细胞工程论文摘要及总结.doc

河北师范大学 细胞工程 论文摘要及总结姓 名 曾 媛 班 级 08级生技 学 号 2008011175 小鼠与人类ES细胞的分离与培养论文摘要昆明小鼠ES细胞的分离培养及鉴定张廷龙1,董雅娟1*,柏学进1,赵晶1,2,岳福杰3,史文升1,龚宜超1(1.青岛农业大学动物胚胎工程中心,山东青岛266109;2.西北农林科技大学,陕西杨凌712100; 3.荣成市出入境检验检疫局,山东荣成265300)摘要目的摸索一种适合昆明小鼠ES细胞分离培养的方法,以提高昆明小鼠ES细胞的建系率。方法分别采用5种方法分离ICM和ES细胞集落,摸索了以BMSCs作为饲养层时,丝裂酶素处理的合适时间。结果连续消化法要明显好于其他4种方法。MMC处理BMSCs 1、1.5、2 h时效果较好,3个时间点无显著差异(P0.05)。mMEF作为饲养层与BMSCs作为饲养层时获得5代ES细胞相比差异不显著(P0.05)。结论连续消化法分离ES细胞效率较高,饲养层采用MMC处理12 h的BMSCs饲养层或常规mMEF饲养层对昆白小鼠ES细胞无明显影响。关键词:昆明小鼠; ES细胞; BMSCs; ICM昆明小鼠胚胎干细胞的分离、培养与鉴定作者:赵晶 （西北农林科技大学）摘要本研究的目的是建立有效的昆明小鼠胎儿成纤维细胞（MEF）饲养层培养体系，用于分离和培养小鼠胚胎干细胞（ES细胞）的研究，筛选出更适合于昆明小鼠ES细胞的克隆培养体系，为进一步建立昆明小鼠ES细胞系奠定基础。1分别选取妊娠11 d，14.5 d和16 d的孕鼠胎儿分离成纤维细胞，比较分离不同日龄胎儿的成纤维细胞增殖及纯化情况，以及观察不同接种密度，成纤维细胞体外培养生长状况。选择成纤维细胞分离培养的最适胚龄和最佳培养条件。试验结果显示，14.5 d的孕鼠胎儿分离培养成纤维细胞效果最好。可分离到大量的成纤维细胞，杂细胞少，并且细胞增殖快。生长旺盛、已纯化的第三代成纤维细胞最适宜做ES细胞的饲养层。2采用全胚培养法，比较桑椹胚和囊胚对ES细胞分离培养的影响。将桑椹胚和囊胚分别培养在饲养层上，桑椹胚的贴壁率、内细胞团（ICM）增殖率和ES细胞集落的出现率均显著低于囊胚（P0.01）。由此看出，试验中获得的小鼠囊胚更适合作为ES细胞分离克隆的材料，并且妊娠第4天采集的胚胎囊胚率高。3比较三种不同浓度消化液：0.25%胰酶+0.04%EDTA，0.05%胰酶+0.04%EDTA和0.02%胰酶+0.01%EDTA，对内细胞团、ES细胞集落分离克隆的影响。试验结果显示，0.02%胰酶+0.01%EDTA是较好的ES细胞消化液，对细胞的损伤力小，且传代后ES细胞集落形成能力也较高；对没有消化离散的大的细胞团块，吸出后用机械法切割。4选取生长旺盛并且已纯化的第3代成纤维细胞，经丝裂霉素-C处理后，用细胞计数板计算细胞数，分别按密度为1104个/mL，1106个/mL和1108个/mL接种，制备饲养层，观察不同密度饲养层的生长状况和不同密度饲养层上囊胚、内细胞团及胚胎干细胞的克隆生长情况。试验结果表明，囊胚在密度为1106个/mL的饲养层上，贴壁率和内细胞团孵出率分别为(97.03.606)%，(96.32.887)%，显著高于其他两组；密度为1106个/mL饲养层上的ES细胞克隆形成率高于密度为1104个/mL（差异极显著，P0.01）和1108个/mL饲养层上的ES细胞克隆形成率差异显著（P0.05）；而1104个/mL饲养层和1108个/mL饲养层上的ES细胞克隆形成率差异不显著（P0.05）。因此，以1106个/mL密度接种的成纤维细胞作为饲养层，最适合用于分离培养昆明小鼠ES细胞，有利于囊胚的发育、内细胞团的增殖、促进ES细胞的增殖，并起到抑制其分化的作用。5对分离克隆出的ES细胞进行细胞形态、集落形态、碱性磷酸酶测定、核型分析以及体外分化能力加以鉴定。分离得到的ES细胞有典型的形态学特征，集落呈鸟巢状，边缘清楚，表面平滑，结构致密，其隆起生长，细胞之间界限不清楚；碱性磷酸酶染色呈强阳性，ES细胞集落具有正常的二倍体核型，可形成拟胚体。表明培养扩增后的细胞具有ES细胞的典型特征。关键词：昆明小鼠；胎儿成纤维细胞；胚胎干细胞；饲养层昆明系小鼠胚胎干细胞分离培养的优化白莹敏1,李跃民1*,邱小燕1,肖雄1,武建中2(1.西南大学动物胚胎工程试验室,重庆北碚400716; 2.宜春学院生命科学与资源环境学院,江西宜春336000)摘要:以昆明系小鼠为对象,经过丝裂霉C处理成纤维细胞(Mouse embryonic fibroblast,MEF)制备饲养层,对影响小鼠胚胎干细胞(Embryonic stem cell,ES细胞)分离培养的相关因素进行研究。分别收集小鼠3.5 d的囊胚(扩张囊胚)和4.5 d囊胚(孵化囊胚)进行培养,比较扩张囊胚和孵化囊胚的贴壁率、原代克隆率及传代率的情况。收集3.5 d胚龄的囊胚,通过全胚法和免疫外科法对内细胞团(Inner cell mass,ICM)进行分离培养ICM集落,确定离散ICM的适宜时间。用0.25%胰酶+0.04%EDTA,0.125%胰酶+0.02%EDTA和0.25%胰酶+1%小鸡血清等方法对小鼠ES细胞集落进行传代,观察不同酶浓度对ES细胞分离克隆的影响。结果显示,孵化囊胚的贴壁率高于扩张囊胚(P0.05),但传代率则相反(P0.05);一般ICM增殖培养23 d(免疫外科法)或45 d(全胚培养法)后,出现典型的克隆集落,再挑取ICM;0.125%胰酶+0.02%EDTA及0.25%胰酶+1%小鸡血清,形成ES原代克隆率较高,2组没有显著性差异(P0.05);结果表明,分离得到的ES细胞经形态学观察,AKP染色,体外分化试验等表明其具有胚胎干细胞的特性。关键词:干细胞;胚胎干细胞;培养;传代;小鼠小鼠MEF的制备及ES细胞的分离培养试验*杨恕玲1,董雅娟1,柏学进1, 2,李建栋1,程明1(1.