人气管粘膜上皮细胞抗菌多肽探讨

用酶低温消化获得的人气管上皮细胞,气-液界面无血清培养,细胞呈现复层生长,细胞角蛋白免疫组化染色呈阳性反应,可确认为上皮细胞。琼脂糖弥散法显示培养细胞表面分泌物对绿脓杆菌有明显的抗菌活性。随底层培养基NaCl浓度的增高,杀菌环直径逐渐减小。当NaCl浓度分别为0、20、40、80、160、320mmol／L时,     

人气管粘膜上皮细胞抗菌多肽探讨

目的：探讨人支气管肺泡灌洗液和体外气液界面无血清培养的人气管上皮细胞分泌液对绿脓杆菌的抗菌作用。方法：采用酸性尿素聚丙酰胺凝胶电泳（ＡＵ－ＰＡＧＥ）分析人支气管肺泡灌洗液阳离子蛋白成分：气液界面无血清培养人气管上皮细胞;电泳凝胶琼脂糖弥散法和琼脂糖弥散法检测支气管肺泡灌洗液及培养细胞分泌液的抗菌活性。结果：人支气管肺泡灌洗液和培养气管上皮细胞分泌液对绿脓杆菌有很强的抗菌作用;随着盐浓度增加,抗菌作     

人细支气管上皮细胞的ROCK激酶抑制剂培养体系扩增及气液相模型的建立

背景：人类原代肺脏上皮细胞在体外难以分离培养,表现为组织来源有限、细胞存活率低、增殖速度慢,缺乏上皮细胞表型分化能力等。目的：体外扩增人细支气管上皮细胞,建立其气液相分化模型,用于肺脏上皮细胞功能研究。方法：采用Pronase和DnaseⅠ联合消化法分离人细支气管上皮细胞。利用ROCK激酶抑制剂培养体系对其进行扩增,免疫荧光染色鉴定细胞类型。建立气液相培养模型,扫描电镜、相差显微镜和免疫荧光染色鉴定细胞分化类型。结果与结论：通过ROCK激酶抑制剂培养体系,体外成功培养扩增了人细支气管上皮细胞。扩增细胞经免疫荧光染色鉴定绝大部分都表达基底细胞标记角质蛋白14,提示人细支气管的基底细胞可能是肺脏上皮干细胞的主要亚群。同时,扩增细胞在气液相培养条件下分化成纤毛细胞和无纤毛柱状细胞,表明ROCK激酶抑制剂培养体系扩增的上皮细胞仍然保持干细胞的增殖分化能力,体外气液相培养模型可以促进人细支气管上皮细胞分化。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

人脐血干细胞分化为鼻黏膜纤毛上皮细胞

BACKGROUND:Damage to nasal ciliated epithelial cells can lead to a severe injury in nasal biological function. Compared with other adult stem cells, human umbilical cord blood stem cells have better differentiation potential. OBJECTIVE:To explore the feasibility of human umbilical cord blood stem cells differentiating into nasal ciliated epithelial cells through in vitro culture and induction techniques. METHODS: Normal and healthy umbilical cord blood samples were collected to isolate human umbilical cord blood stem cells, followed by identification and subculture in vitro. Umbilical cord blood stem cells at passage 3 were infected with recombinant adeno-associated virus carrying enhanced green fluorescent protein and cultured using air liquid interface culture method. Thereafter, PCR assay was employed for detecting MUCS expression in cultured stem cells at 1 and 2 weeks after induction, and immunofluorescent staining for FOXJ1 was performed at 3 weeks. RESULTS AND CONCLUSION:After subculture, passage 3 umbilical cord blood stem cells that could express stem cell surface markers were visible in a uniform shape and had good refraction. After 3 hours of gene transfection, green fluorescence issued from the passage 3 cells were visible, and the cell positive rate was up to 96.2% until 48 hours, indicating good transfection efficiency. RT-PCR findings showed that MUC8 mRNA had no expression in the umbilical cord blood stem cells, but expressed strongly in the nasal ciliated epithelial cells, whose expression was weak at 1 week of culture and increased at 2 weeks. Additionally, the positive expression of FOXJ1 red fluorescence was observed under the transfection of green fluorescent protein. These results suggest that human umbilical cord blood stem cells could differentiate into nasal epithelial cells under suitable conditions.     

