CN1548529A - Separation method of buffering stem cell in human placenta - Google Patents

Separation method of buffering stem cell in human placenta Download PDF

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CN1548529A
CN1548529A CNA031235042A CN03123504A CN1548529A CN 1548529 A CN1548529 A CN 1548529A CN A031235042 A CNA031235042 A CN A031235042A CN 03123504 A CN03123504 A CN 03123504A CN 1548529 A CN1548529 A CN 1548529A
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cells
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msc
cell
placenta
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毅 张
张毅
李长东
江小霞
何津
刘元林
张双喜
吴英
毛宁
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中国人民解放军军事医学科学院基础医
中国人民解放军军事医学科学院基础医学研究所
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Abstract

The present invention discloses the separation method of mesenchymal stem cell in human placenta. On the basis of past separation of tissue cell, the present inventor separates from placenta mesenchymal stem cell with high purity via perfusion process based on the special anatomical structure of placenta. Identification result shows that the mesenchymal stem cell separated from placenta has the biological characteristic and polydirectional differentiation capacity as the reported mesenchymal stem cell of marrow. Owing to the infantile cell component and wide source of placenta, like cord blood, the present invention will have wide clinical application foreground.

Description

一种人胎盘间充质干细胞的分离方法 Mesenchymal stem cells from one human placental separation method

技术领域 FIELD

本发明涉及一种细胞的分离方法,具体地说涉及一种人胎盘间充质干细胞的分离方法。 It relates to a cell separation method according to the present invention, particularly to the separation method for inter human placenta mesenchymal stem cells.

背景技术 Background technique

人间充质干细胞(mesenchymal stem cell,MSC)是最早自骨髓中分离出的一类具有多向分化潜能及自我更新能力的组织干细胞,来源于胚胎发生期的中胚层,在体内和体外特定条件下可向成骨细胞、软骨细胞、脂肪细胞、肌细胞、肌腱细胞、肝细胞甚至神经胶质细胞分化(1.Caplan AI.Mesenchymalstem cells.J Orthop Res.1991,9:641-650.2.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999;284:143-147.)。 Human mesenchymal stem cells (mesenchymal stem cell, MSC) is the first from bone marrow isolated a class of tissue stem cells have pluripotency and self-renewal capacity, mesoderm derived embryogenic stage, in vivo and in vitro specific conditions to differentiate into osteoblasts, chondrocytes, adipocytes, muscle cells, tendon cells, hepatocytes even glial cells (1.Caplan AI.Mesenchymalstem cells.J Orthop Res.1991,9: 641-650.2.Pittenger MF, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999; 284: 143-147).. MSC易于从骨髓中分离纯化,进行有效的体外扩增,对其生物学作用研究结果表明,MSC具有改善骨髓微环境,促进造血移植后造血重建的作用(1.Kadereit S,Deeds LS,Haynesworth S,et al.Expansion ofLTC-ICs and Maintenance of p21 and BCL-2 Expression in Cord BloodCD34+/CD38-Early Progenitors Cultured over Human MSCs as a Feeder Layer.Stem cell.2002;20:573-582.2.Koc ON,Gerson SL,Cooper BW,et al.Rapidhematopoietic recovery after coinfusion of autologous-blood stem cells andculture-expanded marrow mesenchymal stem cells in advanced breast cancerpatients receiving high-dose chemotherapy.J Clin Oncol.2000;18:307-316.3.Klyushnenlova E,Mosca J,McIntosh K.Human mesenchymal stem cells suppressallogeneic T cell responses in vitro:implications for allogeneic transplantation.Blood.1998;92:642a.)。 MSC easily isolated and purified from bone marrow, effectively amplified in vitro studies showed that its biological role, MSC having improved bone marrow microenvironment, promote (1.Kadereit S, Deeds LS hematopoietic reconstitution after hematopoietic transplantation, Haynesworth S , et al.Expansion ofLTC-ICs and Maintenance of p21 and BCL-2 Expression in Cord BloodCD34 + / CD38-Early Progenitors Cultured over Human MSCs a Feeder Layer.Stem cell.2002 as; 20: 573-582.2.Koc ON, Gerson SL , Cooper BW, et al.Rapidhematopoietic recovery after coinfusion of autologous-blood stem cells andculture-expanded marrow mesenchymal stem cells in advanced breast cancerpatients receiving high-dose chemotherapy.J Clin Oncol.2000; 18: 307-316.3.Klyushnenlova E, Mosca J, McIntosh K.Human mesenchymal stem cells suppressallogeneic T cell responses in vitro: implications for allogeneic transplantation.Blood.1998; 92: 642a).. 免疫特性方面,MSC不表达或仅表达可忽略水平的MHC II类分子,不相关供者的MSC不引起异体淋巴细胞反应,能下调异体免疫反应(1.McIntosh K,Klyushnenkova E,Shustova V,et al.Suppression ofalloreactive T cell reaponse by human mesenchymal stem cells involves CD8+cells.Blood.1999;94:133a.2.Klyushnenkova E,Shustova V,Mosca J,et al.Human mesenchymal stem cells induce unresponsiveness in preactivated but notnative alloantigen specific T cells.EXP Hematol.1999;27:122.)。 Immune characteristics, the MSC is not expressed or only negligible expression levels of MHC II class molecule, the MSC unrelated donor allogeneic lymphocytes do not cause reactions, alloimmunization reactions can be reduced (1.McIntosh K, Klyushnenkova E, Shustova V, et al.Suppression ofalloreactive T cell reaponse by human mesenchymal stem cells involves CD8 + cells.Blood.1999; 94: 133a.2.Klyushnenkova E, Shustova V, Mosca J, et al.Human mesenchymal stem cells induce unresponsiveness in preactivated but notnative alloantigen specific T cells.EXP Hematol.1999; 27: 122).. 基于上述特征,使其从发现后迅速成为在细胞治疗、基因治疗中有效发挥作用的理想工程细胞,以及组织工程尤其是骨或软骨组织损伤修复中重要的种子细胞。 Based on the above features, from the discovery that it is rapidly becoming an effective role in cell therapy, gene therapy ideal engineered cells, and tissue engineering in particular bone or cartilage repair tissue damage important seed cells.

