含蟾酥胶囊血清诱导人肝癌细胞株凋亡及机制探讨

目的观察含蟾酥胶囊（CC）血清诱导人肝癌细胞株BEL-7402凋亡作用,并探讨其机制。方法体外培养的BEL-7402加入含不同浓度CC血清,分别孵育24、48h,显微镜下观察细胞形态;MTT比色测算细胞抑制率;流式细胞术测算细胞凋亡率;琼脂糖凝胶电泳测定其DNA梯状条带;免疫细胞化学染色检测细胞中Bcl-2表达变化。结果与不加CC血清比较,加入含CC血清的BEL-7402呈凋亡细胞形态学改变;细胞生长抑制率、凋亡率升高;孵育48h时,琼脂糖凝胶电泳呈现DNA梯状条带;细胞Bcl-2表达明显降低。结论CC可以诱导BEL-7402凋亡,其作用机制可能与下调细胞Bcl-2表达有关。     

阿司匹林对宫颈癌Hela细胞的增殖抑制作用

目的 探讨阿司匹林对宫颈癌Hela细胞增殖的影响.方法 体外培养Hela细胞,分别以不同浓度阿司匹林干预.应用Giemsa染色观察细胞形态学的改变;琼脂糖电泳法观察凋亡细胞DNA Ladder现象;采用MTT法测定细胞增殖抑制率.结果 Giemsa染色后可见阿司匹林组细胞体积缩小,凋亡小体形成,且随着浓度增加,凋亡率也增加;不同浓度阿司匹林作用Hela细胞48 h后,提取DNA电泳,可见明显的DNA裂解带;MTT结果显示,在1.0～10.0 mmol/L浓度区间内,随浓度增加和时间的延长,阿司匹林对细胞的抑制率逐渐增加.结论 阿司匹林在体外可抑制人宫颈癌Hela细胞增殖,其机制可能为抑制肿瘤细胞DNA合成有关.     

五味子多糖对人结肠癌HT29细胞增殖的抑制作用及其机制

目的探讨五味子多糖含药血清对人结肠癌细胞株HT29体外增殖的影响及作用机制。方法采用人结肠癌细胞株HT29作为研究对象,MTT法检测不同浓度的五味子多糖含药血清对细胞增殖的抑制作用;倒置显微镜和荧光显微镜观察细胞形态和细胞核的改变;流式细胞仪分析细胞凋亡的情况。结果五味子多糖含药血清对HT29细胞的增殖有抑制作用而且呈剂量和时间依赖性。五味子多糖含药血清作用72 h后,荧光显微镜可见凋亡细胞及凋亡小体;流式细胞仪检测凋亡。结论五味子多糖含药血清对HT29结肠癌细胞的增殖有抑制作用,其作用机制与诱导细胞凋亡有关。     

复肝春6号抗H22肝癌作用机制的实验研究

目的探讨复肝春6号（FGC-6）抗肝癌作用机制。方法建立荷H笠肝癌小鼠模型，以DNA琼脂糖凝胶电泳观察FGC-6对肿瘤细胞诱导凋亡的作用，以Western blot检测肿瘤组织中bcl-2、bax基因的蛋白表达水平；制备FGC-6含药血清，观察其对体外培养H22细胞的增殖抑制作用。结果FGC-6可诱导肿瘤组织DNA片段化，琼脂糖凝胶电泳结果可见梯状条带出现；Western blot结果显示FGC-6使肿瘤组织中bcl-2基因表达量明显降低，而bax基因的表达水平却明显升高；FGC-6含药血清对体外培养H22细胞增殖有明显抑制作用。结论FGC-6的抗肝癌作用可能与调节bcl-2和bax基因的表达量，诱导肝癌细胞凋亡有关。     

夏枯草提取物对人淋巴瘤Raji细胞增殖的影响及机制探讨

目的 观察夏枯草提取物对人Burkitt淋巴瘤Raji细胞增殖的影响,并探讨其机制.方法 Raji细胞分别加入不同浓度夏枯草提取物进行培养,MTT法检测Raji细胞增殖抑制率,倒置显微镜观察细胞学形态,琼脂糖凝胶电泳观察Raji细胞DNA凋亡情况;流式细胞仪检测Raji细胞凋亡率.结果 不同浓度的夏枯草提取物对Raji细胞增殖有明显的抑制作用,并呈剂量依赖性,半数抑制浓度(IC50)为(18.01±0.92)μg/ml;随夏枯草提取物作用时间的延长Raji细胞DNA凋亡条带增宽变亮,细胞凋亡率升高.结论 夏枯草提取物可抑制Raji细胞增殖,其机制可能与诱导细胞凋亡有关.     

