大鼠肝细胞的微载体黏附培养及其功能测定

目的探讨一种简单实用地获取大量高活性,高密度肝细胞的培养方法。方法用我们自行设计的半原位酶消化技术对成年 SD 大鼠肝细胞进行分离与微载体黏附培养,连续观察肝细胞的形态,检测肝细胞的白蛋白分泌功能和糖合成功能。并同时与贴壁培养的肝细胞比较。结果微载体黏附培养的肝细胞在存活率,保持正常形态和白蛋白分泌功能,葡萄糖合成功能方面都保持较高水平,明显优于贴壁培养的肝细胞。结论微载体培养能提供长时间保持高活性,高密度生长的肝细胞,为肝细胞移植,肝病防治以及生物人工肝等领域的研究和广泛应用提供了基础。     

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。     

旋转生物反应器内微载体大规模扩增肝细胞

目的 评估旋转生物反应器(RCCS)内肝细胞微载体大规模培养的可行性.方法 为获取足够数量活性良好的肝细胞种子细胞.本实验探索在RCCS内应用微载体技术快速扩增肝细胞的方法 .将cL-1细胞和HepFMMU应用Cytodex-3微载体在RCCS内进行动态培养,观测肝细胞的生长情况和细胞代谢率.结果肝细胞在Cytodex-3微载体上生长迅速、生长倍增时间缩短,并保持很高的生物活性.培养至第5天,细胞数量可达最初接种的23倍.细胞的数量在第5天左右达到最高,然后开始下降.功能学检查、倒置显微镜、扫描电镜及MTT均提示细胞功能良好.结论 RCCS微载体肝细胞大规模培养是一种可行的细胞快速扩增方法 ,但对于人工肝庞大的细胞数量和功能需求,应进一步研究,通过优化营养和废物排除,模拟肝脏生理结构,提高细胞培养规模和功能.     

荧光染色法对分枝杆菌活力鉴定及细菌计数技术的研究

本文应用FDA/EB荧光染色法,对BCG等分枝杆菌的活力鉴定作了深入的研究。在菌团分散的方法、60℃水浴对分枝杆菌活力的丧失和其它分枝杆菌适用性方面进行了探讨。本文还详细介绍了一种大头针微滴涂膜的数菌方法。FDA/EB荧光染色和数菌方法联合应用,可用来检测分枝杆菌的活菌数和总菌数,适用于BCG等分枝杆菌菌苗生产和实验室研究。     

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     

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。     

微载体培养人肝细胞及肝非实质细胞

目的研究用微载体培养人肝细胞及肝非实质细胞的方法．方法采用体外简易两步灌流和差速离心法获取胎肝细胞和肝非实质细胞，在综合限定条件下进行微载体ｃｙｔｏｄｅｘ３粘附培养，并对培养肝细胞的形态及合成葡萄糖和白蛋白的功能进行动态测定．结果分离肝细胞及肝非实质细胞在接种时即呈明显的聚集倾向，将其与微载体混合振荡孵育２０ｍｉｎ后，二者极易相粘附．在被覆聚羟乙基异丁烯酸的培养瓶内，使用激素限定条件培养液培养约４８ｈ，典型的多细胞聚集球形体得以形成．该形态特征以及白蛋白、葡萄糖合成能力可保持１月结论使用微载体培养人肝细胞和肝非实质细胞具有多种优点，有广泛的应用价值．     

原代猪肝细胞的分离获取及培养

目的研究猪肝细胞的获取率、活率．观测普通培养过程中肝细胞形态变化过程．方法取用本地杂种小猪作为肝细胞供体．用Ｓｅｇｌｅｎ胶原酶二步灌注法,原位灌注分离获取肝细胞悬液,ＴＢ染色法计算细胞数及细胞活率．在含１００ｍＬ／Ｌ胎牛血清及其他附助因子的ＤＭＥＭ培养基中培养,光镜下观察培养过程中肝细胞形态变化过程,检测不同时期培养上清中清蛋白、尿素的浓度．结果平均猪肝重４３４ｇ,每只猪肝平均可获取２１８×１０１０个肝细胞,平均活力为９４６％．在ＤＭＥＭ培养基中普通培养可存活４ｗｋ～５ｗｋ,并具有生物活性．结论本方法分离获取肝细胞产率高、活性高,可作为人工肝较好的生物材料．     