人α—防御素HNP1基因在气管上皮细胞传染的表达研究

目的 探讨人α－防御素ＨＮＰ１基因在气管上皮细胞转染表达的可行性。方法 采用脂质体转染法将ＨＮＰ１ ｃＤＮＡ重组真核表达质粒ｐＢａｂｅ－Ｎｅｏ－ＨＮＰ１导入无血清培养的人、兔气管粘膜上皮细胞，利用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）法及免疫组化法，分别在核酸水平及蛋白质水平检测ＨＮＰ１的表达。结果 用ＲＴ－ＰＣＲ在人和兔的转染的上皮细胞总ＲＮＡ提取物中均扩增出１条３１９ｂｐ的ＤＮＡ片段，与ＨＮＰ１ｃ     

胰酶冷消化加刮刷法分离培养气管上皮细胞的实验研究

目的:改进气管上皮细胞的培养技术。方法:采用胰酶冷消化加刮刷法分离兔气管上皮细胞, 测定分离细胞的数量、纯度及成活率, 并与机械剥离加酶消化法、酶温消化加刮刷法进行比较。同时比较无血清与有血清培养方法对上皮细胞纯度、融合时间的影响。结果:(1)胰酶冷消化加刮刷法分离所得上皮细胞的数量[(8.217±0.443)×10⁶ cells/只]明显高于机械剥离加酶消化法[(2.198±0.533)×10⁶ cells/只], P＜0.01;成活率(91.730%±1.532%)明显高于机械剥离加酶消化法(86.29%±0.46%)和胰酶温消化加刮刷法(83.07%±1.80%), 均P＜0.05;纯度(94.08%±2.70%)接近机械剥离加酶消化法(97.02%±0.51%), P＞0.05, 但明显高于胰酶温消化加刮刷法(88.900%±1.304%), P＜0.05。(2)无血清组在培养第3d(95.0%±0.0%)、第5d(95.3%±0.3%)、第7d(95.3%±0.3%)气道上皮细胞纯度显著高于有血清组(90.1%±0.8%, 84.7%±1.6%和83.0%±0.9%), P＜0.01;且细胞生长融合时间(6-7d)明显早于有血清组(12-14d)。结论:胰酶冷消化加刮刷法是一种经济、简便、高效的兔气管上皮细胞体外分离培养方法, 无血清培养可以提高培养上皮细胞的纯度, 促进细胞融合。     

人支气管肺泡灌洗液抗菌多肽探讨

用酶低温消化获得的人气管上皮细胞,气-液界面无血清培养,细胞呈现复层生长,细胞角蛋白免疫组化染色呈阳性反应,可确认为上皮细胞。琼脂糖弥散法显示培养细胞表面分泌物对绿脓杆菌有明显的抗菌活性。随底层培养基NaCI浓度的增高,杀菌环直径逐渐减小。当NaCl浓度分别为0、20、40、80、160、320mmol/L时,     

人支气管上皮细胞的体外培养

背景：人支气管上皮细胞培养在呼吸系统疾病研究中应用越来越广泛。 目的：探索美国典型培养物保藏中心人支气管上皮细胞体外培养方法的可行性。 方法：对人支气管上皮细胞培养条件进行反复摸索,最终确定应用含体积分数20%胎牛血清的DMEM/F12培养基进行培养。 结果与结论：实验培养的人支气管上皮细胞扁平,呈多边形,长满后呈“铺路石”样分布,经免疫组化检测发现培养的细胞表达上皮细胞标志物细胞角蛋白。在支气管扩张症患者痰液上清刺激下,细胞表达的核因子κB、肿瘤坏死因子α和白细胞介素8明显增强,证实培养的人支气管上皮细胞获得成功。提示不必拘泥于美国典型培养物保藏中心推荐的培养条件,改进的培养方法简便易行,有应用价值。     

人α-防御素HNP1基因在气管上皮细胞转染的表达研究

目的　探讨人α防御素ＨＮＰ１ 基因在气管上皮细胞转染表达的可行性。方法　采用脂质体转染法将ＨＮＰ１ ｃＤＮＡ 重组真核表达质粒ｐＢａｂｅＮｅｏＨＮＰ１ 导入无血清培养的人、兔气管粘膜上皮细胞,利用逆转录聚合酶链反应（ＲＴＰＣＲ） 法及免疫组化法,分别在核酸水平及蛋白质水平检测ＨＮＰ１ 的表达。结果　用ＲＴＰＣＲ 在人和兔的转染上皮细胞总ＲＮＡ 提取物中均扩增出１ 条３１９ｂｐ 的ＤＮＡ 片段,与ＨＮＰ１ ｃＤＮＡ 片段大小一致, 而在未转染的上皮细胞总ＲＮＡ 中没有扩增出相应ＤＮＡ片段。免疫组化法检测到转染细胞呈强阳性反应,未转染细胞呈阴性反应,与ＲＴＰＣＲ 法检测的结果一致。结论　实验在ｍ ＲＮＡ 和蛋白质水平上均提示重组真核表达质粒ｐＢａｂｅＮｅｏＨＮＰ１ 转染气管粘膜上皮细胞后,能有效表达ＨＮＰ１ 分子。     