山茱萸水提取物对Hela细胞体外增殖的影响

目的探讨山茱萸水提取物对人宫颈癌细胞株Hela的体外增殖的抑制作用。方法采用MTT法及DNA琼脂糖凝胶电泳法检测山茱萸水提取物对人宫颈癌细胞增殖的抑制作用并观察细胞形态变化。结果山茱萸水提取物对Hela细胞抑制作用较强，量效关系和时效大系良好；经不同浓度山茱萸水提取物作用的Hela细胞电泳均出现相差约200bp的DNA梯子状条带，形态学观察到细胞凋亡。结论山茱萸水提取物对人宫颈癌细胞株Hela的细胞增殖有较强的抑制作用，能诱导其细胞凋亡。     

刚地弓形虫培养上清对乳腺癌MCF-7细胞增殖及凋亡的影响

目的观察体外培养条件下弓形虫培养上清对乳腺癌细胞MCF-7细胞增殖及其诱导其凋亡的情况。方法取对数生长期的MCF-7细胞(浓度为5×104和5×105)分别接种于不同细胞培养板,加入相同体积不同浓度弓形虫速殖子培养上清(速殖子浓度分别为2×107/mL、4×107/mL、6×107/mL、8×107/mL)与培养液共同作用12h、24h、48h、72h,四甲基氮噻唑蓝(MTT)法检测吸光度(A490值)并计算抑制率;Annexin-v-FITC/PI染色细胞后上流式细胞仪检测细胞凋亡率;AO/EB染料染色作用后细胞在荧光显微镜下观察细胞形态改变情况并拍照;琼脂糖凝胶电泳观察细胞凋亡DNA条带。结果MTT法检测结果显示MCF-7细胞增殖抑制率随着上清浓度和作用时间增加均明显增大,各实验组抑制率与对照组比较以及对照组之间比较差异均有统计学意义(P<0.05)。流式细胞仪检测显示MCF-7细胞凋亡率随着上清浓度和作用时间增加明显增大,48h达到凋亡最高值(19.79%),48h后凋亡率开始下降,死亡率增加;荧光显微镜观察实验组培养48h的MCF-7细胞,出现细胞核固缩及凋亡小体,对照组未出现凋亡小体。琼脂糖凝胶电泳见DNA梯形条带。结论弓形虫速殖子对体外培养MCF-7细胞增殖有明显的抑制作用,并可诱导MCF-7细胞凋亡。     

维生素A对Hela细胞凋亡的影响

目的研究维生素A对Hela细胞凋亡的影响。方法应用光学显微镜、电子显微镜观察维生素A对Hela细胞凋亡形态的影响;分别应用细胞DNA琼脂糖凝胶电泳法及流式细胞仪分析维生素A诱导Hela细胞凋亡的生物化学特征、细胞特征及凋亡细胞百分率。结果光学显微镜下可见凋亡小体;电子显微镜下可见细胞固缩,核膜扭曲,核染色体聚集成块并靠近核膜等凋亡细胞特征;在琼脂糖凝胶电泳中由于细胞DNA被降解而呈现典型的“阶梯状”图谱;流式细胞仪检测结果显示二倍体核型的特征,在DNA直方图上,G1峰左侧出现亚二倍体细胞群的峰型;维生素A诱导Hela细胞凋亡具有时间依赖性。结论维生素A可诱导Hela细胞凋亡。     

犀黄丸诱导Bel-7402细胞凋亡及其细胞内钙离子浓度的变化

研究犀黄丸(含药血清)诱导肝癌Bel—7402细胞凋亡的作用及凋亡过程中细胞内游离钙离子浓度的变化。DNA琼脂糖凝胶电泳法检测细胞凋亡，流式细胞仪检测凋亡过程中细胞内游离钙离子浓度的变化。DNA琼脂糖凝胶电泳法显示有明显的DNA梯带，同时在细胞凋亡过程中细胞内游离钙离子浓度明显升高，以凋亡早期较为明显。犀黄丸能诱导肝癌细胞凋亡，细胞内游离钙离子浓度升高可能是其机制之一。     