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。     

Staining tissue-derived Mycobacterium leprae with fluorescein diacetate and ethidium bromide

A fluorescent staining procedure incorporating the use of fluorescein diacetate (FDA) and ethidium bromide (EB) has previously been shown to accurately measure the viability of saprophytic mycobacterial cells. Green-stained cells were shown to be viable and red-stained cells, dead. Staining Mycobacterium leprae cells with FDA/EB, however, was complicated by interfering tissue components which masked the presence of stained bacteria. A petroleum ether separation technique enables M. leprae to be segregated from armadillo liver tissue components and permitted M. leprae to be stained qualitatively equal to the saprophytic mycobacteria. An alternative and technically simpler method of staining M. leprae from human skin biopsies and mouse foot pads was developed which permitted the initiation of a clinical assessment of the staining method. Preliminary data indicate that patients who have undergone three or 24 months of chemotherapy possess a significantly lower percentage of green-stained M. leprae in their tissues than untreated patients. This would be expected if the FDA/EB staining method was providing an accurate measure of viability. M. leprae cells obtained from mouse foot pads which were harvested 5-13 months post-infection displayed more than 90% green-stained cells. There was no correlation between the FDA/EB staining method and the morphological index.     

溶癌水泡性口炎病毒体外诱导人肝癌细胞HepG2凋亡的作用机制

目的:研究一株实验室减毒水泡性口炎病毒(VSV)对HepG2细胞的凋亡诱导作用,并探讨其可能的作用机制.方法:首先将水泡性口炎病毒以1.0感染复数(MOI)的接种密度感染HepG2细胞,同时设接种相同体积培养液的Mock感染组,在不同时间点收集细胞,MTT法检测细胞增殖活性;AO/EB结合Hoechst/PI染色观察细...     

MORF4基因诱导HeLa细胞衰老

目的建立MORF4基因高表达诱导HeLa细胞衰老模型,探索MORF4基因对HeLa细胞衰老的影响。方法分别将pcDNA3.1(+)/Flag-MORF4和pcDNA3.1(+)空载质粒转染人宫颈癌HeLa细胞株。SA-β-Gal染色检测细胞衰老,MTT法及流式细胞术检测细胞周期变化及细胞的增殖活力,两者共同确定MG-132处理的最适条件。Western印迹检测MORF4、PCNA基因的表达。结果质粒经酶切和测序鉴定,证明pcD-NA3.1(+)/Flag-MORF4质粒中含有目的基因序列;经浓度梯度1.25、2.5、5、10μmol/L MG-132处理转染细胞2、4、24、48 h后,SA-β-Gal染色和MTT检测结果显示10μmol/L MG-132处理24 h的转染细胞明显衰老,细胞活力降低;细胞周期被阻滞在G0/G1期,S期细胞明显减少,增殖受到抑制;Western印迹检测结果显示MORF4蛋白在细胞中的含量明显升高,而与增殖相关基因PCNA表达下调。结论构建的pcDNA3.1(+)/Flag-MORF4质粒在HeLa细胞中表达;并在MG-132作用下,使MORF4基因的表达产物积累,从而影响PCNA蛋白的表达量,抑制HeLa细胞的增殖活性,阻滞细胞周期的进行,导致细胞进入衰老状态。     

Apoptotic lymphocytes and CD34+ cells in cryopreserved cord blood detected by the fluorescent vital dye SYTO 16 and correlation with loss of L-selectin (CD62L) expression

