不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长曲线和细胞形态的影响

目的：通过探讨不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长曲线和细胞形态的影响，了解它们对成纤维细胞体外生存和生长的情况。方法：采用体外细胞培养技术进行小鼠成纤维细胞L929细胞，并采用磺基罗丹明B（sulforhodamineB，SRB）酶标仪法（A490nm波长下）测量各孔的吸光值（A值）并转换为小鼠成纤维细胞L929细胞系生长曲线；同时，分别采用HE（苏木精-伊红）染色法、、吖啶橙荧光染色和Hoechst33342荧光染色法进行小鼠成纤维细胞L929细胞系形态学观察。结果：小鼠成纤维细胞L929细胞系不同浓度的血清分别在3种不同的介质（RPMI1640培养基、0．9％生理盐水和5％葡萄糖生理盐水）中表现出不同的生长和生存情况及细胞形态学改变。其中，在不同血清浓度（0％、10％、20％、30％、40％、50％、60％、70％、80％、90％、100％）的同一介质中，血清浓度为0％和100％浓度组的小鼠成纤维细胞L929细胞生长明显抑制及吸光度（A）值呈逐步减少的趋势，而除此之外的其它血清浓度组对小鼠成纤维细胞L929细胞的影响，基本表现为随着血清浓度的升高，吸光度（A）值呈逐步增加及促进细胞生长的趋势。而某些相同血清浓度在不同介质中对小鼠成纤维细胞L929细胞系生长和生存及形态学的有利影响，则基本表现为RP—M11640培养基作为介质的细胞培养效果优于0．9％生理盐水，而0．9％生理盐水作为介质的细胞培养效果又优于5％葡萄糖生理盐水。结论：不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长和生存及细胞学形态均有明显影响。提示在进行体外细胞培养时应充分考虑细胞培养介质及所添加血清的浓度。     

血清介质对体外培养L929系小鼠成纤维细胞表达产物Ⅰ、Ⅲ型胶原及其比值的影响

目的 探讨不同浓度血清、不同体外培养时间对L929系小鼠成纤维细胞表达产物Ⅰ、Ⅲ型胶原及其比值的影响.方法 采用双抗体夹心法检测细胞培养液中Ⅰ、Ⅲ型胶原的含量,并同时计算出Ⅰ/Ⅲ型胶原的比值.结果 除0%血清浓度组的Ⅰ、Ⅲ型胶原呈现无明显变化的微弱升高趋势外,其余各血清浓度组的Ⅰ、Ⅲ型胶原均处于增加状态,且在6~8 d时增加最为明显;另外,Ⅰ/Ⅲ型胶原比值随着血清浓度和细胞生长时间不同,表现出不同的变化趋势.其中20%和40%血清浓度组随着培养细胞生长时间延长,Ⅰ/Ⅲ型胶原比值逐渐增加;而其他血清浓度组,随着细胞培养时间的延长,Ⅰ/Ⅲ型胶原比值增加并不明显.结论 L929细胞在不同体外培养时间和不同浓度血清下其表达产物Ⅰ、Ⅲ型胶原含量及其比值有一定变化,为今后优化体外细胞培养条件提供了相关的实验数据.     

体外原代培养脐带基质间充质细胞和人皮肤成纤维细胞

目的探讨体外原代培养脐带基质间充质细胞(UMC)和人皮肤成纤维细胞(HSF)的方法。方法用Ⅳ型胶原蛋白酶、Ⅰ型脱氧核糖核酸酶和0.25%胰蛋白酶消化法原代分离培养UMC细胞,用组织块贴壁法原代分离培养HSF细胞,镜下观察细胞的培养过程和生长状态。结果脐带组织分离培养第3天,有少量UMC贴壁生长,细胞膜周围有折光性,形态类似于成纤维细胞;皮肤组织块贴壁培养第7天,有HSF爬出,呈长梭形、不规则三角形。随着细胞培养时间延长,UMC和HSF数量逐渐增多,细胞生长状态良好。结论应用酶消化法和组织块贴壁法可成功分离培养出UMC和HSF。     

人成纤维细胞的体外分离、纯化培养及细胞鉴定

目的对人皮肤成纤维细胞进行分离、纯化、培养及细胞鉴定,探讨一种高效的分离及纯化方法,为细胞移植提供种子细胞。方法使用酶消化法原代提取人成纤维细胞,快速贴壁法纯化细胞,对细胞进行形态学观察、HE染色,免疫细胞化学鉴定细胞标志物Vimentin。结果利用倒置显微镜及HE染色,可见细胞为散在分布的梭形贴壁细胞,当细胞汇流时,呈鱼群样或漩涡状排列,且细胞在15代内形态保持不变。结论成功地发现快速提取成人成纤维细胞的方法,为成人自体细胞移植的研究奠定了基础。     