目前所报道的MSC主要来源于骨髓,采用密度梯度离心法获得。 MSC currently reported mainly derived from bone marrow obtained by density gradient centrifugation. 虽然分离方法简便,但供者取髓需经历一个比较痛苦的手术,并在取材过程中及取材后会有很高的感染机会;由于人体骨髓中MSC的含量极其稀少,每105~106个单个核细胞中大约只有1个,且随着年龄的增加,骨髓中MSC的数量、增殖和分化能力均显著下降(Rao MS,Mattson MP.Stem cells and aging:expanding the possibilities.Mech Ageing Dev.2001;122:713-734.),使其在研究和应用尤其是临床应用中受到限制。 Although the separation method is simple, but to be experienced marrow donors to take a more painful surgery and drawn during and after have drawn a very high chance of infection; due to the content of human bone marrow MSC is extremely rare, 105 to 106 per individual nucleated cells only about 1, and with increasing age, the number of bone marrow MSC, proliferation and differentiation were significantly decreased (Rao MS, Mattson MP.Stem cells and aging: expanding the possibilities.Mech Ageing Dev.2001; 122: 713-734), it is limited in research and clinical applications, especially applications.

最近,已有具有干祖细胞特性的间充质细胞自包括骨、软骨、肌肉、腱等组织中分离出来(1.Ralf H.Isolation of Primary and Immortalized CD34-Hematopoietic and Mesenchymal Stem Cells from Various Sources.StemCells.2000;18:1-9.2.Deans RJ,Moseley AB.Mesenchymal stem cells:biology andpotential clinical uses.Exp Hematol.2000;28:875-884.),这些组织有一个共同的特征就是来源于胚胎发育期的中胚层。 Recently, there are stem and progenitor cells having the characteristics of mesenchymal cells isolated from the group comprising (1.Ralf H.Isolation of Primary and Immortalized CD34-Hematopoietic and Mesenchymal Stem Cells from Various Sources of bone, cartilage, muscle, tendon and other tissues. StemCells.2000; 18: 1-9.2.Deans RJ, Moseley AB.Mesenchymal stem cells: biology andpotential clinical uses.Exp Hematol.2000; 28:. 875-884), these organizations have a common feature is derived from the embryo mesoderm period.

