新型编织型生物反应器内大量乳猪肝细胞的组织化培养

目的:使反应器内培养的肝细胞能在较长时间保持肝细胞的特殊功能和活力,提高生物反应器的功效并为反应器内储存和运输肝细胞提供可能。方法:将新生实验型小猪肝细胞与微载体共同培养,待肝细胞与微载体充分粘附形成微载体-球形聚集体后,将其置入新型编织型生物反应器的外腔,用培养液循环式人工毛细管培养系统进行培养,在培养液内加入氯化氨、醋胺酚检测生物反应器的转化功能,行无血清培养检测肝细胞的白蛋白的合成功能,观察肝细胞的酶漏出量。锥虫蓝染色观察肝细胞的活力,电镜观察其超微结构。结果:生物反应器内培养的肝细胞在1周内能保持较高的活力,并保持着较高的氯化氨、醋胺酚的生物转化及白蛋白合成功能,酶的漏出量也少。肝细胞超微结构示内质网、线粒体等细胞器丰富,核内染色体分布均匀,肝细胞间的微绒毛形成胆小管样结构。结论:肝细胞与微载体及肝细胞之间的聚集,形成直径大小不等的肝细胞-微载体球形聚集体,这种由肝细胞重新结合而成的聚集体类似于体内的肝组织结构,在新型编织型生物反应器内的中空纤维网架的支持下,肝细胞聚集体均匀地分散其中,为肝细胞的生长提供了一个类似体内内环境的三维空间,因而在无氧合器供氧的情况下,肝细胞也能在1周内保持较好的形态和功能。     

两种人工肝生物反应器内乳猪肝细胞培养的比较研究

目的 采用普通型中空纤维及编织型中空纤维两种人工肝生物反应器,比较其内培养的肝细胞生存时间及功能,为生物反应器的设计提供新的方法和思路.方法 将新生中国实验小型猪的肝细胞与微载体共同培养,待肝细胞与微载体充分粘附形成微载体-球形聚集体后,将其注入生物反应器的外腔,在培养液内加入氯化氨、醋胺酚检测生物反应器的生物转化和代谢功能,行无血清培养检测肝细胞白蛋白的合成功能,并检测肝细胞酶的漏出量.锥虫蓝染色测定肝细胞的活力,透射电镜观察其超微结构.结果 肝细胞与微载体相互聚集,形成了具有一定结构层次的肝细胞聚集体,重构了肝细胞之间的相互连接;接种入生物反应器后,在编织型生物反应器中肝细胞1周内保持较高的活力,至第7天肝细胞活力仍高达(75±6.3)%,对醋氨酚和氯化铵有良好的代谢、生物转化功能,白蛋白合成在第7天为(0.57±0.13)mg/106个肝细胞,酶的漏出量也少,各项指标均明显优于普通型生物反应器组(P＜0.05).结论 编织型生物反应器为肝细胞的生长、增殖提供一个类似体内的三维立体环境,是一种性能良好的生物反应器.     

微载体培养人肝细胞用于人工肝的初步研究

探讨微载体培养人肝细胞用于人工肝的可能性。方法 用微载体Ｃｙｔｏｄｅｘ３培养人体肝细胞，将其置于中空纤维型生物反应器构成人工肝装置，在体外循环实验中进行生物功能及细胞活力的观察与评价。结果 体外两步法分离的人肝细胞，在辅助促聚条件下有效地粘附于微载体上，呈悬浮生长状。     

正常原代猪肝细胞方瓶微载体静止培养

目的初步探讨微载体培养方法作为人工肝中生物材料培养方法的可能性．方法将５×１０７的肝细胞及２ｇ／Ｌｃｙｔｏｄｅｘ３微载体连同５０ｍＬ含１００ｍＬ／Ｌ猪门静脉血清及其他附助因子的ＤＭＥＭ培养基加入１００ｍＬ的培养方瓶中培养．光镜及电镜下观察细胞生长情况,检测培养上清中Ａｌｂｕｍｉｎ,Ｕｒｅａ浓度．结果接种的肝细胞在接种时即呈明显的朝Ｃｙｔｏｄｅｘ３聚集,二者极易相粘贴．在培养后２４ｈ～４８ｈ,可见典型的多细胞聚集球形成,该形态特点及Ａｌｂｕｍｉｎ,Ｕｒｅａ合成能力可保持８ｗｋ左右．结论使用微载体培养猪肝细胞具有多种优点．微载体培养的肝细胞可望作为生物人工肝的生物材料     