绵羊羊膜上皮细胞分离培养及鉴定*☆

背景：目前有研究发现人羊膜和大鼠羊膜分离消化培养后,可成功获得具有干细胞特性的人羊膜上皮细胞和鼠羊膜上皮细胞,在体外一些外源因子作用下具有向三胚层细胞分化的潜能。 目的：获取绵羊羊膜上皮细胞,并对其干细胞特性进行鉴定。 方法：采用机械方法剥离绵羊羊膜组织,运用低速旋转TrypLE(胰酶替代物)消化法对绵羊羊膜进行分离培养获得绵羊羊膜上皮细胞。 结果与结论：细胞免疫荧光检测结果表明绵羊羊膜上皮细胞表达Oct-4、SSEA-1、SSEA-3、SSEA-4、TRA-1-60、TRA-1-81等胚胎干细胞标记蛋白;RT-PCR技术鉴定结果显示绵羊羊膜上皮细胞的Oct-4、Sox-2、Rex-1全能性相关基因表达,而Nanog基因不表达。检测结果提示实验成功获得具有干细胞特性的绵羊羊膜上皮细胞。      

Serum-free cell culture of normal human urothelium

Summary A simple and effective method is described for obtaining epithelial cell cultures from normal human urothelium (bladder or ureter epithelium) using a new serum-free medium, HMRI-2. Tissue explanted in HMRI-2 exclusively produced epithelial outgrowths that could be subcultured once or twice and cryopreserved in liquid nitrogen in the complete absence of serum.     

Culture of conducting airway epithelial cells in serum-free medium

Summary Reproducible techniques for the isolation and culture of conducting airway epithelial cells from various species including human are described. Tissues recovered after necropsy or surgery are treated with 0.1% protease at 4°C overnight. The epithelial lining cells are liberated from adventitia by flushing with ice-cold minimal essential medium containing 10% fetal bovine serum. The resulting suspension of single cells and cell clusters is centrifuged and then plated on type 1 collagen gel substratum in a serum-free F12 medium supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, hydrocortisone (or dexamethasone), bovine hypothalamus extract, and retinol. Increased calcium concentration in the medium to 1 to 3 mM augments in vitro mucous cell differentiation. Both a squamouslike and mucociliary differentiation of the cultured epithelium can be identified by biochemical, immunohistologic, and morphologic means. The polarity of epithelial cell cultures is promoted by use of biphasic culture methods, in which epithelial cells are fed basally and are in direct contact with the air phase.     

Isolation and culture of colonic epithelial cells in serum-free medium

Summary Techniques are described for the dissociation, fractionation through Percoll, and in vitro maintenance of mucosal epithelia from the human or guinea pig large bowel. Tissue recovered after surgery is predigested with trypsin-citrate and treated subsequently with a mixture of trypsin, citrate, and collagenase. The resulting suspension of single cells, cell clusters, and partially digested crypts is suspended over a Percoll solution and enriched in multicellular elements by two sequential centrifugations. The recovered multicellular complexes are inoculated to specially treated culture vessels in a serum-free medium supplemented with epidermal growth factor, insulin, transferrin, selenium, and bovine pituitary extract. Epithelia, characterized as such by transmission electron microscopy, adhere to the substrate, form colonies, and can be maintained routinely for study for at least 10 wk.     

Serum-free medium for hybridoma and parental myeloma cell cultivation: a novel composition of growth-supporting substances

Serum-free chemically defined medium for hybridoma and parental myeloma cultivation was developed on the basis of testing of individual substances supporting hybridoma growth under serum-free conditions. Optimized concentrations of transferrin, insulin, ethanolamine, linoleic acid, serum albumin, ascorbic acid, hydrocortisone, and trace elements could substitute serum. Developed serum-free hybridoma (SFH) medium differs from analogous previously described media mainly by a more complete combination of growth-supporting supplements and by the presence of ascorbic acid and hydrocortisone. Growth comparable with that in the medium supplemented with 10% bovine serum was achieved with four hybridomas and two myelomas. SFH medium was also suitable for long-term cultivation of hybridomas without cessation of monoclonal antibody production. Growth potency and the specific growth requirements of hybridomas in serum-free medium are, to a large degree, determined by parental myeloma.     