莱阳农学院动物胚胎工程中心,山东青岛266109; 2.西北农林科技大学)摘要:本试验采用两种方法制备小鼠胎儿成纤维细胞饲养层(mouse embryonic fibroblast, MEF),两种方法的差别在于用丝裂霉素-C(mitomycine-C,MMC)处理后,一种是用PBS-洗净MMC后直接使用;另一种是丝裂霉素-C处理后,用胰酶消化后重新接种,待细胞融合后再使用。将获得的小鼠胚胎直接接种在MEF上,分别在56d后取增殖的ICM团块,消化再接种培养,观察胚胎干细胞(embryonic stem cel,l ES细胞)样集落的生长情况,并对获得的ES细胞样集落进行碱性磷酸酶(AKP)染色鉴定。结果发现两种处理方法得到的MEF培养体系均具有促进ES细胞增殖以及抑制ES细胞的分化的作用,而且得到的ES细胞样集落符合ES细胞的形态特征,AKP呈强阳性。采用本试验的第一种方法的优点是简化了胎儿成纤维细胞饲养层的制备过程,并且适合做ES细胞的饲养层。关键词:小鼠; MEF; ES细胞; 分离培养小鼠胚胎干细胞体外分离培养影响因素的实验研究*刘登华肖文伍陈红张苏明华中科技大学同济医学院附属同济医院神经内科,武汉430030摘要目的为了更高效地分离胚胎干细胞(ES),对几种影响分离ES细胞的因素进行探讨。方法通过胚泡Hoechst33342染色及胚泡培养,研究超排卵对胚泡贴壁率及ES细胞分离效率的影响。并从3种不同培养体系中寻找较为理想的ES细胞分离体系。结果超排卵获得的胚泡的细胞数与正常胚泡之间差异无显著性意义。超排卵对胚泡的72 h贴壁率及ES细胞克隆率无显著影响。在ES细胞培养过程中,有饲养层体系优于无饲养层体系。结论超排卵获得胚泡的方法在小鼠ES细胞分离培养过程中值得采用。人胚胎成纤维细胞饲养层在ES细胞分离过程中具有较好的作用。关键词：小鼠;胚胎干细胞;分离培养改进的培养体系在小鼠体细胞核移植及重构胚ES细胞分离培养中的应用曹鸿国,刘俊平,张涌*(西北农林科技大学生物工程研究所,陕西杨凌712100)摘要取8周后的雌性昆明小鼠进行超排,取卵母细胞用作核受体,收集卵母细胞周围的卵丘细胞作核供体,进行体细胞核移植。核移植重构胚经SrCl2激活处理6 h后,与改良的M16培养液和小鼠输卵管上皮细胞共培养;将发育到早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加含心肌细胞培养液的ES细胞培养液;把孵出的ICM进行消化接种培养,对孵出的ES细胞集落进行鉴定培养。结果显示,以小鼠卵丘细胞为核供体,体细胞核移植重构胚激活率为6523%,囊胚发育率为1169%; 9个核移植重构囊胚中分离出ES细胞集落,分离率为277%;分离出的核移植ES细胞集落具有岛屿状团状隆起结构、碱性磷酸酶染色呈阳性,体外分化可形成类胚体,并能分化成上皮样或梭形细胞。ES细胞集落经常规冻存和复苏后,显示出同冻存前相似的集落形态,并具有较强的增殖能力。实验证实小鼠输卵管上皮细胞、改良的M16培养液及含心肌细胞培养液的ES细胞培养液可以更为成功地运用于小鼠的体细胞核移植及ES细胞的分离培养研究。关键词:心肌细胞; 输卵管上皮细胞; 改良培养液; 小鼠; 体细胞核移植; ES细胞人类羊水源类ES细胞多能性干细胞的分离培养华进联，刘雨潇，董武子，窦忠英(西北农林科技大学陕西省干细胞工程技术研究中心，陕西杨凌712100)摘要干细胞具有自我更新和多向分化潜能，是形成机体多种组织器官的祖细胞。按其来源，可分为胚胎干细胞和成体千细胞。胚胎千细胞(Embryonic stem ce11s,ES细胞)是由早期胚胎内细胞团(Inner cell mass,ICM)或原始生殖细胞(Primordial germ cells, PGCs)分离而来的多潜能细胞，ES细胞是研究哺乳动物早期胚胎发生、细胞组织分化、基因表达调控等发育生物学基础研究的一个非常理想的模型系统和非常有用的工具，也是进行动物胚胎工程研究、生产和临床医学研究的一个重要途径。千细胞可望用于治疗中风、帕金森氏症、阿尔茨海驮氏症、脊位损伤、精尿病等重大疾病。但由于ES细胞研究的伦理学、免疫排斥问题和建系难等问题制约着ES细胞的应用，需要探寻机体其他来源的多能性干细胞。羊水细胞培养是产前诊断胎儿先天性遗传疾病的经典方法。羊水细胞的培养已经成功，最近，研究表明可能含有一些具有多能性干细胞特性的细胞，羊水细胞可用于矫正某些类型的出生后胎儿的先天性缺陷疾病，还可能提供胎儿干细胞供研究和治疗之用。 无菌收集怀孕20-30周龄胎儿羊水，采用类似人类ES细胞的分离培养方法，即以小鼠胎儿成纤维细胞( MEF)或人胎儿成纤维细胞( HEF )等作饲养细胞层，Knock-out DMEM(Gibco)或DMEM(高精)+15%KSR(Knock-out serum replacement)或15%FBS(胎牛血清)+0.1 mmol/L 2-流基乙醉等为基础培养液，添加LIF. SCF, bFGF为培养液，分离培养的部分羊水细胞具有ES细胞特性，细胞克隆形成集落，细胞结构致密，界限不清，部分细胞AP, OCT-4, SSEA-1免疫组化染色阳性.AP, OCT-4, SSEA-1等均为胚胎性多能性干细胞的特异性标志，尤其oct-4为维持千细胞多能性的特异性标记.结果表明，羊水细胞可能是胚胎性多能性干细胞的又一个潜在来源和途径，其可能来源于脱离正常迁移定居路途的原始生殖细胞(PGCs )或为间质干细胞或上皮千细胞的祖细胞。羊水源胚胎性多能性千细胞的发现可以避免人类ES细胞研究的论理学问题，可为胎儿和人类重大疾病的治疗提供大l的种子细胞。