利用气液界面无血清培养原代兔气管上皮细胞的研究

用低温酶消化法分离兔气管上皮细胞,具有细胞损伤小,活力及纯度高的优点,成纤维细胞污染极低。人胎盘胶原提高了气管上皮细胞贴壁性。无血清培养基能促进细胞增殖,分化和成熟。气液界面培养方式更好地模拟了气管上皮细胞的天然生长环境,在膜上呈复层生长,有利于细胞的分化成熟及功能表达。光镜下细胞形态及免疫组化细胞角蛋白染色阳性证实培养细胞为气管上皮细胞。本文所建立的兔气管上皮细胞体外气液界面无血清培养方法为研究气 管上皮细胞的生理和病理提供了一个十分有用的模型。     

猪呼吸道上皮细胞体外分化模型的建立及其应用研究

猪是许多呼吸道病毒感染的天然宿主,其与人类在肺生理学、呼吸道形态学和呼吸道细胞类型以及受体分布都有相似之处。为了解呼吸道病毒感染机制和筛选呼吸系统疾病药物,选择以猪肺组织为原料采用无血清气液界面培养法构建一种猪呼吸道上皮细胞(Porcine airway epithelial cell,PAEC)体外分化模型,然后通过扫描电子显微镜、电生理和免疫组织化学等方法鉴定该模型。采用三质粒共转染法构建重组腺相关病毒rAAV6-GFP(rAAV6-green fluorescent protein,rAAV6-GFP),通过顶端表面感染PAEC模型,探讨AAV6在PAEC模型中基因治疗领域的应用。结果显示分化成熟的PAEC模型为多层上皮结构,顶端表面含有纤毛细胞、黏液分泌细胞和基底细胞。rAAV6-GFP能通过顶端表面感染PAEC,有效地介导外源基因的长期表达。本文建立的猪呼吸道上皮细胞体外分化模型为呼吸道病原体感染和呼吸系统疾病药物筛选和基因疗法等研究奠定了良好的基础。     

呼吸道上皮细胞模型的研究进展

呼吸道病毒严重威胁人类的健康。呼吸道上皮细胞是机体防御呼吸道病毒和细菌等外来病原体的第一道防线,因此建立呼吸道上皮细胞体外分化模型,为研究呼吸系统疾病发病机理、筛选有效的治疗药物奠定基础。本文主要介绍人呼吸道上皮细胞原代培养技术的研究进展及其在呼吸道病毒和呼吸系统疾病方面的应用。     

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。     

鸡输卵管上皮细胞体外分离培养的研究

鸡输卵管上皮细胞是卵清蛋白的主要分泌细胞,是研究输卵管特异表达蛋白调控的重要工具。在以往的研究中,多采用普通DMEM培养液对鸡输卵管上皮细胞进行分离与培养,容易造成其自身特性在体外培养过程中的改变。本研究我们优化了细胞分离方法,发现从输卵管漏斗部组织分离的输卵管上皮细胞增殖较快;用鸡输卵管上皮细胞培养基相比DMEM更适合促进细胞生长;与胰酶相比,用Accutase消化酶进行细胞传代,有利于输卵管上皮细胞特性维持。对所获得的输卵管上皮细胞鉴定发现,己烯雌酚能促进卵清蛋白的表达,说明分离培养的细胞保持了鸡输卵管上皮细胞特性。本研究建立的方法为输卵管特异表达蛋白调控以及家禽生物反应器的研究奠定了基础。     

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。     

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。     

兔羊膜上皮细胞的体外培养和增殖

研究妊娠晚期兔羊膜上皮细胞(amniotic epithelial cells, AECs)在体外生长和增殖特性.取妊娠晚期兔(27-28E) AECs进行体外培养, 光镜、扫描电镜下观察后, 利用免疫组化单克隆抗体AE1/AE3、AE5检测培养的AECs中细胞角蛋白的表达, 并采用流式细胞仪检测表皮生长因子(epidermal growth factor, EGF)和血清对AECs细胞周期的影响.结果表明妊娠晚期兔AECs在体外培养条件下生长良好、增殖旺盛; 单克隆抗体AE1/AE3、AE5染色阳性; 血清组、EGF组和联合应用组分别与对照组比较, 各周期细胞比例发生变化, G0/G1期减少, S期、G2/M期增加, 细胞增殖指数(PI)增加, P<0.01, 联合应用组分别与血清组、EGF组比较, P<0.05.说明妊娠晚期兔AECs表达细胞角蛋白CK3, 血清和EGF均能通过改变妊娠晚期兔AECs的细胞周期而促进AECs增殖, 两者联合应用对促进AECs的增殖更为显著.     

