产物］picornavirus 小rna 病毒picrotoxin印防己毒素，木防己苦毒素piericidin粉蝶霉素，杀粉蝶菌素pigementation 色素形成pilin菌毛蛋白pilocarpine毛果（芸香）碱pilot experiment 预试验pilot protein先导蛋白pilus菌毛pimaricin匹马菌素22pimelate庚二酸；庚二酸盐、酯、根pimelic acid 庚二酸pin technology大头针技术［采用特制聚乙烯大头针作支持体进行超微量固相多肽合成的技术，可用作表位作用］pinacol频哪醇pinane蒎烷pineal松果体的pineal body 松果体分子生物学词汇(Pl)相关内容：23。