Discrimination between live and apoptotic cells is important for accurate determination of viable CD34(+) cells in hematopoietic stem cell transplant products. SYTO16 is a sensitive fluorescent dye for discriminating live from apoptotic leukocytes. The incidence of apoptotic leukocytes in paired samples of fresh and cryopreserved-thawed cord blood (CB) was determined by the SYTO16/7-AAD flow cytometric assay. Cell migration and expression of the cell homing molecule L-selectin (CD62L) was determined in relation to SYTO16 staining. SYTO16 detected significant proportions of apoptotic lymphocytes and CD34(+) cells in fresh and thawed CB buffy-coat samples that were not detected by 7-AAD. Compared to fresh CB, the proportion of apoptotic lymphocytes and CD34(+) cells significantly increased following thawing. Significantly higher proportions of live SYTO16(bright) lymphocytes and CD34(+) cells were found in the migrated cell population compared to the non-migrated population. Significantly fewer lymphocytes and CD34(+) cells expressed CD62L following thawing. Absence of CD62L expression was strongly correlated with apoptotic/SYTO16(dim) lymphocytes and CD34(+) cells. Cryopreserved-thawed CB contains significant proportions of apoptotic lymphocytes and CD34(+) cells that are not detected by 7-AAD. SYTO16 offers a sensitive method for discrimination of live from apoptotic leukocytes and assists in accurate assessment of CB quality and suitability for use in clinical transplantation.     

Effects of Genotypically Different Strains of Helicobacter pylori on Human Microvascular Endothelial Cells In Vitro

Helicobacter pylori induces a number of disturbances in rodent gastric microcirculation in vivo. These events may result from direct necrotic or apoptotic damage to endothelial cells. This study therefore aimed to investigate the effects of genotypically different H. pylori strains on microvascular endothelial cell (MVEC) viability in vitro. Four H. pylori extracts were prepared from strains with different cagA or vacA status. MVECs were plated into 96-well plates and coincubated with 50 l of extract or vehicle for 24, 48, 72, or 96 hr. An MTT assay quantified overall MVEC viability. The dual labeling of MVECs with propidium iodide and Hoechst 33342 distinguished between necrotic and apoptotic cell death, respectively, and allowed total number of viable cells to be determined. All strains of H. pylori decreased cell viability after 72 and 96 hr. Neither necrosis or apoptosis was observed. Counting total number of viable cells revealed decreased cell proliferation with all strains when compared to controls, again reaching significance at 72 and 96 hr. In conclusion, both the MTT assay and the direct cell counting technique demonstrated that all H. pylori strains induced cytostatic but not cytotoxic effects on MVECs. This suggests that microcirculatory disturbances observed in vivo may not be the result of direct endothelial cell damage. However, inhibition of angiogenesis may explain why ulcer healing is delayed in H. pylori-infected patients.     

Effects of incubation temperature and time after thawing on viability assessment of peripheral hematopoietic progenitor cells cryopreserved for transplantation

Three widely used viability assessments were compared: (1) membrane integrity of nucleated cells using trypan blue (TB) exclusion and a fluorometric membrane integrity assay (SYTO 13 and propidium iodide), (2) enumeration of viable CD34+ cells, and (3) clonogenic assay (granulocyte-macrophage colony-forming units, CFU-GM). Post thaw peripheral hematopoietic progenitor cells (HPC) were incubated at 0, 22, and 37 degrees C for 20-min intervals before assessment. The recovery of viable nucleated cells assessed by TB and SYTO/PI decreased significantly with time at incubation temperatures of 22 and 37 degrees C (P<0.05), and correlated with the concentration of mononuclear cells (MNC) (r=0.936, P<0.05). The decrease in recovery of viable nucleated cells was slower when thawed cells were incubated at 0 degrees C compared with 22 degrees C or 37 degrees C. The recovery, measured by absolute viable CD34+ or CFU-GM, was not affected by 2 h post thaw incubation (P>0.05) at 0, 22, and 37 degrees C (P>0.05). There were no significant differences in the measured recovery of viable CD34+ cells and CFU-GM at all incubation times (P>0.05) and temperatures (P>0.05). Both CFU-GM and absolute CD34+ cells can be used as post thaw viability assays for HPC cryopreserved for transplantation.     