起源于胚胎发育期胚外中胚层的胎盘是由间质、血管及滋养细胞组成,含有大量的间充质成分。 Placental origin extraembryonic mesoderm in the developing embryo are stroma, blood vessels and trophoblast cells, it contains a large amount of mesenchymal components. 在胚胎发育时期,胚外中胚层的多能干细胞可能有少量的未被招募发生分化,而以静止形式存在于胎盘组织中,如果能从胎盘中分离培养出MSC,将为实验研究和临床应用开辟一个崭新而丰富的来源。 During embryonic development, extraembryonic mesoderm pluripotent stem cells may not offer a small amount of differentiation occurs, and is present in the placenta in an immobilized form, if isolated from the placenta cultured the MSC, for experimental and clinical study open up a new and rich source. 2000年美国血液学年会,Jaroscak等报道采用酶消化法,将胎盘组织用手术剪刀剪碎,加组织酶消化一定时间后,过滤获得细胞悬液,接种于培养基中,经过一段时间培养后,可见培养瓶中有贴壁细胞,继续培养,获得的胎盘贴壁细胞采用流式细胞术检测细胞表面标志,结果显示获得的胎盘贴壁细胞与骨髓MSC在细胞表面标志染色特性上没有明显区别,但胎盘贴壁细胞是异质性的,细胞表面标志SH2,SH3,SH4等为阳性;不表达CD45,PECAM,提示其为非造血细胞及内皮细胞,说明胎盘中存在MSC成分(Jaroscak J,Smith T,Haynesworth S,et al.Preliminary characterization of the surface stainingof placental derived adherent cells:A potential new souece of stroma for umbilicolcord blood(UCB)expansion.THE AMERICAN SOCIETY OFHEMATOLOGY,December 1-5,2000.)。 2000 Annual Meeting American blood, Jaroscak reported by enzyme digestion, the placental tissue minced with surgical scissors, plus some time organized enzymatic digestion, the cell suspension obtained was filtered, seeded in the culture medium, the culture over time, flasks have seen adherent cells, cultured, placental adherent cells obtained by flow cytometry cell surface markers, the results obtained show placental adherent cells with bone marrow MSC no significant difference on the cell surface marker staining characteristics, However, placental adherent cells are heterogeneous cell surface markers SH2, SH3, SH4, etc. positive; do not express CD45, PECAM, suggesting that non-hematopoietic cells and endothelial cells, indicating that MSC component (Jaroscak J placenta present, Smith T, Haynesworth S, et al.Preliminary characterization of the surface stainingof placental derived adherent cells: A potential new souece of stroma for umbilicolcord blood (UCB) expansion.THE AMERICAN SOCIETY OFHEMATOLOGY, December 1-5,2000).. 2002年日本学者Watanabe等采用酶消化法或组织培养法结合流式细胞术分选法从胎盘中分离出MSC,具体方法主要为将胎盘组织剪碎,组织酶消化或直接组织块培养,流式细胞仪分选所需细胞,继续培养一段时间后,进行细胞生物学鉴定并诱导细胞向成脂肪、成骨和成软骨分化,进一步诱导细胞向神经细胞分化(Watanabe N,Igura K,Nagamura-Inoue T,et al.Multilineage potential of human placenta-derivedmesenchymal cells.THE AMERICAN SOCIETY OF HEMATOLOGY,December 1-5,2002)。 Japanese scholar Watanabe et al., 2002 using a combination of flow cytometry sorting out the MSC isolated from the placenta, the placenta is a particular method mainly minced tissue, tissue enzymatic digestion or explant culture directly, or flow enzyme digestion tissue culture after the desired cell sorting cytometer, cultured for a period of time, for cell biology to identify and induce adipogenic, osteogenic and chondrogenic differentiation, induce further differentiation of the cells into neural cells (Watanabe N, Igura K, Nagamura-Inoue T, et al.Multilineage potential of human placenta-derivedmesenchymal cells.THE AMERICAN SOCIETY oF HEMATOLOGY, December 1-5,2002). 但是上面所述的分离方法均较为繁琐。 However, the above separation process are more complicated.

发明内容 SUMMARY

本发明的目的是公开一种简便的分离人胎盘间充质干细胞(MSC)的方法,该方法包括以下步骤: Object of the present invention is disclosed a simple method for isolating human placenta mesenchymal stem cells (MSC), the method comprising the steps of:

按照胎盘的解剖结构,在无菌条件下,自脐血管插入导管形成循环液体灌流系统。 According to the anatomy of the placenta, under sterile conditions, insertion of the catheter is formed from the umbilical cord perfusion liquid circulation system. 用含肝素的DMEM或IMDM培养基作为灌流液。 With DMEM or IMDM medium containing heparin as a perfusate. 先用灌流液通过动静脉循环将残留的血液洗出,然后用灌流液孵育。 First perfusate circulating through the arteriovenous washed out residual blood, and then incubated with the perfusate. 采用密度梯度离心法从灌流液中分离单个核细胞,洗涤后用MSC培养基悬浮所获细胞,进行培养。 By density gradient centrifugation mononuclear cells from perfusate, cells were washed with MSC medium suspension obtained, cultured. 待散在细胞形成克隆后,将各克隆细胞挑出用MSC培养基分别培养,细胞80~90%融合后,用胰酶消化传代,将传至3代以上的细胞于液氮中冻存,并进行细胞的生物学特性及多向分化潜能鉴定。 After the formation of scattered cells after cloning, the cells of each clone were picked culture with MSC medium, cells were 80-90% confluent, trypsinized passage, spread over 3 generations of cell cryopreservation in liquid nitrogen, and and the biological properties of the cell pluripotency identified.

细胞的生物学特性鉴定进行了细胞生长特点及形态学特点鉴定,流式细胞术鉴定MSC表面标志,胎盘MSC细胞周期的分析,胎盘MSC生长曲线的绘制及对数生长期倍增时间的测定。 Biological Characterization of cell growth cells were identified characteristics and morphological characteristics, surface marker rendering MSC, MSC cell cycle analysis placental, placental MSC measured growth curve of the logarithmic growth phase and the doubling time of the identification by flow cytometry.