关键词:人;羊水;胚胎(ES)干细胞:OCT-4;多能性Culture pH and osmolality influence proliferation and embryoid body yields of murine embryonic stem cells Muhammad A. Chaudhrya, b, Bruce D. Bowenb and James M. Pireta, baMichael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada bDepartment of Chemical and Biological Engineering, University of British Columbia, 2360 East Mall, Vancouver, BC V6T 1Z3, Canada Received 16 October 2008; revised 27 February 2009; accepted 6 March 2009. Available online 19 March 2009. AbstractThe realization of many stem cell based regenerative therapies will depend on culture production. Murine embryonic stem cells (mESC) provide a practical model for stem cell process research as they can be readily obtained at relatively high numbers and purities. However, an understanding of their environmental tolerance ranges is still lacking. These tolerance ranges were explored using an embryoid body (EB) formation assay as a functional measure of mESC maintenance. The mESC were exposed to wide ranges of initial medium glucose, glutamine, ammonium, lactate, pH and osmolality conditions. Within the common ranges of conventional maintenance cultures, the only environmental variables that significantly influenced EB yields were pH and osmolality. Doseresponse experiments with these two variables revealed that, within 48h, the EB yield was, for example, 3-fold decreased (p0.05) when mESC were cultured either at an initial pH of 7.0 or an osmolality of 400mOsm/kg compared to a medium at pH 7.3 and 300mOsm/kg. This decline was due to decreases in both the growth rate and in the fraction of EB-forming cells More extreme pH (6.7 or 7.75) as well as osmolality (200 or 500mOsm/kg) conditions reduced the EB formation potential to even lower levels. The decreased growth rates and EB forming potential of mESC cultured at pH 6.7 or 7.75 for 24 or 48h, when returned to pH 7.3 medium, recovered to control levels within 96144h. These studies provide guidance in developing optimal environmental tolerance ranges within which both the mESC output from maintenance cultures as well as the subsequent EB yields can be maximized.Keywords: Embryonic stem cells; Cell therapies; pH; Osmolality; Kinetics;Cell culture engineeringCharacterization and Culture of Human Embryonic Stem Cells Andrew L Laslett a, Adam A Filipczyka and Martin F PeraaaMonash Institute of Reproduction and Development, Monash University, Clayton, Victoria, AustraliaAvailable online 25 September 2003. AbstractHuman embryonic stem (ES) cells are cultured cell lines derived from the inner cell