EGF和TGF-a对体外培养大肠癌细胞生长的调控

本应用^3H-TDR渗入试验、受体结合分析方法定量观测了EGF和TGF-a对大肠癌细胞HT-29与HR8348生长的影响。结果二株癌细胞均可自分泌低水平的EGF，且可同时表达高亲和力的EGF-R，外源性的EGF和TGF一均可刺激HT-29细胞的生长，而加入抗EGF．R抗体则可抑制HT-29细胞的生长．提示EGF／TGF-与EGF-R自分泌环在大肠癌的发展中起一定作用。     

维拉帕米对体外培养神经瘢痕成纤维细胞形态学的影响

目的 观察钙通道阻滞剂维拉帕米对体外培养神经瘢痕成纤维细胞形态学的影响.方法 Wistar大鼠10只,制作双侧坐骨神经损伤模型,术后 2 w取损伤处神经瘢痕,按组织块法培养神经瘢痕成纤维细胞,实验所用细胞为4～8代,分2组,维拉帕米组加入含维拉帕米的DMEM培养液,浓度为100 μmol/L,对照组不加药.48 h后进行Giemsa染色及制备扫描电镜标本,观察细胞形态学的变化.结果维拉帕米组成纤维细胞生长稀疏,胞体呈圆形,胞浆内有较多囊泡,细胞突起细小,网状物质较少;对照组成纤维细胞生长稠密,形态多样,多呈梭形或多角形,细胞间界限不清,互相连接或重叠,胞间网状基质较多,细胞突起粗大,胞间突起互相交联,胞浆内囊泡少见.结论维拉帕米能使体外培养神经瘢痕成纤维细胞趋于球形变,可能同时对成纤维细胞的增殖及胶原蛋白的分泌具有抑制作用.     

激素和生长因子对无血清培养成纤维细胞调控作用的实验研究

研究激素和生长因子对体外无血清培养３Ｔ３和Ｃ３Ｈ１０Ｔ１／２成纤维细胞增殖的调控作用，发现加胰岛素，胰高血糖，表皮生长因子（ＥＧＦ）和成纤维细胞生长因子（ＦＧＦ）各组细胞数均明显高于单纯无血清对照组，且加激素各组＾３Ｈ－ＴｄＲ掺入值均高出对照组３～６倍（Ｐ＜０．０５），其中ＥＧＦ＋ＦＧＦ以及上述四种激素混合使用，二细胞＾３Ｈ－ＴｄＲ掺入值超过对照组３０倍以上。     

人肛瘘管壁成纤维细胞的体外分离培养和鉴定

通过组织块法分离并原代培养人肛瘘管壁组织,用胰蛋白酶和乙二胺四乙酸（EDTA）消化成纤维细胞、上皮细胞,获得更加纯化的成纤维细胞;以DMEM培养液为基础培养蒸,添加胎牛血清（体积分数10％）、青霉素（100U／L）和硫酸链霉素（100U／L）,置37℃、5％CO2培养箱中培养。倒置相差显微镜和细胞爬片HE染色。观察成纤维细胞形态结构,并对培养成纤维细胞行角蛋白、波形蛋白免疫细胞化学鉴定．结果分离后的成纤维细胞在第4～12h于体外快速贴壁,第2～4天进入对数期生长、增殖期。细胞起源鉴定波形蛋白免疫细胞化学染色为阳性,角蛋白免疫细胞化学染色为阴性。提示该方法所获得的肛瘘管壁成纤维细胞可在体外稳定培养,不含有杂质细胞。可为在细胞分子水平上研究肛瘘瘘管愈合的机制提供可靠的细胞模型。     

In vitro influence of lectins and neoglycoconjugates on the growth of three human sarcoma cell lines

Purpose : The aim of our study is to investigate the in vitro effects of plant lectins, galectins and neoglycoconjugates on the proliferation of three human sarcoma cell lines. Methods : Proliferation was assessed by means of the tetrazolium derivative reduction (MTT) assay. In addition, glycohistochemistry was used to make visible the plant-lectin-specific binding sites; the intensity of the lectin binding pattern was quantified by means of image analysis. Results : Depending on the cell lines, the staining intensity and the percentage of labelled cells were different. With respect to growth modulation, the cell lines also responded differently to the probes used. Besides a predominant inhibitory effect elicited by the probes at 50 μg/ml, dose-dependent effects, including growth stimulation, were detectable in several instances. These effects relate to the animal galectins tested and several neoglycoconjugates, e.g. the lactose- and blood-group-A-trisaccharide-bearing probes. Conclusions : Endogenous lectins and lectin-reactive cellular glycoconjugates can apparently affect the regulation of the growth of human sarcoma cells. We suggest that these results are relevant for further histopathological monitoring in correlation with prognosis and in vitro assays to reveal possible clinical applications. Received: 4 May 1998 / Accepted: 7 December 1998     