紫杉醇对肝癌HepG2细胞增殖及凋亡的影响

目的观察紫杉醇对肝癌HepG2细胞增殖及凋亡的影响。方法取对数生长期人肝癌HepG2细胞,分别置入5、10、20 nmol/L紫杉醇共同培养48 h（实验组）,另设阴性对照组。采用MTT法测定细胞增殖活性;Ho-echst33342荧光染色法观察紫杉醇处理后细胞形态的变化;DNA琼脂糖凝胶电泳检测DNA梯状条带;免疫组织化学法检测细胞中凋亡相关蛋白cFLIP、Survivin、Caspase-3的表达。结果紫杉醇浓度分别为5、10、20 nmol/L时,细胞增殖活性分别为0.51±0.02、0.49±0.05、0.38±0.09,与阴性对照组（0.61±0.07）比较P〈0.05或〈0.01;荧光显微镜观察可见典型的细胞凋亡形态学改变,以20 nmol/L紫杉醇作用最明显;DNA琼脂糖凝胶电泳显示,实验组细胞均可见典型的＂梯状＂条带;与阴性对照组比较,实验组能显著抑制cFLIP、Survivin蛋白的表达,促进Caspase-3蛋白的表达（P〈0.05或〈0.01）。结论紫杉醇可能通过下调cFLIP、Survivin蛋白的表达及上调Caspase-3蛋白的表达诱导人肝癌HepG2细胞凋亡。     

蛴螬提取物抗肿瘤作用的体外血清药理学实验

目的 探讨蛴螬提取物对人肺癌A549和人宫颈癌HeLa细胞株抗肿瘤作用.方法 采用血清药理学实验方法,观察含药血清体外对人肺癌A549和人宫颈癌HeLa细胞株抗增殖作用.结果 血清药理学实验结果显示,终浓度分别为20%、25%的灭活的蛴螬提取物含药大白鼠血清对A549细胞株的增殖抑制率分别为18.92%、37.54%;终浓度分别为10%、20%、25%的灭活的蛴螬提取物含药血清对HeLa细胞株的增殖抑制率分别为39.86%、24.86%及26.60%.结论 蛴螬提取物含药血清对人肺癌A549和人宫颈癌HeLa细胞株增殖有抑制作用.     

Serum-free culture of H pylori intensifies cytotoxicity

AIM： To perform a long culture passage of H pylori without serum, taking into account its cytotoxicity and the presence of the probable new cytotoxic factor.
METHODS： One sample of H pylon 60190 （ATCC 49503） was grown on Brain Heart Infusion （BHI） agar containing 0.5% 2,6-di-O-methyl-β-cyclodextrin without any serum, being passaged 70-100 times every 3-4 d for approximately 2 h, while another sample of H pylori contained 70 mL/L fetal calf serum without 2,6-di-O- methyl-β-cyclodextrin. Their supernatant and extract after 16 h in culture were evaluated for changes in cell morphology and for cell viability using HeLa cells. Furthermore, the characteristics of the probable cytotoxic factor in the extract were examined on partial purification studies and its oytotoxicity was evaluated in various human cells.
RESULTS： The supernatant and the extract of the bacterium grown on serum-free medium had strong cytotoxicity compared with those grown on serumcontaining medium. They irreversibly damaged HeLa cells without vacuolation that was altogether different from that of the bacterium when grown with serum. Their cytotoxicity was easily measured by cell viability assay. The probable cytotoxic factor partially purified and detected by chromatography had characteristics difference from that of vacuolating toxin and a broad cytotoxicity toward various cell lines.
CONCLUSION： Serum-free long culture method of H pylorl makes its supernatant and its extract cytotoxic enough to be easily measured by cell viability assay. The probable cytotoxic factor has a unique characteristic and might be a new cytotoxin.     

消瘤汤含药血清对人肝癌细胞HepG2凋亡相关因子表达的影响

[目的]观察消瘤汤含药血清对人肝癌细胞HepG2凋亡相关因子表达的影响,进一步证实该方对肿瘤细胞的抑制作用,并初步探讨其诱导肝癌细胞凋亡的作用机制。[方法]给予昆明种小鼠中药煎剂灌胃3d后,制备消瘤汤鼠含药血清,将其作用于HepG2细胞,用四氮唑盐法（MTT法）检测含药血清对HepG2细胞增殖的抑制作用,通过流式细胞技术测定肿瘤细胞的凋亡情况,应用免疫组化法检测半胱氨酸蛋白酸3（Caspase-3）、B细胞淋巴瘤2（Bcl-2）蛋白表达水平。[结果]消瘤汤鼠含药血清作用于HepG2细胞72h后,对肿瘤细胞生长有明显抑制作用。流式细胞仪检测可见含药血清组在细胞G1／G0期前出现明显的凋亡峰,即“亚二倍体峰”。凋亡率为22．3％,明显高于对照组的1．05％（P〈0．05）。免疫组化结果表明Caspase-3在含药血清干预后,表达明显增加,而Bcl-2在含药血清干预后,表达明显降低。[结论]消瘤汤可能通过激活Caspase-3及抑制Bcl-2的活性,从而诱导肝癌细胞凋亡,有效地抑制肝癌细胞生长,达到抗肿瘤的作用。     