A serum-free method for culturing normal human bronchial epithelial cells at clonal density

Summary A technique for the isolation and culture of normal human bronchial epithelial (NHBE) cells is described. This procedure involves first explanting fragments of large airway tissue to initiate fibroblastic-cell-free outgrowths of epithelial cells and subsequently using a serum-free medium (LHC-9) to propagate the NHBE cells at clonal density.     

正常人乳腺上皮细胞的原代培养

目的研究正常人乳腺上皮细胞原代培养的可行性,以及激素对其增殖的影响。方法从乳房区段切除术切除组织中获取正常人乳腺组织,用胶原酶溶液消化乳腺组织分离得到较纯净的上皮细胞,用烧灼法结合酶消化法解决细胞纯化问题。细胞形态观察和电镜检测鉴定细胞。结果正常人乳腺上皮细胞的原代培养是可行的。1×10-5的雌二醇和孕酮混合液可以促进细胞增殖。     

Serum-free medium cultivation to improve efficacy in establishment of human embryonic stem cell lines

BACKGROUND: Serum-containing and serum-free media were used to derive human embryonic stem (HES) cells from donated oocytes and embryos. METHODS and RESULTS: Inner cell masses (ICM) were isolated by immunosurgery. The HES cells were found to be easily obtained and expanded in a serum-free medium. The efficacy in establishing human embryonic stem cell lines improved in a serum-free medium compared with that in serum-containing media. Four HES cell lines were derived from 13 isolated ICM on mouse embryonic fibroblast feeder layers. All four cell lines possess the same characteristics and differentiating potency: normal 46, XX or 46, XY karyotype; and expressing a series of surface markers such as APase, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, but not SSEA-1. They can form embryoid bodies in suspension culture and develop teratomas comprising derivatives of three embryonic germ layers when injected into severe combined immunodeficient mice. CONCLUSION: These preliminary results suggest that serum-free cultivation may be superior to serum-containing cultivation for deriving human embryonic stem cells.     

利用基因转染技术长期培养人甲状腺细胞

本工作采用Ｃａ３（ＰＯ４）２ＤＮＡ共沉淀法，将含有编码ＳＶ４０病毒早期基因的ｐＳＧ５质粒转染原代培养的人甲状腺上皮细胞。在重复进行的１２次转染中，有１次细胞生存达２００ｄ，传了２６代。检测证实，转染后的细胞仍能保持少量Ｔ４的分泌和Ｔｇ的合成；细胞经长期培养后形态无异常，Ｇ带染色正常，对裸鼠无致瘤性。甲状腺滤泡上皮细胞长期培养模型的建立，为进一步研究细胞免疫在自身免疫性甲状腺炎中的作用打下了基础。     

改良组织块培养法体外培养人角膜上皮干细胞

文题释义：角膜上皮干细胞：属于单能干细胞,具有细胞周期长、低分化状态、增殖潜力大、不对称分裂等特点,定位于角膜缘基底细胞层,又称之为角膜缘干细胞,对角膜上皮细胞更新及维持角膜透明起着重要作用。角膜缘干细胞的体外培养方法：主要包括酶消化培养法和组织块培养法。酶消化培养法是利用DispaseⅡ酶破坏角膜缘上皮细胞与基底膜之间的半桥粒连接,然后剥取角膜缘上皮层,再使用胰酶将其消化为单个细胞进行培养。组织块培养法没有经过酶的双重消化,将剖取的角膜缘组织块进行贴壁,细胞游离出组织块进行贴壁生长,需要一个漫长的过程。  摘要背景：角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手。目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向。目的：探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性。方法：人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于 37 ℃、体积分数为5%CO₂细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养。组织块周边有细胞游离出贴壁生长记作“培养第1天”,每日相差显微镜下观察细胞生长变化。利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达。结果与结论：①改良组织块培养组出膜时间明显短于常规组织块培养组(P < 0.05),出膜率明显高于常规组织块培养组(P < 0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞。ORCID: 0000-0001-8370-174X(许中中) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程