胎盘MSC多向分化潜能鉴定进行了成脂肪诱导、成骨诱导和成软骨诱导。 Multi MSC placenta were adipogenic differentiation potential identification, osteogenic and chondrogenic.

实验结果说明,从胎盘中分离的贴壁细胞与以往报道的骨髓间充质干细胞具有相同的生物学特性和多向分化能力,即从人胎盘中成功分离、纯化、获得间充质干细胞。 Experimental results show that, isolated from the placental adherent cells with conventional bone marrow reported mesenchymal stem cells have the same biological characteristics and differentiation capacity, i.e., successfully isolated from human placenta and purified to obtain mesenchymal stem cells.

目前MSC主要采用手术法抽取供者骨髓,密度梯度离心分离单个核细胞,接种于MSC专用培养基中培养获得。 The main surgical methods currently MSC donor bone marrow extraction, density gradient centrifugation, mononuclear cells were inoculated in culture medium obtained dedicated MSC. 该法操作繁琐,供者在取髓中和取髓后均有感染的可能。 The method complicated operation, takes the donor marrow and cord may have taken after infection. 发明人在总结以往分离组织细胞的基础上,利用胎盘特殊的解剖结构,采用灌流法,成功自胎盘中分离获得纯度较高的MSC。 On the basis of previous inventors isolated tissue cells, the placenta using specific anatomical structure, using the perfusion method, successfully isolated from placental obtain high purity MSC.

该法简便易行,且由于胎盘与脐血一样,细胞成份较幼稚,来源广泛,方便易得,因此本发明的方法在临床上将具有广泛的应用前景。 The method is simple, and because the placenta and umbilical cord blood, as compared with naive cell components, widely available, conveniently available and therefore the method of the present invention have a wide application prospect in clinical on.

附图说明 BRIEF DESCRIPTION

图1为细胞生长形态学镜下观察。 FIG 1 is a cell growth morphology were observed. 其中A为培养3d后可见散在的贴壁细胞B为7~10d形成克隆 Wherein A is scattered after 3d culture adherent cells B is 7 ~ 10d formed Cloning

C为经筛选克隆形成致密贴壁细胞D为瑞氏姬姆萨染色图2为流式细胞术鉴定MSC表面标志结果。 C to form a dense clones screened adherent cells D of Wright Giemsa staining flow cytometry Figure 2 is the results of surface markers identified MSC.

图3为胎盘MSC细胞周期的分析结果。 3 is the analysis result MSC placental cell cycle.

其中P3为培养第三代细胞的DNA含量,分析细胞周期,可见大部分细胞处于静止期(G0/G1期,96.66%),极少细胞处于增殖期(S期,3.25%)。 Wherein P3 is a third generation culture cell DNA content, cell cycle analysis, showing that most of the cells in stationary phase (G0 / G1 phase, 96.66%), few cells in the proliferative phase (S phase, 3.25%).

P6为培养第六代的细胞的DNA含量,分析细胞周期,可见大部分细胞处于静止期(G0/G1期,96.35%),极少细胞处于增殖期(S期,2.54%)。 P6 is the sixth generation of DNA content in the culture of cells, cell cycle analysis, showing that most of the cells in stationary phase (G0 / G1 phase, 96.35%), few cells in the proliferative phase (S phase, 2.54%).

图4为细胞生长曲线图。 FIG 4 is a graph showing the cell growth.

图5为胎盘MSC多向分化潜能鉴定的成脂肪诱导细胞图。 FIG 5 is a placental MSC pluripotent cells identified adipogenic FIG.

其中A为诱导3d,对照组细胞形态B为诱导3d,实验组细胞形态发生改变CD为诱导7d,可见胞浆内脂滴形成EF为油红染色脂滴被特异性染成红色图6为胎盘MSC多向分化潜能鉴定的成骨诱导细胞图。 Wherein A is induced 3d, group B was induced cell morphology 3d, experimental group induced morphological changes as CD 7d, visible lipid droplets within the cytoplasm red staining is formed as an oil EF is specifically stained lipid droplets red placental 6 FIG MSC plurality induce cell differentiation identified osteogenic potential.

其中A为诱导1w,对照组细胞形态B为诱导1w,实验组细胞形态发生改变C为诱导2w,原位染色显示碱性磷酸酶表达阳性D为诱导2w,涂片染色显示碱性磷酸酶表达阳性EF为诱导4w,von Kossa染色显示骨结节形成图7为胎盘MSC多向分化潜能鉴定的成软骨诱导细胞图。 Wherein A is induced 1w, B cell morphology in the control group as induced 1w, experimental group C is induced morphological changes 2w, in situ expression of alkaline phosphatase positive staining of induced D 2w, expression of alkaline phosphatase staining smears to induce positive EF 4w, von Kossa staining osteonodosity FIG. 7 MSC plurality FIG induce cell differentiation potential identification cartilage placenta.