mass of the blastocyst that can be grown indefinitely in their undifferentiated state, yet also are capable of differentiating into all cells of the adult body as well as extraembryonic tissue. Detailed investigation of the properties of embryonal carcinoma cells of both the mouse and human as well as mouse and primate ES cells led to the initial isolation and subsequent culture of human ES cells The methodologies that were developed to culture and characterize these cell lines have provided a template for the development of human ES cells The existing data illustrate a number of important differences and similarities between human ES cells and the other cell lines. This review aims to provide a brief historic account of the development of the mammalian pluripotent stem cell field; describe how this led to the isolation, culture, and characterization of human ES cells; and discuss the potential implications of recent advancesCulture of mouse embryonic stem cells on photoimmobilized polymers Tomohiro Konnoa, Naoki Kawazoeb, Guoping Chenb and Yoshihiro Itoa, caRegenerative Medical Bioreactor Project, Kanagawa Academy of Science and Technology, KSP East 309, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan bBiomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan cNano Medical Engineering Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Received 27 March 2006; accepted 3 July 2006. Available online 18 November 2006. AbstractMouse embryonic stem (ES) cells were cultured on four types of polymer with different surface properties. The polymers were poly(acrylic acid), polyallylamine, gelatin, and poly(2-methacryloyloxyethyl phosphorylcholine-co-methacrylic acid) (PMAc50), and were coupled with azidophenyl groups and photoimmobilized on conventional polystyrene cell-culture dishes. Mouse ES cells were cultured on the immobilized polymer surfaces, and cell morphology, cell growth, staining for alkaline phosphatase, activation of the transcription factor stat3, and expression of the octamer-binding protein 3/4 (Oct3/4) transcription factor and the zinc finger-containing transcription factor (GATA4) were observed. Morphology and growth rate were significantly affected by the polymer surface properties. The ES cells attached to gelatin or polyallylamine surfaces; however, colonies formed on the former but not the latter. In addition, significant enhancement of growth was observed on the gelatin surface. In contrast, ES cells aggregated to form