新生大鼠小肠上皮细胞分离培养研究

本实验比较了4种分离大鼠IEC的方法,结果显示联合应用粗胶原酶和中性蛋白酶分离效果最好,细胞贴壁生长能力强。胶原涂膜改善玻璃培养瓶或盖玻片表面的性状有利于细胞贴壁生长。细胞的增殖依赖于培养液的质量、成分及细胞间的相互作用。培养细胞一般1～2天贴壁,7～8天明显增殖,10～14天汇合成片。培养细胞细胞角质蛋白、碱性磷酸酶染色阳性,光镜和电镜检查均显示为IEC。本文所建立的新生大鼠IEC体外培养方法为研究IEC生理和病理提供了一个十分有用的实验模型。     

牦牛输卵管上皮细胞分离培养和纯化鉴定

为建立牦牛输卵管上皮细胞原代培养及纯化方法,通过选取牦牛输卵管,运用机械刮取法和0.25%胰蛋白酶消化两种方法分离上皮细胞进行体外培养。对不同分离方法的培养效果比较,培养细胞进行形态学观察与传代培养、MTT比色检测细胞活力并制定生长曲线,原代及传代上皮细胞的免疫组织化学鉴定,冷冻解冻后经台盼蓝排斥试验检测活细胞数。结果表明该试验分离出的原代细胞,纯化后传代培养,经鉴定为牦牛输卵管上皮细胞,培养的细胞生长状况良好,建立了一套牦牛输卵管上皮细胞分离培养及纯化鉴定的方法。     

奶山羊乳腺上皮细胞的分离、培养及鉴定

应用组织块培养法高密度培养、连续传代法建立西农萨能奶山羊乳腺上皮细胞体外培养体系,通过生长曲线绘制、核型分析、免疫荧光染色 (角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白)、油红染色及β-酪蛋白基因的RT-PCR分析进行培养细胞鉴定。实验结果表明细胞生长曲线为典型的S型,染色体数目众数为60,细胞角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白表达均呈阳性,油红染色后可见细胞质内的脂滴,且细胞表达酪蛋白mRNA。说明运用本方法培养的细胞为正常的乳腺上皮细胞,并具有一定的泌乳功能。     

成人宫颈上皮细胞的原代培养及鉴定

目的:探索采用无血清培养基原代培养成人宫颈上皮细胞的方法。方法:以成人的宫颈上皮组织为研究对象,采用胰蛋白酶-EDTA消化法获得宫颈上皮细胞悬液,于上皮细胞专用无血清培养基中培养,采用免疫细胞化学法测定细胞中角蛋白及波形蛋白的表达,对细胞纯度进行鉴定。结果:原代培养10-15天细胞融合达60%,传代至4-6代,细胞出现生长衰退。早期细胞生长状态良好,细胞纯度在90%以上。结论:采用酶消化法及K-SFM无血清培养基培养可获得纯度高的成人宫颈上皮细胞。     

谷氨酰胺对体外培养人小肠上皮细胞缺氧复氧损伤的保护作用

应用原代培养人胎小肠上皮细胞（ＩＥＣ）,观察了谷氨酰胺（ＧＬＮ）对缺氧复氧（Ａ／Ｒ）损伤人ＩＥＣ的影响。结果：缺氧６０ｍｉｎ复氧３０ｍｉｎ后,细胞内乳酸脱氢酶（ＬＤＨ）漏出量显著上升,细胞存活率显著下降。预先应用１～５ｍｍｏｌ／ＬＧＬＮ可使Ａ／Ｒ损伤ＩＥＣ细胞存活率升高和细胞内ＬＤＨ漏出量减少,ＧＬＮ作用的最佳剂量为２ｍｍｏｌ／Ｌ。提示ＧＬＮ对人ＩＥＣ具有直接的保护作用,这可能是其整体保护作用机制之一。     

分步消化法原代培养鼠阴道黏膜上皮细胞的研究

目的探讨大鼠阴道黏膜上皮细胞的体外培养和扩增技术,为构建组织工程化阴道动物模型提供种子细胞。方法取大鼠阴道全层组织,经Dispase酶和胰酶分步消化后,接种于无血清角化细胞培养液中连续培养,观察细胞形态、体外生长特性和超微结构,绘制生长曲线,免疫组化鉴定。结果原代细胞培养24-36 h后开始贴壁,7-10d约80%融合,呈铺路石样外观,可连续传5-6代;扫描电镜下细胞表面可见微绒毛嵴;角蛋白染色阳性,细胞纯度98%;第五代细胞为正常二倍体核型。结论该方法培养的阴道上皮细胞增殖状态良好,细胞纯度高,扩增迅速,可在较短时间内获得大量细胞用于组织工程学研究。     