其中A为诱导2w,对照组细胞Alcian blue染色B为诱导2w,实验组细胞Alcian blue染色 Wherein A is induced 2w, control cells is induced Alcian blue staining B 2w, Alcian blue stained cells in the experimental group

CD为高倍镜下可见II型胶原形成细胞外基质被特异性染成蓝色具体实施方式实施例一 胎盘MSC分离培养方法按照胎盘的解剖结构,在无菌条件下,自脐血管(2条脐动脉和1条脐静脉)插入聚乙烯导管形成循环液体灌流系统,用含肝素的DMEM或IMDM培养基作为灌流液。 CD is a high-powered microscope, type II collagen is an extracellular matrix formed specifically stained blue DETAILED DESCRIPTION Example embodiments placental MSC Isolation and culture of placenta according to the anatomical structure, under sterile conditions, from the umbilical cord (umbilical 2 an artery and umbilical vein) form a polyethylene catheter inserted into the perfusion liquid circulation system, using DMEM or IMDM medium containing heparin as a perfusate. 先用灌流液通过动静脉循环将残留的血液在产后0.5~2.5h内洗出,然后用100~250ml灌流液20~25℃孵育12~24h。 Blood perfusion fluid through the first with arteriovenous loop remaining within 0.5 ~ 2.5h post-natal washed out, followed by 100 ~ 250ml perfusate 20 ~ 25 ℃ incubated for 12 ~ 24h. 采用密度梯度离心法从灌流液中分离单个核细胞,洗后用MSC培养基悬浮所获细胞,37℃、5%CO2全湿条件下培养24~48h后,换液去除未贴壁悬浮细胞,继续培养,每隔3~4d换液一次。 By density gradient centrifugation mononuclear cells from perfusate, washed with MSC medium obtained cell suspension, 37 [deg.] C, 5% CO2 for culturing all wet condition after 24 ~ 48h, the medium was changed removal of non-adherent cells in suspension, culture was continued every 3 ~ 4d medium was changed. 待早期散在细胞形成克隆后,将各克隆细胞挑出用MSC培养基分别培养,细胞80~90%融合后,用0.25%胰酶消化,1∶3或1∶4传代,将传至3代以上的细胞用于以下实验,并于液氮中冻存部分细胞以备后用。 After the dispersion is formed early in the cell clones, the cells of each clone were picked culture with MSC medium, the cells were 80-90% confluent, was digested with 0.25% trypsin, 1:3 or 1:4 passage, spread substituting 3 cells used in the above experiments, some cells and frozen in liquid nitrogen for later use.

实施例二 胎盘MSC的生物学特性鉴定一、细胞生长特点及形态学特点通过实施例一方法培养,约需3d,镜下可见散在呈纺锤形贴壁细胞,7~10d左右形成放射状克隆,将各克隆分别挑出至24孔板单独培养。 Biological characteristics of the second embodiment of placental a MSC identification, morphological characteristics of cell growth characteristics and a method of culture by way of example, about 3d, microscope, scattered fusiform adherent cells, cloning is formed radially about 7 ~ 10d, the individual clones were picked into 24 well culture plates alone. 待细胞纯化至3代后,用于细胞生物学特性检测。 After cells were purified through 3 generations, cell biology for feature detection. 培养过程中,发现这种细胞形态相对均一,增长速度快,贴壁速度快,易被胰酶消化,传代至15代以上,其形态及生长特点亦无明显改变。 During the culture, cell morphology was found that a relatively uniform, rapid growth, high speed adherent, susceptible to trypsin digestion, passaged over 15 passages, morphology and growth characteristics which no significant change. 见图1。 see picture 1.

二、流式细胞术鉴定MSC表面标志分别取第3、6、9、12、15代细胞,流式细胞术检测表面标志,动态观察培养过程中表面标志的变化。 Second, the identified MSC surface markers by flow cytometry 3,6,9,12,15 take the first passage cells were detected by flow cytometry surface markers, the dynamic changes of surface markers was observed during the culture. 消化收集细胞,计数后取5×106个,分装10管;PBS洗一次,1500rpm离心10min;弃上清,残留100~200μl,吹打混匀细胞;加入PE标记的CD34、CD73、CD166抗体和FITC标记的CD45、CD105、HLA-DR、UEA-1抗体和间接标记的CD29、CD44抗体各10μl,并设一管为空白对照,一管为二抗对照;4℃,避光反应30min;PBS洗一次,1500rpm离心10min;直接标记的细胞弃上清,加入200μl PBS吹打混匀细胞,200μl的1%多聚甲醛固定,置4℃待测,3d内上流式细胞仪检测。 Cells were harvested, counted and taken 5 × 106 th, dispensing 10; PBS wash, centrifugation 1500rpm 10min; the supernatant was discarded, the residue 100 ~ 200μl, cells were mixed by pipetting; of CD34 PE-labeled added, CD73, CD166 antibody, and CD29, CD44 antibodies of each 10μl FITC-labeled CD45, CD105, HLA-DR, UEA-1 antibody and the indirect labeling, and a set of blank, a secondary antibody control; 4 ℃, dark reaction 30min; PBS washed once by centrifugation 1500rpm 10min; directly labeled cell supernatant was discarded, cells were mixed by pipetting 200μl PBS was added, 200 l of 1% of paraformaldehyde, at 4 ℃ measured by flow cytometry on the 3d. 间接标记的细胞需与二抗反应后,重复上述步骤后上机检测。 After testing on the machine for an indirect labeling cells with secondary antibody reaction, repeat the above steps.

