体外人表皮细胞对胰蛋白酶耐受的探讨

目的:观察人表皮细胞对胰酶消化的耐受能力,通过不同细胞恢复贴壁时间不同来探索分离和纯化表皮细胞的新方法,并探讨胰酶耐受细胞的干性表达,及其与muse细胞的可能相关性。方法:中性蛋白酶及胰酶消化获取表皮细胞,用0.25%的胰酶悬浮表皮细胞,以3.0×105/m L的细胞密度种植于12孔板,每间隔半小时或1小时终止胰酶1孔,其中胰酶作用时间最长达46小时。记录不同细胞贴壁时间、生长状态,并在接种后第7天,对贴壁细胞进行Nestin、Sox10抗体免疫细胞化学染色。结果:随着胰酶作用时间的延长,贴壁细胞数目递减,细胞贴壁所用时间也延长。所有孔中最早出现的贴壁细胞为树突状细胞,这些细胞开始生长缓慢,大约4天后生长迅速,10天后部分孔出现鱼群样细胞团。部分孔经Nestin、SOX10抗体的免疫细胞化学染色结果均为阳性,其中以Nestin抗体较明显。结论:人表皮细胞对胰酶消化的耐受能达46小时,从形态学观察判断,黑素细胞贴壁早于角质形成细胞,大多数贴壁后细胞增殖力强,干细胞表面标记显示部分阳性。     

钙离子对鼠角质细胞生长和分化的影响

用无血清培养基培养角质细胞,研究了Ca²⁺对鼠角质细胞生长和分化的影响。实验结果表明,培养基中钙离子最佳浓度为0.2mmol/L。在此浓度下,细胞克隆形成率达到10.8%,细胞的贴壁率达到28.7%,细胞的分化比例和老化比例分别为5.4%和26.3%;当Ca²⁺浓度达到0.6mmol/L以上时,则会引起角质细胞显著的分化和老化。     

东方田鼠皮肤成纤维细胞的分离培养及生物学特性

目的探索和建立东方田鼠皮肤成纤维细胞体外分离、培养的技术方法并观察其生物学特性。方法采用含10%小牛血清的Dulbecco改良Eagle培养液(DMEM)和1640两种培养体系,运用组织块贴壁法和胰酶消化法,分别对出生后1、3 d和5 d的东方田鼠乳鼠皮肤成纤维细胞进行原代分离、培养。苏木素-伊红染色及倒置相差显微镜下观察成纤维细胞形态和生长特性。结果 0.25%胰酶消化分离出生后1 d和3 d东方田鼠乳鼠皮肤较出生后5 d组织可获得较多数量细胞,成纤维细胞在体外快速贴壁生长,一般6～7 d长满培养瓶,细胞纯度高,HE染色细胞呈长梭形,胞核明显;DMEM和1640两种培养液均可用于东方田鼠皮肤成纤维细胞的培养,但细胞传代后生长趋缓,只可传代2～3次。本实验运用组织块贴壁法未能培养出皮肤成纤维细胞。结论确定了有效分离东方田鼠皮肤成纤维细胞的日龄和方法,为进一步深入研究提供了技术方法和操作依据。     

培养基血清浓度对人表皮干细胞增殖分化的影响

目的:比较不同血清浓度培养体系对表皮干细胞增殖分化的影响.方法:采用两步酶消化法和IV型胶原差速贴壁相结合的方法获得人原代表皮干细胞,分别以0%、5%、10%、15%和20%血清浓度的培养基在96孔板中进行培养.观察表皮干细胞形态,克隆形成及增殖的情况,应用四甲基偶氯唑蓝(MTT)比色法检测各组细胞存活和生长情况,分析量效和时效关系;持续传代培养细胞,每次传代的同时取适量细胞,用免疫细胞化学的方法进行表皮干细胞和表皮细胞相应标志物(K19、K14和K10)的测定.结果:表皮干细胞在各种血清浓度的培养基内均能形成克隆,增殖良好.用四甲基偶氮唑蓝(MTT)比色法测定,所得相同时间点各组OD值在统计学上没有差异(P>0.05),表皮干细胞生长速度各组间无差异.第1代表皮干细胞K19均有表达,而K14和K10表达均为阴性;其后高血清浓度(15%、20%)培养基中细胞较低血清浓度(0%、5%)先出现K14、K10蛋白的表达;培养至第10代是各组细胞均出现K10高表达,而K19、K14表达阴性.结论:在低血清浓度(0%、5%)的培养基中表皮干细胞生长良好,且能够相对较好保持表皮干细胞的特性.     