人肝细胞的微载体培养及其意义

目的：探讨大量培养高活性人肝细胞的理想方法，为肝脏病研究提供良好的细胞材料。方法：在限制贴壁、定时振荡条件下，采用载体ｃｙｔｏｄｅｘ３为材料进行人肝细胞的微载体培养。结果：预先将体外两步灌流分离的人肝细胞振荡３０分钟后，肝细胞易与微载体相粘附．在被覆聚羟乙基异丁烯酸的培养瓶中继续间歇性振荡２４小时，约７０％的肝细胞附着于微载体之上。在使用激素限定条件培养液培养的１５天内，肝细胞保持蛋白分泌功能和多细胞粘附聚集球形体形态特征。结论：本法培养人肝细胞具有细胞数量多、密度大和活性好的特点，适宜于体外生物人工肝支持系统、肝细胞移植以及培养肝细胞因子的提取。     

明胶、丝素-壳聚糖微载体培养肝细胞的比较研究

目的自制丝素-壳聚糖多孔微载体,在模拟微重力条件下接种培养肝细胞,并与多孔微载体Cultispher S上的细胞生长状况进行比较。方法油包水乳化后以冻干法制作丝素-壳聚糖多孔微载体。培养体积50ml,以旋转培养系统分别对接种至丝素-壳聚糖多孔微载体和Cuhispher S的CL-1细胞进行培养,并动态观察形态学、细胞计数,检测人源性白蛋白分泌和尿素合成功能。结果CL-1细胞在Cuhispher S上呈单层生长,在丝素-壳聚糖多孔微载体上呈现多层生长。第9天细胞计数结果达到峰值,丝素-壳聚糖多孔微载体的细胞密度可达到5．7×10^6个／ml,明显高于Cuhispher S组（P〈0．01）。自培养第10天起,丝素-壳聚糖多孔微载体组上清中的人源性白蛋白和尿素水平均明显高于Cuhispher S组（P〈0．01）。结论相对Cuhispher S,丝素-壳聚糖多孔微载体更适合在微重力条件下培养CL-1细胞。     

中空纤维生物反应器的建立及其功能的初步鉴定

目的构建生物反应器并评估其生物学功能。方法采用自主建立的永生化HepG2细胞系为生物材料,以中空纤维构建生物反应器,观察肝细胞在生物反应器内生长情况,同时进行肝细胞功能检测,以肝衰竭患者血浆置换出的废弃血浆作为试验因素,试验性行生物人工肝治疗。结果该系统内肝细胞生长良好,每个生物反应器培养72h后细胞数量达7．60×10^8个,肝细胞活力保持90％以上。AST、LDH低水平波动;利多卡因转化实验良好;对细胞培养液进行细菌培养监测及支原体检测,均未发现异常。应用肝衰竭患者血浆500ml培养肝细胞6h,总胆红素平均下降98．6μmol／L。结论建立的永生化肝细胞系应用中空纤维构建生物反应器,肝细胞功能良好,经进一步改进有望应用于生物人工肝治疗。     

在线微载体肝细胞荧光示踪研究

目的拟探讨SYTO/EB在微载体细胞培养中活体细胞原位示踪的应用价值。方法采用活细胞核酸染色剂(SYTO13)和溴化乙啶(EB)2种核酸染色剂对微载体生长的细胞进行双重染色,荧光显微镜下通过颜色不同判断细胞的活力状态并计算出活力百分比。以间接活力测定的四氮唑盐法同时对肝细胞进行活力的检测,并将结果进行比较。结果 2种方法测定的结果均可显示微载体生长的肝细胞。但SYTO/EB法更灵敏,可精确计数。结论 SYTO/EB测定微载体肝细胞的活力快速、灵敏且简便,并可原位观察细胞的活力,不失为一种可取的微载体活力检测方法。     

微载体粘附培养的肝细胞腹腔内移植治疗急性肝功能衰竭大鼠的实验研究

目的　评价微载体粘附培养的肝细胞腹腔内移植对D 氨基半乳糖盐酸盐诱导的大鼠急性肝功能衰竭的治疗作用。方法　胶原酶灌注分离大鼠肝细胞 ,行Cytodex 3粘附培养 ,移植 1× 10 7个微载体粘附肝细胞至D 氨基半乳糖盐酸盐诱导的急性肝功能衰竭大鼠腹腔内 ,比较各组间生存率、肝功能及肝脏病理改变情况以评估疗效。结果　微载体移植组大鼠存活率明显高于空载体组 ,移植 5d后两组比较差异显著 (P <0 .0 5 ) ;空载体组大鼠肝功能及肝脏修复明显迟于微载体组。结论　微载体粘附培养的肝细胞腹腔内移植可对急性肝衰竭大鼠提供代谢支持作用 ,提高急性肝衰竭大鼠存活率。     