流式细胞仪检测细胞的表面标志,动态观察来源于各克隆的第3、6、9、12、15代细胞,无明显改变。 Flow cytometry of cell surface markers, each clone derived from the dynamic observation of 3,6,9,12,15-generation cells, no significant change. 不表达造血细胞表面标志即CD34(HSPC及内皮细胞阳性)、CD45(白细胞阳性)、HLA-DR(MHC-II类分子)持续阴性,CD29和CD44(纤维蛋白和透明脂酸盐的受体,基质细胞表达),CD73(即SH-3、4)、CD105(即SH-2)、CD166(间充质细胞表达)和UEA-1(内皮细胞的表面标志)持续为阳性。 Did not express hematopoietic cell surface markers i.e. CD34 (HSPC and positive endothelial cells), CD45 (leukocyte positive), HLA-DR (MHC-II molecules) continuously negative, CD29 and of CD44 (fibrin transparent and lipid acceptor salt, stromal cells), CD73 (i.e., SH-3,4), CD105 (i.e., SH-2), mesenchymal cells express CD166 (room), and UEA-1 (endothelial cell surface marker) continue to be positive. 经3代以上传代后,细胞成分均一,纯度在95%以上。 By substituting the passaged 3, uniform cellular components, more than 95% purity. 见图2。 See Figure 2.

三、胎盘MSC细胞周期的分析细胞长至80-90%融合时,消化收集细胞约1×106个,PBS洗一次,加入70%的乙醇固定,4℃待测。 Third, analysis of cell cycle placental MSC cells grown to 80-90% confluence, the cells were harvested from about 1 × 106 th, PBS washed once, fixed with 70% ethanol, 4 ℃ tested. 检测时,先离心去乙醇,再用PBS洗一次,加入RNaseI 500u,37℃反应30min,PBS洗一次,加入碘化丙啶(PI,终浓度50μg/ml)1ml,室温避光反应20min,上机检测细胞DNA含量。 Detecting, prior to ethanol by centrifugation, washed once with PBS, was added RNaseI 500u, the reaction 30min 37 ℃, PBS wash, propidium iodide was added (the PI, final concentration 50μg / ml) 1ml, dark at room temperature the reaction 20min, the detects cellular DNA content.

经测定第3代和第6代细胞的DNA含量,细胞周期分析,G0/G1期、S期和G2M期所占比例分别为96.35%、96.66%,1.11%、0.09%,和2.54%、3.25%。 Determination by DNA 3rd and 6th generation, cell cycle analysis, G0 / G1 phase, S phase, and G2M phase proportion were 96.35%, 96.66%, 1.11%, 0.09% and 2.54%, 3.25 %. 结果提示体外培养的细胞具有典型的干细胞增殖特点,即只有少数细胞处于活跃的增殖期(1.11%、0.09%),大部分的细胞处于静息期(96.35%、96.66%)。 Cells cultured in vitro results suggest that the typical characteristics of stem cell proliferation, i.e., only a few cells in the active proliferation stage (1.11%, 0.09%), most of the cells are in a resting phase (96.35%, 96.66%). 见图3。 See Figure 3.

四、胎盘MSC生长曲线的绘制及对数生长期倍增时间的测定取对数生长期细胞,消化计数,以10%FBS的LG-DMEM培养基制成细胞悬液(2×104/ml),24孔板中每孔接种0.5ml,37℃,5%CO2,饱和湿度下培养。 Fourth, the growth curve plotted placental MSC and measuring the doubling time of the logarithmic growth phase logarithmic growth phase cells, digested count to LG-DMEM medium with 10% FBS cell suspension (2 × 104 / ml), 24 well plates seeded per well 0.5ml, 37 ℃, 5% CO2, saturated humidity culture. 每天取3复孔,台盼蓝染色后计数活细胞数,计算平均值,连续观察7d。 3 wells taken daily, after trypan blue staining viable cells, calculate the average continuous observation 7d. 以培养时间为横轴,细胞数为纵轴,绘制细胞生长曲线。 In incubation time as the horizontal axis, vertical axis is number of cells, cell growth curve. 以Patterson公式计算细胞在对数生长期的倍增时间,即Td=Tlg2/Lg(Nt/No),Td:倍增时间(h),T:细胞由No增至Nt所用的时间(h),N:细胞数。 Patterson formula in cell doubling time of logarithmic growth phase, i.e., Td = Tlg2 / Lg (Nt / No), Td: doubling time (h), T: the cells used Nt No time (h) increase, N : the number of cells.