小鼠表皮干细胞的分离与培养

目的:探讨体外分离和培养小鼠表皮干细胞和分析表皮干细胞克隆形成能力的方法。方法:采用中性蛋白酶和胰酶消化新生小鼠表皮基底层细胞,将细胞直接接种在细胞瓶中,在无滋养层条件下培育;利用表皮干细胞标记物K15和α6整联蛋白进行免疫荧光鉴定;以小鼠胚胎成纤维细胞作为滋养层与成年小鼠角质细胞共培养,进而分析表皮干细胞的克隆形成能力。结果:新生小鼠表皮干细胞克隆在培养2～3 d后开始形成,细胞核质较小,细胞呈小而圆的形态特征;传代后的细胞可以被K15和α6整联蛋白特异性标记。结论:利用该方法能够实现对小鼠表皮干细胞的体外培养和传代。     

介绍一种新的表皮细胞传代方法

我们设计了一种新的传代方法,在传代之前,先用 DispaseⅡ酶,将培养的表皮细胞片整个消化下来,再用0.25—0.3%胰酶冷消化将表皮细胞驱散,进行1:2传代获得成功,传代59次成功率为71.2%。若在培养后7—16天传代,成功率可达80%。本文讨论了此种传代方法比单纯用胰蛋白酶或用胰蛋白酶和EDTA 混合传代方法优越。     

抗氧化剂对皮肤角质细胞体外寿命的影响

考察了抗氧化剂对鼠角质细胞体外培养寿命的影响。实验发现在角质细胞的体外培养过程中添加抗氧化剂有利于延长细胞的寿命,其中效果最好的是巯基乙醇,其次为过氧化氢酶和SOD,但在体外培养过程中,角质细胞生长速率仍然逐渐下降。实验还发现,添加抗氧化剂可在一定程度上提高角质细胞的克隆形成率,减缓细胞衰老速率。同时,通过考察鼠表皮角质细胞衰老动力学,获得了对应于不同抗氧化剂的细胞衰老动力学常数。      

豚鼠肾小管上皮原代细胞的培养

目的建立一种简便易行的豚鼠原代肾小管上皮细胞培养方法。方法运用筛网分离法和多种酶消化法获取高纯度的肾小管上皮细胞。利用免疫组化法和形态学观察法鉴定培养的肾小管上皮细胞性质及纯度。结果通过肾小管节段贴壁,胶原酶消化组织节段和细胞等方法,有效地促进肾小管原代细胞增殖;胰酶节段消化法的细胞贴壁效果稍差,细胞传代状态不理想;胰酶消化法则细胞贴壁较少,细胞生长状态较差。结论培养豚鼠原代。肾小管上皮细胞是可行的。     

仔猪睾丸支持细胞的体外生物学特性研究

为研究仔猪睾丸支持细胞生物学特性,以3~5周龄仔猪睾丸为材料,优化消化和差异贴壁方法,分离纯化支持细胞,研究其体外培养特性包括增殖活力、标志分子和细胞因子的表达。结果显示,先以0.25%胶原酶IV和0.1%透明质酸酶消化60 min,再以0.25%胰蛋白酶和0.1%DNase I消化20 min,有利于获得支持细胞;支持细胞接种后贴壁时间主要集中在3~5 h;纯化后细胞胞体宽大,呈梭形或不规则形;GATA4染色呈阳性,碱性磷酸酶染色呈阴性;RT-PCR结果表明,细胞表达GATA4、SOX9和INHB,不表达CYP17、3β-HSD和α-SMA;ELISA结果表明,细胞表达GDNF、LIF、CSF-1、EGF和FGF2,符合支持细胞的基本特征。     

牛肾组织及其培养物的不同消化法效果评价

为了比较不同消化液及消化方式对牛肾皮质组织和培养后形成单层细胞的消化分散效果并确定最适消化液和消化方式,分别用0.25%胰蛋白酶和0.25%胰蛋白酶-0.02%EDTA两种消化液,消化牛肾皮质组织及其培养形成的单层牛肾皮质细胞。牛肾皮质组织采用热(37℃)和冷(4℃)两种消化方式;经培养形成单层的牛肾皮质细胞采用室温(25℃)消化。结果显示,用0.25%胰蛋白酶-0.02%EDTA消化液热消化牛肾组织时,分散获得牛肾皮质细胞的活细胞数、存活率、贴壁率均优于其他消化方法,差异显著(P0.05)。培养形成的单层牛肾皮质细胞用0.25%胰蛋白酶-0.02%EDTA消化液的消化速度明显快于用单一0.25%胰蛋白酶消化液的消化速度,统计学分析显示具有显著性差异(P0.01),两种消化液消化所得细胞的存活率及贴壁率前者要更高,差异显著(P0.05)。     