2种气管黏膜上皮细胞体外培养技术的研究

目的为以猪气管黏膜上皮为细胞模型的研究奠定物质基础,进一步探讨猪气管黏膜上皮细胞的体外传统培养和气液界面培养技术,从而使2种培养技术优势互补。方法对猪气管上皮分离、纯化、培养和传代,并探索上皮细胞最佳冻存复苏条件;复苏后的气管上皮细胞进行气液界面培养,绘制细胞生长曲线和观察细胞纤毛生发情况。利用免疫组化法鉴定上皮细胞。结果4步纯化法可以得到高纯度的气管上皮细胞。使用胎牛血清、DMEM/F12培养液和DMSO的体积分数为50%、40%和10%的冻存体系保存的气管上皮,复苏后细胞存活率平均可达89%。优化后的传统方式培养的上皮细胞可连续传代到第8代,但从第2代开始便观察不到纤毛,转换成气液界面连续培养2代后重新生发纤毛,细胞存活期延长。免疫组化结果显示分离培养细胞为上皮细胞。结论成功建立了2种猪气管上皮细胞培养技术,并找到适宜的气管上皮细胞冻存条件,节省了不断原代取材的成本和时间,并成功实现传统培养细胞冻存复苏后很快适应气液界面的培养并恢复细胞的天然结构,为猪气管黏膜上皮相关研究提供丰富的细胞来源。     

猪原始生殖细胞的分离、培养与鉴定

Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ trans-plantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24- 26 days old porcine embryos were treated in 0.02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco‘‘s modified Eagle‘‘ s medium (DMEM) supplemented with 20 % Buffalo rat lever (BRL) cell-conditioned medium, 15 % fe-tal bovine serum, 1 mmol L-glutamine, 0.1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics.The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff‘‘ s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cul-tured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain.These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had success-fully isolated EG-like cells from oorcine PGCs.     

狗小肠消化间期复合肌电的微机分析

本文报道利用微机处理狗小肠消化间期复合肌电（ＩＭＣ）周期，定量地分析了１４只狗十二指肠和空肠各个时相中所含锋电位簇数、锋电位数、平均每簇所含锋电位数和各时相所占时程。并比较了十二指肠和空肠的差异。以上定量化的分析为生理、药理等领域有关肠道运动功能的研究提供了精确的定量化的客观依据。     

大鼠前列腺上皮细胞培养鉴定及体外炎症模型建立初探

目的:在体外培养大鼠前列腺上皮细胞,建立脂多糖和蛇毒诱导的大鼠前列腺上皮细胞的体外炎症模型。方法:采用WAJC404培养基在体外培养大鼠的前列腺上皮细胞,通过免疫细胞化学方法对其进行鉴定,加入脂多糖和蛇毒用以建立体外的前列腺炎症模型。结果:在体外培养了大鼠的前列腺上皮细胞,泛细胞角蛋白染色阳性,脂多糖和蛇毒可损伤体外培养的前列腺上皮细胞,但不影响体外培养的前列腺上皮细胞培养液中细胞因子TNF-α和IL-1β及免疫球蛋白IgG、IgA和IgM的含量。结论:在体外培养了大鼠的前列腺上皮细胞,对大鼠前列腺体外炎症模型的建立进行了初步探索。     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

家鸽小肠绒毛微血管铸型的扫描电镜观察

用扫描电镜观察了ＡＢＳ丁酮溶液灌注的家鸽小肠绒告发同血管构筑情况。家鸽小肠绒毛血管丛由输入沁动脉、毛细血管网和输出小静脉组成,小肠绒毛血管丰富,并相到吻合成单层密集网;办入小动脉既可从肠腺周围血管丛发出,也可直接由粘膜下去一发出,绒毛下部血管表现为微直血管形态,可能部分具有门静脉性质。     

