中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2007年
24期
4824-4827
,共4页
背景:一些研究提示,胎盘可以作为一种新的间充质干细胞来源.胎盘组织中分离出的贴壁细胞与骨髓间充质干细胞有相似形态和细胞表面标志,具有分化为成骨细胞以及神经细胞的能力.目的:探索一种新的获取间充质干细胞的来源及方法.设计:观察性实验.单位:无锡市第三人民医院.材料:胎盘(我院妇产科剖腹产手术中胎盘,经家属同意并经院伦理委员会通过签署知情同意书).培养基;sABC试剂盒、DAB显色试剂盒;羊抗鼠-FITC;重组人碱性成纤维细胞生长因子;兔抗人神经元特异性烯醇化酶;兔抗人胶质纤维酸性蛋白.方法:实验于2005-05/2006-08在无锡第三人民医院细胞与分子生物学研究室完成.消化胎盘实体组织,贴壁培养细胞,观测形态,绘制出各代PDMCs的生长曲线.取原代培养细胞1和7 d时的上清液,用化学发光法测定β-HCG含量.检测细胞表面抗原表达以及分化潜能.培养细胞在诱导分化24 h(神经细胞),2周(成骨细胞)后采用常规方法免疫细胞化学染色.显色后在常规显微镜或荧光显微镜下观察.主要观察指标:①形态学以及生长曲线的观察;②多分化潜能细胞培养上清液β-HCG测定、流式细胞仪检测多分化潜能细胞抗原表达;③多分化潜能细胞诱导分化和诱导分化后细胞免疫组织化学分析.结果:①多分化潜能细胞的原代培养情况:胎盘组织消化后获得细胞中仅有少量的贴壁细胞,经两周后逐渐形成扁平单层细胞,呈漩涡状生长或成簇生长,随着细胞密度的增加,胞体变得细长,形态类似成纤维细胞.②多分化潜能细胞的生长曲线分析结果:细胞在接种后的2～8 d为生长的潜伏期,细胞逐渐开始出现贴壁,无明显扩增;8 d以后细胞进入对数生长期,此时细胞增殖活跃,相差显微镜下观察细胞突起向周围伸展,出现两个核细胞分裂相的间充质干细胞多见,细胞密度增大,彼此相连;11～14 d,生长曲线逐渐进入平台期,MSCs铺满瓶底,细胞扩增趋缓,原代培养结束.③β-HCG测定结果:两个时间点培养上清液中均未检测到β-HCG表达.④多分化潜能细胞的表面抗原特性:多分化潜能细胞表达CD29,CD44和CD105,不表达CD34,CD45,CD19和CD106.⑤多分化潜能细胞的诱导分化结果:向神经细胞诱导24 h后,细胞形态明显改变,胞体回缩,胞核部分折光性增强,出现类似于树突及轴突样结构,染色可见神经元特异性烯醇化酶、胶质纤维酸性蛋白阳性.结论:胎盘组织中含有的多分化潜能细胞与骨髓间充质干细胞形态功能相似,胎盘可以作为获取间充质干细胞的一种有效来源.