通过每天细胞计数的结果绘制细胞生长曲线,计算倍增时间。 Results daily cell counts cell growth curve, doubling time is calculated. 由细胞生长曲线可以看出,细胞在第2-4d处于指数生长期。 As it can be seen from the growth curve, the first cell in the exponential growth phase 2-4d. 根据公式计算出第5代细胞在指数生长期的倍增时间分别为22.6h。 Doubling time is calculated according to the formula in the fifth generation of the exponential growth phase cells were 22.6h. 见图4。 See Figure 4.

实施例三 胎盘MSC多向分化潜能鉴定一、成脂肪诱导3代以上MSC,按1×105/孔接种于六孔板,标准培养基培养24h后换用含10%经筛选FBS的高糖DMEM,并加入地塞米松1μM、消炎痛200μM、IBMX 0.5mM、胰岛素10μg/ml,每3d半量换液,共诱导2w,油红染色鉴定脂滴形成。 Third Embodiment placental MSC identified a pluripotent, three or more generations adipogenic MSC, by 1 × 105 / well were seeded in six-well plates, standards were cultured in high glucose DMEM transducer after 24h with 10% FBS was screened , and [mu] M dexamethasone, indomethacin 200μM, IBMX 0.5mM, insulin 10μg / ml, medium was changed every half-3d, were induced 2w, oil red staining lipid droplets.

在含10%经筛选FBS的DMEM-HG,加入地塞米松1μM、消炎痛200μM、IBMX 0.5mM、胰岛素10μg/ml培养3d,细胞即发生形态改变,由纺锤形的成纤维细胞样逐渐收缩变短,90%以上细胞成为立方形或多角形;连续培养7d,镜下可见细胞内有微小脂滴出现,随着培养时间的延长,脂滴逐渐增大并融合,至培养2w时,可见融合成团的脂滴充满整个细胞。 In DMEM-HG containing 10% FBS was screened, [mu] M dexamethasone, indomethacin 200μM, IBMX 0.5mM, Insulin 10μg / ml culture 3d, i.e., cell morphology changes from fusiform fibroblast-like gradually shrink and short, more than 90% of the cells become cubic or polygonal; 7D continuous culture, the cells have micro microscope lipid droplet, with time culture, and fusion of lipid droplets is gradually increased, to the culture 2w, visible fusion lipid droplets agglomerated fill the cells. 油红O染色可见细胞内产生的脂肪被特异性染成红色。 Oil Red O staining produced in fat cells are specifically stained red. 见图5。 See Figure 5.

二、成骨诱导3代以上MSC,按1×105/孔接种六孔板,用标准培养基培养24h后换用含10%经筛选FBS的DMEM-HG并加入地塞米松0.1μM、抗坏血酸磷酸盐50μM、β-磷酸甘油10mM,每3天半量换液,共诱导2-4周。 Two, three or more generations osteogenic the MSC, by 1 × 105 / well were seeded in six-well plates, cultured with standard medium 24h after switching to DMEM-HG containing 10% FBS was screened and 0.1 uM dexamethasone, ascorbic acid salts 50μM, β- 1OmM glycerophosphate, medium was changed every 3 days and a half, a total of 2-4 weeks induction. 碱性磷酸酶染色鉴定成骨细胞形成,Von Kossa染色鉴定骨结节形成。 Alkaline phosphatase staining of osteoblasts formed, Von Kossa staining bone nodule formation.

在含10%经筛选FBS的DMEM-HG,加入地塞米松0.1μM、抗坏血酸磷酸盐50μM、β-磷酸甘油10mM培养1w,细胞形态发生明显的改变,由纺锤形的成纤维细胞样变为多角形,类似于神经元细胞样,细胞周边出现长丝状突出,并可向周围延伸。 In DMEM-HG containing 10% FBS was screened, 0.1 uM dexamethasone, ascorbic acid phosphate 50μM, β- glycerophosphate 10mM culture 1w, cell morphology changed obviously, the fusiform fibroblast-like multi becomes angled, similar to neuron-like cells, cells appear around filamentary projections, and may extend to the periphery. 继续培养2w以上后,细胞基质中出现钙化斑,矿化物逐渐出现,并且开始形成多层小结结构,至培养4w后,可见明显钙化结节。 After culture was continued 2w above, matrix calcification occurred, mineralization occurs gradually, and nodules started to form a multilayer structure, to the culture after 4w, calcified nodules were. 2w碱性磷酸酶染色呈强阳性反应,达到95%以上,而未加以诱导的对照组则大部分为阴性,只有不到5%显示为弱阳性,表明细胞已向成骨细胞转化。 2w alkaline phosphatase staining was strongly positive reaction, more than 95%, whereas the control group were induced to be mostly negative, less than 5% showed weakly positive, indicating that the cells had been transformed osteoblasts. von Kossa染色可将骨结节中沉积的钙染成黑色,诱导组可见大量的黑色骨结节,有明显的立体结构,而对照组在任何时间都没有阳性反应。 The von Kossa staining of calcium deposition in bone nodule dyed black, black shows a large group induced bone nodule, a clear three-dimensional structure, while the control group at any time no positive reaction. 见图6。 See Figure 6.