三氧化二砷选择性诱导人肝癌细胞凋亡及 相关基因的实验研究

目的 明确三氧化二砷对人肝癌细胞的诱导凋亡作用及其分子机制。方法 用四甲基偶氮唑蓝法、AO/EB荧光染色法、透射与扫描电镜观察、DNA电泳、流式细胞术、TUNEL法及免疫组织化学等方法,观察了三氧化二砷对体外培养的人肝癌细胞株QGY-7701、QGY-7703和正常人肝细胞株L-02的生长活力、细胞凋亡、细胞周期及其相关基因表达的影响。结果 三氧化二砷能显著地抑制人肝癌细胞QGY-7701和QGY-7703的生长,用药后诱导其出现了典型的细胞凋亡形态学和生化学改变,并使细胞周期阻滞于S期,且bcl-2基因表达明显下降,bax和Fas基因表达显著增强;三氧化二砷对正常人肝细胞株L-02无明显作用。结论 三氧化二砷对人肝癌细胞有显著的选择性诱导凋亡作用,且受到多种基因调控,这一结论为应用三氧化二砷治疗原发性肝癌提供了可靠的实验依据。     

Islet cell metabolism is reflected by the MTT (tetrazolium) colorimetric assay

Summary Insulin secretion depends critically on glucose metabolism. We investigated whether a rapid viability test could be established for assessing glucose metabolism in insulin secreting cells. The MTT (C,N-diphenyl-N′-4,5-dimethyl thiazol 2 yl tetrazolium bromide) colorimetric assay (reduction of tetrazolium salt to formazan) was applied to rat islets and rat insulinoma cell lines. It was found that the rate of formazan production correlated with glucose oxidation and glucose utilization at glucose concentrations which also stimulated insulin secretion. In differentiated insulinoma INS-1 cells, salt reduction paralleled the insulin release, at glucose concentrations of up to 8.3 mmol/l. The glucose-induced formazan production in INS-1 cells and islets was abolished by exposure to the Beta-cell cytotoxic agents, streptozotocin or alloxan. The MTT assay thus provides a convenient tool for the rapid assessment of Beta-cell metabolism and viability.     

脂联素对结肠癌HT-29细胞的增殖及凋亡研究

目的探讨脂联素对结肠癌HT-29细胞的增殖、凋亡的影响。方法以不同浓度脂联素干预细胞,MTT法检测细胞增殖能力;AnnexinV／PI双染后流式细胞仪检测细胞凋亡。结果随着脂联素浓度的不断升高和培养时间的延长,HT-29细胞的生长逐渐受到抑制,脂联素可以诱导细胞凋亡。结论脂联素可抑制HT-29细胞增殖,诱导细胞凋亡。     

藤梨根提取物对大肠癌LoVo细胞增殖的抑制作用及诱导凋亡的影响

目的:研究藤梨根提取物(ethanol extract from radix of actinidia chinensis,EERAC)对人大肠癌LoVo细胞增殖和凋亡的影响.方法:提取藤梨根抗癌有效活性成分(EERAC),按浓度分为4处理组(10、40、160、320mg/L)和空白对照组(0mg/L).各实验组经作用24、48、72h后,进行一般形态学和AO/EB荧光染色观察;MMT法检测细胞增殖的抑制情况;免疫组织化学(immunohistochemistry,IHC)法测定LoVo细胞中凋亡相关基因Bcl-2、Bax、Caspase-3的蛋白表达变化.结果:与空白对照组比较,一般形态学显示EERAC处理组能使细胞密度减低,增殖变慢;细胞逐渐变大,细胞间接触变松,胞浆中颗粒增多,细胞脱壁现象和周围碎片增多;荧光染色观察可见处理组细胞呈橙红色荧光,细胞核出现碎片状或固缩状的凋亡特征学形态改变,凋亡现象与EERAC的浓度呈正相关性;MTT法检测显示,EERAC处理组对LoVo细胞的最佳作用时间为72h,最大抑制率为79.48%,具有浓度和时间的依赖性(P<0.01);IHC检测结果显示EERAC作用LoVo细胞24h后,Bcl-2表达明显减弱,Bax、Caspase-3表达水平明显增高,Bcl-2/Bax比值下降,差异具有统计学意义(P<0.05),其效应与浓度相关.结论:EERAC具有明显抑制LoVo细胞增殖的作用,其机制可能与降低Bcl-2表达,上调Bax、Caspase-3的表达水平,激活线粒体凋亡途径有关.