一种原代肺上皮细胞体外培养方法

目的:建立一种操作简便、重复性好的体外培养BALB/c小鼠原代肺上皮细胞(AEC)的方法,探究不同发育阶段小鼠AEC中柯萨奇病毒和腺病毒受体(CAR)表达量的变化,及其对小鼠原代AEC贴壁的作用。方法:手术获取小鼠肺组织,机械法剪碎肺组织,PBS缓冲液清洗肺组织块数次去血,联合使用链霉蛋白酶和胶原酶Ⅰ消化、分离肺组织块获得单细胞悬液,差速离心逐步清除其他种类的细胞,达到纯化AEC的作用。细胞在Ⅰ型鼠尾胶原蛋白包被过的细胞板中培养,光学显微镜下观察不同发育阶段AEC贴壁、生长状态;免疫荧光法鉴定AEC,检测AEC中CAR的表达量。结果:体外获得不同发育阶段BALB/c小鼠的原代AEC;胎鼠、幼鼠AEC贴壁并开始增殖所需时间较成体鼠短;胎鼠及幼鼠AEC中CAR的表达量明显较成体鼠高。结论:建立了稳定可重复的分离、纯化、体外培养小鼠原代AEC的方法,证明了AEC中的CAR可以促进原代AEC贴壁,为完善原代细胞培养方法提供了科学依据。     

牛乳腺上皮细胞的分离培养及其生物学特性

采用胶原酶消化法和胰蛋白酶选择性消化法分离、培养和纯化牛乳腺上皮细胞。形态学观察表明,培养的细胞具有典型的上皮细胞形态特征;染色体分析结果表明,培养的细胞具有正常的染色体数目。通过荧光免疫细胞染色方法鉴定了培养的细胞表达上皮细胞特异的角蛋白5和8。该细胞在添加胰岛素、氢化可的松以及羊催乳素的无血清培养液中诱导培养时,用RT-PCR方法检测到了β-酪蛋白基因的转录。这些结果表明,分离培养的细胞是乳腺上皮细胞,这些细胞在诱导培养的条件下能够转录表达β-酪蛋白。     

转溶菌酶基因山羊乳腺上皮细胞的原代培养及特性分析

采用组织块法、胰酶差速消化法分离了人溶菌酶(hLYZ)转基因山羊的乳腺上皮细胞,建立了转基因羊乳腺上皮细胞(TGMEC)的体外培养体系,并对其生长与体外分泌特性进行了分析.研究显示其体外生长曲线符合典型的S型,传代期间二倍体染色体数正常;通过免疫荧光染色、RT-PCR发现该细胞能表达上皮细胞所特有的角蛋白18,并能表达内源性乳蛋白(酪蛋白与乳球蛋白);进一步通过酶联免疫试验发现该乳腺上皮细胞在体外培养中可持续稳定地分泌重组人溶菌酶.由此获得的人溶菌酶转基因羊乳腺上皮细胞系为乳腺生物反应器研究提供了一个重要细胞膜型.     

新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析

利用骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)治疗疾病已经逐渐成为现实,但是作为被移植的种子细胞,BMSCs体外传代能力非常有限,种子细胞来源极为贫乏。本研究通过差速贴壁筛选的方法分离出一种猪BMSCs的衍生细胞株,命名为猪骨髓间充质干细胞衍生细胞(Bone mesenchymal stem-derived cells,BMSDCs)。分别对BMSDCs与BMSCs细胞进行细胞生物学特性分析,探讨其体外诱导分化特性,并应用流式细胞术测定细胞表面标记物。结果表明,BMSC和BMSDCs细胞倍增时间分别为31.3 h和30.3 h,平均传代时间分别为3-5 d和2-3 d;两种细胞均阳性表达CD34、CD90,阴性表达CD44、CD45;经体外诱导后均可分化为成脂细胞和成肌细胞。在传代能力上,前者可传代15至20次,后者可长期传代(200次以上)且维持正常染色体特征。研究认为在适宜的实验条件下,体外培养的猪骨髓间充质干细胞的衍生细胞——BMSDCs能够稳定生存增殖并维持BMSCs多向分化潜能,可作为组织工程的理想种子细胞。     

Microbial Metabolites and Intestinal Stem Cells Tune Intestinal Homeostasis

Microorganisms that colonize the gastrointestinal tract, collectively known as the gut microbiota, are known to produce small molecules and metabolites that significantly contribute to host intestinal development, functions, and homeostasis. Emerging insights from microbiome research reveal that gut microbiota‐derived signals and molecules influence another key player maintaining intestinal homeostasis—the intestinal stem cell niche, which regulates epithelial self‐renewal. In this review, the literature on gut microbiota‐host crosstalk is surveyed, highlighting the effects of gut microbial metabolites on intestinal stem cells. The production of various classes of metabolites, their actions on intestinal stem cells are discussed and, finally, how the production and function of metabolites are modulated by aging and dietary intake is commented upon.