腹腔移植微载体粘附人肝细胞对急性肝衰竭小鼠的保护作用

为了探讨腹腔移植微载体粘附培养人肝细胞对D氨基半乳糖(GalN)诱导的急性肝衰竭小鼠的保护作用．本实验采用胶原酶灌流法分离人肝细胞，胶原被覆的微载体Cytodex3加以培养，经腹腔注射肝细胞及载体，观察其对急性肝衰竭小鼠的影响。结果显示．与只接受微载体而无粘附肝细胞的对照组比较，腹腔移植培养肝细胞对肝衰竭小鼠的存活率有明显改善，其血清ALT及总胆红素较低(P<0．05)。肝组织病理改变较轻。该初步观察结果提示．腹腔移植微载体牯附培养肝细胞可提供代谢支持作用，从而使GalN损伤的小鼠肝功能得以恢复。     

Regulation of adrenocortical cell proliferation in culture

The regulation of the proliferation of adrenocortical cells in culture is reviewed. The hormones and growth factors affecting adrenocortical proliferation in culture and their physiological relevance are discussed. The following general conclusions are made: (i) ACTH is growth-stimulatory in vivo, but directly replication-inhibitory both in culture and in vivo, and is therefore an indirect mitogen. (ii) Insulin, IGFs, some pituitary and brain growth factors, and other unknown factors in serum, stimulate growth in culture, but their role in control of adrenocortical size in vivo is unknown. (iii) Pure, known pituitary hormones, other than ACTH, have no effect on adrenocortical proliferation in culture. (iv) Angiotensin is mitogenic in culture and perhaps also in vivo under some circumstances.     

人工肝生物材料人肝细胞系CL-1的微载体培养.

目的使用微载体培养人肝细胞系ＣＬ１,并观察细胞的转化及合成功能．方法使用Ｃｙｏｔｄｅｘ３微载体进行人肝细胞系ＣＬ１的高密度培养,应用光镜及扫描电镜于培养１,３,５,７,９ｄ动态观察细胞的生长情况,并同时检测培养系统的安定转化及清蛋白合成量．结果培养７ｄＣＬ１细胞密度达到最高,为２１６×１０９／Ｌ;安定转化量最高为６１９μｇ;清蛋白合成量最高为７８μｇ．结论使用微载体培养高分化人肝细胞系可达到较高的密度,且具有较好的生物转化及合成功能,可望作为组合性人工肝的生物材料     

Antioxidants stimulate endothelial cell proliferation in culture

The effect of antioxidants on the proliferation of cultured endothelial cells (ECs) were studied. Probucol, a lipid-lowering drug with antioxidant properties, added to the growth medium stimulated the proliferation of ECs. D1-alpha-tocopherol (tocopherol), a natural antioxidant, and butylated hydroxytoluene, a synthetic antioxidant also had the same effect on the proliferation of ECs. There was no significant difference in the content of thiobarbituric acid-reacting substances between the probucol-containing growth medium and control medium during the growth assay. Pretreatment of ECs with probucol or tocopherol also enhanced ECs proliferation in medium without any added antioxidants. The addition of free radical scavengers superoxide dismutase, catalase and mannitol to the growth medium did not enhance the proliferation of ECs. These findings indicate that the stimulatory effect on ECs proliferation of antioxidants in the growth medium appears to be unrelated to preventing free radical reactions in the medium itself, but rather may be related to the uptake of antioxidants by ECs.     

The induction of smooth muscle cell proliferation in vitro using an organ culture system

Agents which promote vascular smooth muscle cell (SMC) proliferation are still unknown and cannot be readily defined in vivo. To examine this problem, we have developed a technique to maintain arteries in culture for more than two weeks without loss of endothelium and without loss of contractility. These vessel segments were maintained in 30% calf serum, and yet SMC replication rate in the media was found to be below 0.1% per day. No loss of medial cells nor medial necrosis was observed. Gentle endothelial denudation did not cause intimal thickening. If, in addition to denudation, direct mechanical injury was applied, then the replication rate of intimal SMC was found to be 60% per day during the first week. These cells were confirmed to be of SMC origin using cell specific antibodies. Since SMC start to proliferate after mechanical injury and not after application of serum, we conclude in our experimental model that exogenous growth factors play a minor role in the process of induction of proliferation of these cells.     