肠绒毛消化法所得仔猪小肠上皮细胞的培养与鉴定

小肠上皮细胞作为肠道的主要功能细胞,在多种肠道疾病和上皮间质转化的研究中发挥着重要的作用。采取组织块消化和肠绒毛消化两种方法对新生仔猪小肠上皮细胞进行分离培养,传代后通过细胞形态学及免疫荧光等方法对其进行鉴定,结果表明:肠绒毛消化法所获得的小肠上皮细胞要远好于组织块消化法所得细胞,细胞在24～48h贴壁,呈现出典型的三角形或多角形样,10～12d细胞汇合成片、单层生长、互不重叠;细胞角蛋白18(cytokeratin-18)和尾型同源盒基因2(Cdx2)阳性,碱性磷酸酶检测阴性,扫描电镜下可以清楚地看到均匀分布的肠绒毛。以上结果表明,该实验成功建立出可连续传代并符合小肠上皮细胞鉴定标准的仔猪小肠上皮细胞。     

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。     

Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes

The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia.     

Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium.     

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

Summary N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2–8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP was consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.     

Comparison of culture methods for human-human hybridomas secreting anti-HBsAg human monoclonal antibodies

Human-human hybridomas which secrete a human monoclonal antibody (h-MoAb) against hepatitis B virus surface antigen showed growth associated production kinetics. The rate of h-MoAb production rapidly decreased after cell growth was arrested in a perfusion culture, even if the perfusion rate was increased. A continuous suspended-perfusion culture, in which both culture broth and culture supernatant are continuously harvested and the same volume of fresh medium is continuously fed into the reactor, was developed to maintain continuous growing conditions during cultivation. In this culture system, the production of h-MoAb continued for more than 50 days with an average productivity of 5.0 mg/l of working volume/day. A semicontinuous immobilized-perfusion culture in which parts of the cells are repeatedly removed from the immobilized reactor was another useful technique for the long term cultivation of these h-h hybridomas. As an average h-MoAb production rate, 62 mg/l of immobilized-bed volume/day was achieved for 65 days of cultivation using a ceramic matrix reactor, and 327 mg/l/day was achieved over 47 days of cultivation using a hollow fiber reactor equipped with Cultureflo M^TM Thus, the antibody productivity per reactor volume per day by the semicontinuous immobilized-perfusion culture was much higher than that of the continuous perfusion culture in an agitation reactor.     

Microalgal mass culture systems and methods: Their limitation and potential

Cultivation of microalgae using natural and man-made open-ponds istechnologically simple, but not necessary cheap due to the high downstream processing cost. Products of microalgae cultured in open-pondscould only be marketed as value-added health food supplements, specialityfeed and reagents for research. The need to achieve higher productivityand to maintain monoculture of algae led to the development of enclosedtubular and flat plate photobioreactors. Despite higher biomassconcentration and better control of culture parameters, data accumulatedin the past 25 years have shown that the illuminated areal, volumetricproductivity and cost of production in these enclosed photobioreactors arenot better than those achievable in open-pond cultures. The technicaldifficulty in sterilizing these photobioreactors has hindered their applicationfor the production of high value pharmaceutical products. The alternativeof cultivating microalgae in heterotrophic mode in sterilizable fermentorshas achieved some commercial success. The maximum specific growth ratesof heterotrophic algal cultures are in general slower than those measured inphotosynthetic cultures. The biomass productivity of heterotrophic algalcultures has yet to achieve a level that is comparable to industrialproduction of yeast and other heterotrophic microrganisms. Mixotrophiccultivation of microalage takes advantage of their ability to utilise organicenergy and carbon substrates and perform photosynthesis concurrently. Moreover, production of some algal metabolites is light regulated. Futuredesign of sterilizable bioreactors for mixotrophic cultivation of microalgaemay have to consider the organic substrate the main source of energy andlight the supplemental source of energy, a change in mindset.     