Electron microscopic-radioautographic study of cultured human epidermocytes

A split thickness skins of burned patients were treated by trypsin and received the epidermocyte suspension. In that suspensions the content of basal keratinocytes was 10 to 70% in dependence from the treating conditions. As a rule, cultivation was a success only in those cases, when among basal keratinocytes of initial suspension were cells, synthesized DNA. Cultures were grown more rapidly under decrease of ion calcium concentration in nutritional medium up to 0.14 mM and in the presence of cell-free surface of substratum. At such conditions connections between cells were weakened, desmosomes disappeared, stratification and colony formation were broken.     

人表皮细胞分离培养的研究

目的探索来源于人的表皮细胞的分离培养方法,为其进一步作为皮肤组织工程中的种子细胞或临床应用奠定前期研究基础。方法取3~9岁健康儿童包皮环切术后包皮,经分离酶处理分离真表皮,再将表皮以胰蛋白酶消化为细胞悬液,分别接种于有血清培养基DMEM和无血清培养基K-SFM中进行细胞培养,观察表皮细胞生长融合情况及克隆形成率。结果表皮细胞在DMEM和K-SFM培养液中均能融合成片,但在K-SFM中的融合成片时间明显短于在DMEM中所需时间;在K-SFM中2周时克隆形成率显著高于在DMEM中的克隆形成率。结论两步酶法分离表皮细胞接种于K-SFM中培养,是一种简便有效的表皮细胞分离培养方法。     

低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制研究

目的初步探讨低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制。方法甲基纤维素集落形成实验检测低浓度丰加霉素对人白血病K562细胞集落形成能力的影响;CCK-8法检测低浓度丰加霉素对K562细胞的生长抑制率;AnnexinV／PI双染流式细胞仪检测低浓度丰加霉素作用下的K562细胞凋亡率;PI单染流式细胞仪检测药物作用后细胞的周期分布改变;Western免疫印迹和实时定量PCR检测周期相关分子表达水平变化。结果低浓度丰加霉素对人白血病K562细胞具有较强的集落形成抑制作用;可明显抑制K562细胞的生长,呈时间一剂量依赖性;尽管短时间（48h）的药物处理仅出现轻度的细胞凋亡和周期阻滞,但10nmol/L和30nmol/L的丰加霉素长时间（7d）作用后,K562细胞G0／G1期比例分别是（62．3±1．7）％和（76．9±0．7）％,与对照组（38．9±1．1）％相比差异具有高度统计学意义（P〈0．01）;低浓度丰加霉素长时间作用后诱导K562细胞周期相关分子P16蛋白水平和转录水平的高表达。结论丰加霉素在低浓度,长时间作用于人白血病K562细胞后,具有较强的集落形成抑制和生长抑制作用,此作用可能与诱导细胞周期相关分子p16高表达,导致细胞G0／G1期阻滞有关。     

Elevation of cell cycle control proteins during spontaneous immortalization of human keratinocytes.

A human line of spontaneously immortalized keratinocytes (SIK cells) has been derived from ostensibly normal epidermis and has proven useful in dissecting molecular changes associated with immortalization. The original cultures had a normal karyotype and a colony forming efficiency of approximately 3% through 10 passages. At passage 15, after which normal strains ordinarily senesce, these cells continued vigorous growth and gradually increased in colony forming efficiency, stabilizing at approximately 30% by passage 40. During the early stage of increasing colony forming efficiency, the cells acquired a single i(6p) chromosomal aberration and 5- to 10-fold increases in expression of the cell-cycle control proteins cyclin A, cyclin B, and p34cdc2. Additional chromosomal aberrations accumulated at later passages (i(8q) and +7), but the i(6p) and the increased expression of cell-cycle proteins were maintained, raising the possibility that these features were important for immortalization. Regulation of cell growth and differentiation in the cultures appeared minimally altered compared with normal keratinocytes as judged by their microscopic appearance and generation of abortive colonies, sensitivity to growth suppression by transforming growth factor-beta and tetradecanoylphorbol acetate, and dependence upon epidermal growth factor for progressive growth.     

CO2/bicarbonate stimulates growth independently of PH in mouse mammary epithelial cells

Mouse mammary cells of the NMuMG line proliferated faster and formed colonies more efficiently when the air above the cells contained 5% CO2. An increase in colony forming efficiency also occurred if the bicarbonate concentration in the medium was higher (44 versus 13 mM). These growth increases induced by the CO2 or bicarbonate occurred even when the control cultures were maintained at the same pH, and they occurred at every pH tested. Both the growth rate and colony forming efficiency of the NMuMG cells were highest at pH 7.0 to 7.3.     