揹景:一些研究提示,胎盤可以作為一種新的間充質榦細胞來源.胎盤組織中分離齣的貼壁細胞與骨髓間充質榦細胞有相似形態和細胞錶麵標誌,具有分化為成骨細胞以及神經細胞的能力.目的:探索一種新的穫取間充質榦細胞的來源及方法.設計:觀察性實驗.單位:無錫市第三人民醫院.材料:胎盤(我院婦產科剖腹產手術中胎盤,經傢屬同意併經院倫理委員會通過籤署知情同意書).培養基;sABC試劑盒、DAB顯色試劑盒;羊抗鼠-FITC;重組人堿性成纖維細胞生長因子;兔抗人神經元特異性烯醇化酶;兔抗人膠質纖維痠性蛋白.方法:實驗于2005-05/2006-08在無錫第三人民醫院細胞與分子生物學研究室完成.消化胎盤實體組織,貼壁培養細胞,觀測形態,繪製齣各代PDMCs的生長麯線.取原代培養細胞1和7 d時的上清液,用化學髮光法測定β-HCG含量.檢測細胞錶麵抗原錶達以及分化潛能.培養細胞在誘導分化24 h(神經細胞),2週(成骨細胞)後採用常規方法免疫細胞化學染色.顯色後在常規顯微鏡或熒光顯微鏡下觀察.主要觀察指標:①形態學以及生長麯線的觀察;②多分化潛能細胞培養上清液β-HCG測定、流式細胞儀檢測多分化潛能細胞抗原錶達;③多分化潛能細胞誘導分化和誘導分化後細胞免疫組織化學分析.結果:①多分化潛能細胞的原代培養情況:胎盤組織消化後穫得細胞中僅有少量的貼壁細胞,經兩週後逐漸形成扁平單層細胞,呈漩渦狀生長或成簇生長,隨著細胞密度的增加,胞體變得細長,形態類似成纖維細胞.②多分化潛能細胞的生長麯線分析結果:細胞在接種後的2～8 d為生長的潛伏期,細胞逐漸開始齣現貼壁,無明顯擴增;8 d以後細胞進入對數生長期,此時細胞增殖活躍,相差顯微鏡下觀察細胞突起嚮週圍伸展,齣現兩箇覈細胞分裂相的間充質榦細胞多見,細胞密度增大,彼此相連;11～14 d,生長麯線逐漸進入平檯期,MSCs鋪滿瓶底,細胞擴增趨緩,原代培養結束.③β-HCG測定結果:兩箇時間點培養上清液中均未檢測到β-HCG錶達.④多分化潛能細胞的錶麵抗原特性:多分化潛能細胞錶達CD29,CD44和CD105,不錶達CD34,CD45,CD19和CD106.⑤多分化潛能細胞的誘導分化結果:嚮神經細胞誘導24 h後,細胞形態明顯改變,胞體迴縮,胞覈部分摺光性增彊,齣現類似于樹突及軸突樣結構,染色可見神經元特異性烯醇化酶、膠質纖維痠性蛋白暘性.結論:胎盤組織中含有的多分化潛能細胞與骨髓間充質榦細胞形態功能相似,胎盤可以作為穫取間充質榦細胞的一種有效來源.
배경:일사연구제시,태반가이작위일충신적간충질간세포래원.태반조직중분리출적첩벽세포여골수간충질간세포유상사형태화세포표면표지,구유분화위성골세포이급신경세포적능력.목적:탐색일충신적획취간충질간세포적래원급방법.설계:관찰성실험.단위:무석시제삼인민의원.재료:태반(아원부산과부복산수술중태반,경가속동의병경원윤리위원회통과첨서지정동의서).배양기;sABC시제합、DAB현색시제합;양항서-FITC;중조인감성성섬유세포생장인자;토항인신경원특이성희순화매;토항인효질섬유산성단백.방법:실험우2005-05/2006-08재무석제삼인민의원세포여분자생물학연구실완성.소화태반실체조직,첩벽배양세포,관측형태,회제출각대PDMCs적생장곡선.취원대배양세포1화7 d시적상청액,용화학발광법측정β-HCG함량.검측세포표면항원표체이급분화잠능.배양세포재유도분화24 h(신경세포),2주(성골세포)후채용상규방법면역세포화학염색.현색후재상규현미경혹형광현미경하관찰.주요관찰지표:①형태학이급생장곡선적관찰;②다분화잠능세포배양상청액β-HCG측정、류식세포의검측다분화잠능세포항원표체;③다분화잠능세포유도분화화유도분화후세포면역조직화학분석.결과:①다분화잠능세포적원대배양정황:태반조직소화후획득세포중부유소량적첩벽세포,경량주후축점형성편평단층세포,정선와상생장혹성족생장,수착세포밀도적증가,포체변득세장,형태유사성섬유세포.②다분화잠능세포적생장곡선분석결과:세포재접충후적2～8 d위생장적잠복기,세포축점개시출현첩벽,무명현확증;8 d이후세포진입대수생장기,차시세포증식활약,상차현미경하관찰세포돌기향주위신전,출현량개핵세포분렬상적간충질간세포다견,세포밀도증대,피차상련;11～14 d,생장곡선축점진입평태기,MSCs포만병저,세포확증추완,원대배양결속.③β-HCG측정결과:량개시간점배양상청액중균미검측도β-HCG표체.④다분화잠능세포적표면항원특성:다분화잠능세포표체CD29,CD44화CD105,불표체CD34,CD45,CD19화CD106.⑤다분화잠능세포적유도분화결과:향신경세포유도24 h후,세포형태명현개변,포체회축,포핵부분절광성증강,출현유사우수돌급축돌양결구,염색가견신경원특이성희순화매、효질섬유산성단백양성.결론:태반조직중함유적다분화잠능세포여골수간충질간세포형태공능상사,태반가이작위획취간충질간세포적일충유효래원.