Inhibited effects of veliparib combined doxorubicin for BEL-7404proliferation of human liver cancer cell line

Objective:To explore inhibition effects of veliparib as PARP inhibitor combined doxorubicin for BEL-7404 proliferation of human liver cancer cell line.Methods:BEL-7404 was taken as the object of study and conventional culture was performed.It waslreated by doxorubicin and(or) veliparib after 24 h.Cell proliferation rate was detected by four methyl thiazolyl tetrazolium(MTT) assay,cell apoptosis was measured with annexin V-F1TC/PI double staining method by flow cytometry,DNA damage degree evaluation by single cell gel electrophoresis assay,and cytosolic C levels of the mitochondrial and cytosol by polyacrylamide gel electrophoresis(Western blotting).Results:Cell proliferation rate of doxorubicin combined veliparib group was lower than that of the control group and doxorubicin alone treated group significantly(P0.01),the apoptosis rate was significantly higher than that of the control group and doxorubicin alone treated group(P0.05).At the same time,DNA damage level of doxorubicin combined with veliparib group was significantly higher than doxorubicin alone treatment group and the control group(P0.01),and cytochrome C in the cytosol was significantly higher than that of control group and doxorubicin alone treated group(P0.01).Conclusions:Veliparib,PARP inhibitor could inhibit PARP activity,block tumor cell DNA repair,and have significant sensitizing effect for hepatocellular carcinoma cell line BEL-7404 treated with doxorubicin.This might provide a new target for clinical treatment of hepatic carcinoma.     

Regulation of human colonic cell line proliferation and phenotype by sodium butyrate

Colonic butyrate may maintain mucosal differentiation and oppose carcinogenesis. We characterized butyrate effects on differentiation, proliferation, and matrix interactions in Caco-2 and SW620 human colonic cells. Differentiation was assessed by brush border enzyme activity and doubling time by serial cell counts. Motility across matrix proteins was quantitated by monolayer expansion and correlated with adhesiveness to matrix. Integrin subunit surface pools were measured by immunoprecipitation. Butyrate-stimulated differentiation inhibited proliferation and was significantly more potent than acetate in this regard. Butyrate also inhibited motility across collagen I, collagen IV, and laminin, as well as decreasing adhesiveness to these matrices and β1, α1, and α2 integrin subunit surface expression. Butyrate acts in cultured cells at clinically relevant concentrations to oppose classical malignant behavior, inhibiting proliferation and motility while promoting differentiation. Since butyrate is derived from fermentation of dietary fiber, such mechanisms may contribute to the apparent protective action of fiber against colon carcinogenesis.     

Telomerase activity in HeLa cervical carcinoma cell line proliferation

Normal human somatic cells in culture have a limited dividing potential. This is due to DNA end replication problem, whereby telomeres shorten with each subsequent cell division. When a critical telomere length is reached cells enter senescence. To overcome this problem, immortal HeLa cell line express telomerase, an enzyme that prevents telomere shortening. Although immortal, the existence of non-dividing cells that do not incorporate ³H-thymidine over 24 h of growth has been well documented in this cell line. Using DiI labeling and high-speed cell sorting, we have separated and analyzed fractions of HeLa cells that divided vigorously as well as those that cease divisions over several days in culture. We also analyzed telomerase activity in separated fractions and surprisingly, found that the fraction of cells that divided 0–1 time over 6 days in culture have several times higher endogenous telomerase activity than the fastest dividing fraction. Additionally, the non-growing fraction regains an overall high labeling index and low SA-β-Gal activity when subcultured again. This phenomenon should be considered if telomerase inhibition is to be used as an approach to cancer therapy. In this paper we also discuss possible molecular mechanisms that underlie the observed results.     

A simple technique for large scale in vitro culture of Plasmodium falciparum.

The large scale in vitro cultivation method of Fairlamb et al (1985) was modified to contain human plasma-supplemented medium in HEPES buffer. After 4 days with no change of medium nor agitation of the culture flask, 27- to 50-fold increase in the starting parasitemia of trophozoites were obtained.