人肝脏再生增强因子对体外肝细胞生长的影响

目的基因工程方法纯化人肝脏再生增强因子（hALR）,研究hALR对体外培养肝细胞生长的影响。方法构建hALR原核表达载体PGEX-3X-hALR,诱导表达、纯化收集GST-hALR,用X因子切去GST,凝胶过滤得到高纯度hALR蛋白;分别用正常肝细胞（L-02细胞）和肝癌细胞（HepG2细胞）分析hALR对其生长的影响。结果hALR原核表达载体pGEX-3X-hALR构建成功,经诱导表达,可纯化得到高纯度的hALR蛋白。hALR蛋白能促进正常肝细胞的生长,并与浓度成正相关;而肝癌细胞的生长却起抑制作用。结论成功表达和纯化重组hALR蛋白,hALR促进正常肝细胞的生长而抑制肝癌细胞生长。     

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum

Purpose Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines.Methods The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS).Results Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1.Conclusions Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.     

The in vitro influence of eight hormones and growth factors on the proliferation of eight sarcoma cell lines

Little is known about the regulation of sarcoma proliferation by hormones and/or growth factors. We therefore characterised the in vitro proliferative influence on eight sarcoma cell lines of the platelet-derived growth factor, the insulin-like growth factor 1, triiodothyronine, the epidermal growth factor, the luteinising-hormone-releasing hormone, progesterone, gastrin and 17 β-oestradiol. The influence of the different factors on the proliferation of sarcoma cell lines was measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Two culture media were studied: (1) a nutritionally poor medium containing 2% of fetal calf serum and (2) a nutritionally rich one containing 5% or 10% FCS both with and without the addition of non-essential amino acids. The results were analysed either by conventional statistical analyses or by a classification method based on a decision-tree approach developed in Machine Learning. This latter method was also compared to other classifiers (such as logistic regression and k nearest neighbours) with respect to its accuracy of classification. Monovariate statistical analysis showed that each of the eight cell lines exhibited sensitivity to at least one factor, and each factor significantly modified the proliferation of five or six of the eight cell lines under study. Of these eight lines one of fibrosarcoma origin was the most “factor-sensitive”. Decision-tree-related data analysis enabled the specific pattern of factor sensitivity to be characterised for the three histological types of cell line under study. The effects of hormone and growth factors are significantly influenced by the type of culture medium used. The method used appeared to be an accurate classifier for the kind of data analysed. Sarcoma proliferation can be modulated, at least in vitro, by various hormones and growth factors, and the proliferation of each histopathological type exhibited a distinct sensitivity to different hormone and/or growth-factors. Received: 14 April 1997 / Accepted: 1 December 1997     

兔外周血与骨髓源性内皮祖细胞培养鉴定及生物学特性的研究

目的:探索并建立兔内皮祖细胞(EPCs)的体外培养、鉴定方法。方法:采用密度梯度分离法分别从兔外周血和骨髓中分离出单个核细胞,接种于预包被明胶的培养瓶,并应用培养基(EBM-2)诱导培养,诱导分化为EPCs;在倒置显微镜下观察两种源性细胞生长过程;台盼蓝法测定细胞传代成活率;通过绘制生长曲线法、MTT法、DNA周期检测比较两种源性获得的EPCs增殖情况;采用免疫荧光染色观察EPCs相关抗原CD34、CD31、CD133及vWF因子的表达。结果:两种源性EPCs均于培养3～4d完全贴壁,呈克隆样生长,7～9d形成类似成熟血管内皮细胞形态,细胞数量逐渐增多,细胞成活率均为95%。两种源性获得的第2代细胞生长曲线近似"s"形、MTT法显示3～5d细胞增殖较明显,外周血源性EPCs G0-G1期和S+G2+M期所占比例分别为96.48%和3.52%,骨髓源性EPCs G0-G1期和S+G2+M期所占比例分别为97.11%和1.84%。第2代EPCs进行免疫荧光鉴定结果显示:两种源性内皮祖细胞均阳性表达CD34、CD133、vWF因子,CD31弱阳性表达。结论:两种源性的EPCs均可高效获得且在体外稳定培养。与传统的骨髓源性EPCs相比较,从外周血源获得的EPCs是一种可靠、简便的培养方法,为组织工程学提供理想的细胞来源。