BACKGROUND: Some researches suggest that placenta is regarded as a new source of mesemchymal stem cells (MSCs). Adherent cells derived from placenta tissue have similar morphological characteristics and surface markers to MSCs; meanwhile, they can differentiate into osteoblasts and nerve cells.OBJECTIVE: To find a new source and way for MSCs separation.DESIGN: Observational study.SETTING: The Third People's Hospital of Wuxi.MATERIALS: Placenta was sourced from cesarean in our department of obstetrics and gynecology and provided confirmed consent from the relatives and ethics committee. The main reagents contained culture medium, sABC kit, DAB staining kit, caprine-anti-rat FITC, recombinant human basic fibroblast growth factor (rh-bFGF), rabbit-anti-human glial fibriliary acidic protein (GFAP).METHODS: The experiment was carried out in the Laboratory of Cells & Molecular Biology, the Third People's Hospital of Wuxi from May 2005 to August 2006. Placenta entity tissue was digested, adherent cells were cultured, morphological characteristics were observed, and growth curves of placenta-derived multipotent cells (PDMCs) in various generations were drawn. On the 1st and 7th days, supernatant was derived from cells in primary culture to measure content of β-glycerophosphate disodium (β-HCG) with chemiluminescence technique. In addition, expression of surface antigen and differentiating potency were detected at the same time. At 24 hours (at phase of nerve cells) and 2 weeks (at phase of osteoblasts) after inducible differentiation, cultured cells were dealt with routine immunocytochemical stain, and then they were observed under routine microscope or fluorescence microscope.MAIN OUTCOME MEASURES: ① Morphological indexes and growth curves of PDMCs; ② measurement of β-HCG in supernatant of PDMCs and expression of antigen of PDMCs with flow cytometer; ③ immunohistochemical analysis of PDMCs during and after inducible differentiation.RESULTS: ① Primary culture of PDMCs: After digestion of placenta tissue, a few of adherent cells were obtained and gradually formed thin and flat monolayer cells two weeks later. The monolayer cells grew like whirlpool or cluster. With the increase of cell density, soma was slenderer and slenderer and like fibroblast. ② Growth curves of PDMCs: Growth latency ranged from 2 to 8 days after cell inoculation. During this period, adherence was observed gradually but not obviously amplified. Eight days later, cells entered log growth phase. During this period, proliferation was active and cell process surrounding extended under phase contrast microscope. A lot of MSCs were observed in division phase between two nuclei. In addition, density was increased and cells connected to each other. Within 11-14 days, growth curve gradually entered platform phase. MSCs covered the bottom of bottle, cells slowly expanded, and primary culture stopped. ③ Measurement of β-HCG: Expression of β-HCG was not detected in supernatant at two time points. ④ Characteristics of surface antigen of PDMCs: PDMCs could express CD29, CD44 and CD105, but not CD34, CD45,CD19 and CD106. ⑤ Inducible differentiation of PDMCs: At 24 hours after inducing to nerve cells, form of PDMCs obviously changed, soma rebounded, refraction of nucleus was partially reinforced, structure was similar to dendrite and axis-cylinder, and positive neuro-specific enolase and GFAP were observed after staining.CONCLUSION:Placenta tissue contains PDMCs whose morphological function is similar to MSCs; in addition, placenta is regarded as an effective source of MSCs.