三、成软骨诱导3代以上细胞,低速离心使细胞在试管中形成微团,在含2.5%FBS的DMEM-HG中加入胰岛素、转铁蛋白、亚硒酸钠各6.25μg/ml,BSA1.25μg/ml,丙酮酸钠1mM/L,抗坏血酸磷酸37.5μg/ml,TGF-β150ng/ml,每3d半量换液,连续培养2w。 Third, more than three generations of chondrogenic cells, the cells by low speed centrifugation micelles formed in the tube, was added in DMEM-HG insulin containing 2.5% FBS, the transferrin, sodium selenite each 6.25μg / ml, BSA1. 25μg / ml, sodium pyruvate 1mM / L, ascorbic acid 37.5μg / ml, TGF-β150ng / ml, the amount was changed every half-3d, continuous culture 2w.

诱导2w后将细胞微团打散涂片,Alcian blue染色可见II型胶原形成细胞外基质呈蓝色,对照组无蓝染。 After induction cells 2w micelles break up smears, Alcian blue staining collagen type II extracellular matrix is ​​formed blue, the blue dye in control group. 见图7。 See Figure 7.

Claims (10)

1.一种分离人胎盘间充质干细胞的方法,包括以下步骤:(1)在无菌条件下,自脐血管插入导管形成循环液体灌流系统;(2)先用灌流液通过动静脉循环将残留的血液洗出,然后用灌流液孵育;(3)从灌流液中分离单个核细胞,进行培养;(4)用胰酶消化传代进行鉴定。 Mesenchymal stem cells isolated human placental 1. A method, comprising the steps of: (1) under sterile conditions, insertion of the catheter is formed from the umbilical cord perfusion liquid circulation system; (2) first with perfusion fluid through the arterial and venous circulation wash out residual blood, and then incubated with a perfusate; (3) isolating mononuclear cells from perfusate, culturing; (4) were trypsinized and passaged identified.
2.根据权利要求1所述的方法,其特征在于所述的灌流液为含抗凝剂的培养基。 2. The method according to claim 1, wherein said perfusate medium containing anticoagulant.
3.根据权利要求2所述的方法,其特征在于所述的抗凝剂为肝素。 3. The method according to claim 2, wherein said anticoagulant is heparin.
4.根据权利要求2所述的方法,其特征在于所述的培养基为DMEM或IMDM培养基。 4. The method according to claim 2, wherein said medium is a DMEM or IMDM medium.
5.根据权利要求1所述的方法,其特征在于从灌注液中分离单个核细胞采用离心的方法实现。 5. The method according to claim 1, characterized in that the method of mononuclear cells from the centrifugal separating perfusate implemented.
6.根据权利要求5所述的方法,其特征在于从灌注液中分离单个核细胞采用密度梯度离心的方法实现。 6. The method according to claim 5, characterized in that the method of mononuclear cells isolated by density gradient centrifugation to achieve the perfusate.
7.根据权利要求1所述的方法,其特征在于从灌注液中分离的单个核细胞用MSC培养基进行培养。 7. The method according to claim 1, characterized in that the perfusate from isolated mononuclear cells were cultured with MSC medium.
8.根据权利要求1所述的方法,其特征在于分离的细胞鉴定包含生物学特性鉴定及多向分化潜能鉴定。 8. The method according to claim 1, characterized in that the identification of cells comprising an isolated biological characterization and identification of pluripotency.
9.根据权利要求8所述的方法,其特征在于分离的细胞的生物学特性鉴定包含细胞生长特点及形态学特点鉴定,流式细胞术鉴定MSC表面标志,胎盘MSC细胞周期的分析,胎盘MSC生长曲线的绘制及对数生长期倍增时间的测定。 9. The method according to claim 8, characterized in that the biological characterization of isolated cells comprising cell identification morphological features and growth characteristics, surface marker analysis MSC, MSC placental cell cycle identified by flow cytometry, placenta MSC determination of growth curve and doubling time of the logarithmic growth phase.
10.根据权利要求8所述的方法,其特征在于分离的细胞的多向分化潜能鉴定包含成脂肪诱导、成骨诱导和成软骨诱导。 10. The method according to claim 8, characterized in that the identification of pluripotent cells comprising the isolated adipogenic, osteogenic and chondrogenic.
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