Development of one-cell ovine embryos in two culture media under two gas atmospheres

The objective of this study was to develop a successful system for culturing one-cell ovine embryos through several cleavage divisions. One hundred and four one-cell embryos were collected from synchronized, FSH-treated ewes 48 hr after the onset of estrus and randomly placed in one of four culture treatments. The effect of glucose supplementation and reduced oxygen tension (20% vs. 5%) on embryo development was studied. Embryo development was quantitated by a cleavage index based on the number of completed cell divisions. The number of embryos completing at least two cell divisions when cultured in Brinster's Pyruvate Medium (BPM) was 7 26 and 9 26 , under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while 22 26 and 20 26 embryos divided when cultured in BPM supplemented with 0.1% glucose (BPM-G) under similar atmospheres. Mean cleavage indices for embryos cultured in BPM were 1.2 and 1.6 under 5% CO(2) in air and 90% N(2), 5% CO(2), 5% O(2), respectively, while embryos cultured in BPM-G had mean cleavage indices of 4.6 and 4.0, respectively. Results of this study indicate that one-cell ovine embryos can be successfully cultured through several cleavage divisions. Glucose supplementation was beneficial for one-cell ovine embryo development. Reducing the oxygen tension from 20 to 5% had no effect on embryo development, and there was no media x gaseous atmosphere interaction.     

肺炎支原体液体培养法检测的实验评价

目的比较液体培养法和固体培养法平行检测肺炎支原体结果的一致性;评价液体培养法检测肺炎支原体的可靠性。方法采用液体培养基和固体琼脂培养基平行检测1 648份临床标本的肺炎支原体,比较同一份标本在2种培养基上的检测结果。结果液体培养法阳性296例,阳性率为18%;固体培养法阳性244例,阳性率为14.8%;液体培养法阳性而固体培养法阴性57例;固体培养阳性而液体培养法为阴性5例。2种方法的阳性检出率比较差异有统计学意义(P<0.05)。结论肺炎支原体快速液体培养法与固体培养有较好的一致性,具有方便、简单、准确且可以用于早期检测等优点,适合临床大批量标本筛查。需结合患者临床症状等排除真菌和耐药菌造成的假阳性。     

Comparative study of two culture conservation methods in medical mycology

The efficiency of two fungal conservation methods was compared: Suspension in sterile distilled water and subcultures on potato dextrose agar (PDA) slants at 4 °C. One hundred and eleven strains corresponding to 84 different-species of microorganisms studied in medical mycology were evaluated. The efficiency of each method was estimated by the survival percentage and the preservation of the morphological features of each strain within a seven-year period. From the 111 strains, 79 (71.2%) were preserved viable in water, compared to 86 (77.5%) strains preserved by subculture on PDA slants. Concerning morphological features 75 of the 79 water viable strains (94.9%) conserved their morphology. In contrast, only 60 of the 86 strains (69.8%) conserved their typical morphology by the PDA subculture method. The water conservation method offers important benefits over serial subculture such as: Minimal pleomorphism, simple, rapid and requiring few materials. Thus, the water conservation method is recommended for laboratories where specialized conservation equipment is not available.     

A comparison of methods for callus culture and plant regeneration of RD25 rice (Oryza sativa L.) in two laboratories

While methodology is transferable from one laboratory to another, an exact transfer does not usually occur and even a nearly exact transfer of methods does not always result in repeatable data. Researchers should not expect that an effort to duplicate a published procedure will necessarily lead to identical results.In attempting to transfer rice tissue culture methods between laboratories in Fort Collins, Colorado, USA and Bangkok, Thailand, we discovered that a combination of the methods of each laboratory produced the best results in term of callus productions and plant regeneration. In the experiments reported here, the type of culture vessel used and the geographical location were also important variables.Supported by the USAID/Cooperative Agreement No DAN-4137-A-00-4053-00.