Stability of enzymes in water/organic co-solvent mixtures

The half lives of polyphenol oxidase and trypsin at 50% (v/v) co-solvent (water miscible) were from 23 to 176 h when polarity index of the co-solvent was >5.8 or if the co-solvent was an alcohol. Polyphenol oxidase, trypsin, chymotrypsin and -glucosidase lost only 10% activity in such water/organic co-solvent mixtures when the organic co-solvents were present in the concentration ranges of 55–90%, 60–78%, 15–70% and 7–25% respectively. © Rapid Science Ltd. 1998     

Effect of Trypsin Treatment on the ATPase Activity of Myofibrils from the Stored Rabbit Muscle

The extent of activation of myofibrillar ATPase activity by trypsin treatment has been measured.

When myofibril (5 mg/ml) was treated with a low concentration of trypsin (2.5 μg/ml), the Mg-modified ATPase activity of myofibrils at a low ionic strength increased appreciably, while the EDTA-enhanced ATPase activity of myofibrils at a high ionic strength did not change with the progress of trypsin digestion.

The dependence of myofibrillar ATPase activity on KCl concentration also became greater with the progress of trypsin digestion.

Trypsin treatment caused 5-fold increase in the Mg-modified ATPase activity of 0-myofibril, when treated with trypsin in a ratio of 1 to 2000 myofibril for 80 min. Under the same condition, the ATPase activity of 1-myofibril increased by about 150%, whereas that of 8-myofibril increased by approximately 50%.

When myofibrils were treated with trypsin in a ratio of 1 to 200 myofibril, the Mg-ATPase activity of 8-myofibril decreased earlier than that of 1-myofibril did by about 20 min.

Experimental results obtained in this study were enough to confirm that the myofibrils from the aged muscle are more susceptible to tryptic action.

An assumption was made that the structural alteration of myofibrils during aging might be attributed to the change in thin filament of myofibrils, including Z-lines, which are mainly due to the change in the native tropomyosin of thin filaments.     

Ciliary elongation in blastulae of Arbacia punctulata induced by trypsin

Hatched sea urchin blastulae, which have primarily short 25-μm cilia except for some long 40-to 70-μm cilia at the apical tuft, were induced to form long (40- to 70-μm) cilia around most of their circumference when treated with trypsin (0.008–0.1%) or concanavalin A. Other animalizing agents did not induce the formation of long cilia when applied to the normal blastulae. The formation of long cilia by trypsin was both time and concentration dependent. The long cilia first appeared around the apical tuft after 6–8 hr in trypsin (21°C), and by 18–22 hr most of the blastula was covered with the long cilia. Length distribution studies on cilia isolated at various times showed that the percentage of long cilia increased from approximately 10% in the normal blastula to over 66% in the 22-hr trypsin-treated embryo, and indicated that the long cilia formed by the elongation of the original short cilia. Only the blastulae and gastrulae could be induced to form long cilia; the prisms and plutei could not. Once development was inhibited by the trypsin and the first long cilia appeared, the trypsin effect could not be reversed. When blastulae with long cilia were removed from the trypsin for 10 hr, the cilia remained long; when the long cilia were detached, the blastulae regenerated long cilia in the absence of trypsin. The induced long cilia moved poorly, similar to the long, apical tuft cilia of normal embryos. The formation of long cilia by trypsin treatment of sea urchin blastulae provides a model system for studying the mechanisms of ciliary length control.     

The inter-relationship between anchorage independence and tumorigenicity in early cultures of oral keratinocytes

This study examines the expression of anchorage independence and tumorigenicity in early cultures of oral rat keratinocytes. The epithelial cell lines originated from the palatal and the lingual mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. The colony forming efficiency (CFE) in gel culture of the cell lines derived from five squamous cell carcinomas of the tongue and palate predominantly increased with passage in culture. Carcinoma-derived cell lines that had a relatively high CFE (greater than 2.5%) formed tumours when transplanted to athymic mice, but cells in which the CFE was less than 2.5% were non-tumorigenic. Keratinocytes from a dysplastic palatal lesion were immortal, anchorage dependent and non-tumorigenic. A lingual papilloma cell line consistently expressed a very low CFE but was tumorigenic at the higher culture passages. The results show that the routine passage of cells in culture leads to the emergence of the anchorage independent and tumorigenic phenotypes in keratinocytes of malignant origin and, further, suggest that anchorage independence and tumorigenicity may exist as distinct phenotypes, with anchorage independence preceding tumorigenicity.