消瘤汤含药血清对人肝癌细胞Bel-7402增殖和凋亡的影响

[目的]观察消瘤汤含药血清对人肝癌细胞Bel-7402增殖和凋亡的影响。[方法]采用消瘤汤含药血清,应用血清药理学方法,选择不同浓度的含药血清体外培养人肝癌细胞Bel-7402 96 h。通过MTT法和细胞形态学观察检测细胞的增殖抑制率和细胞凋亡。[结果]MTT法检测表明,消瘤汤高、中、低剂量组与0.95%氯化钠（对照）组相比,对人肝癌细胞增殖均有抑制作用（P〈0.01）。细胞形态学检测表明消瘤汤各剂量组含药血清均可诱导细胞凋亡。[结论]消瘤汤能明显抑制肝癌Bel-7402细胞的增殖,其抑瘤作用有细胞毒作用和诱导细胞凋亡2种途径。     

Serum enhances the cycling and survival of HeLa cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

We have defined the growth kinetics of HeLa cell populations by determining the frequencies of mitoses and deaths and the lengths of intermitotic intervals. This was done by time-lapse cinemicrography. Untreated control cells proliferated at closely similar rates in medium enriched with 5% or 15% fetal calf serum, with an average of 4% dividing and less than 0.1% dying per hr. The mean intermitotic interval was 16 hr during exponential growth of the control populations. In contrast, in cultures treated with 40 or 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a selective inhibitor of heterogeneous nuclear RNA synthesis, the frequency of mitoses was markedly and directly dependent on serum concentration, whereas the frequency of deaths was inversely dependent. DRB prolonged the intermitotic interval in cells cycling in the presence of the drug, but the effect was less in 15% than in 5% serum. After prolonged treatment of HeLa cells with DRB, the inhibition of heterogeneous nuclear RNA synthesis by DRB appeared to be reduced, which was not due to inactivation of DRB in the culture medium.     

The differential inhibitory effects of genistein on the growth of cervical cancer cells in vitro

The biological effect of genistein on cervical cancer was studied on two cervical cancer cell lines with different cellular characteristics. Here we report that genistein exhibits inhibitory effects on the growth of HeLa and ME-180 cells. The IC50 was 35 microM and 60 microM for HeLa and ME-180 cells, respectively. ME-180 cells showed obvious G2/M arrest with genistein treatment while most of the HeLa cells were accumulated in S phase. The underlying molecular mechanism was further elucidated by apoptosis analysis and expression levels of cell cycle regulatory proteins. Treatment of the cell lines with genistein also resulted in suppression of invasion through a surrogate membrane in a dose-dependent manner, particularly the HeLa cells. While the underlying mechanism needs to be further studied, the higher suppressive effect on invasion of HeLa cells, an adenocarcinoma cell line, are noteworthy. This in vitro observation may have clinical implication to improve the treatment of cervical adenocarcinoma.     

Inhibition of Entamoeba histolytica cytotoxin by alpha 1 antiprotease and alpha 2 macroglobulin

We previously reported partial purification of a proteinaceous substance with cytotoxic and enterotoxic activity isolated from the soluble fraction of sonicated axenically cultivated Entamoeba histolytica trophozoites. Demonstration of cytotoxic activity of the preparation (amebal toxin) was dependent on removal of serum from the tissue culture assay system. The objective of the present study was to identify the factor(s) in non-immune sera responsible for producing in vitro inhibition of amebal toxin cytotoxicity on HeLa cells. Gel filtration of non-immune sera from adult humans or bovines demonstrated that two portions of the eluate had significant inhibitory against the toxin. A high molecular weight inhibitory fraction was identified as predominantly alpha-2 macroglobulin and a low molecular weight inhibitory fraction was identified as predominantly alpha-1 antiprotease. Preparative isoelectric focusing of human serum isolated inhibitory fractions containing these same alpha globulins. Alpha-2 macroglobulin was purified and alpha-1 antiprotease was partially purified from human serum by other methods and shown to have high inhibitory activity against the amebal cytotoxin. Substances that were inhibitory to the cytotoxic activity of the amebal toxin also mediated reattachment of toxin treated HeLa cells. We conclude that the characteristics of the serum inhibitors, especially their ability to reverse the cytotoxic effects of amebal toxin on HeLa cells, suggests that the amebal toxin has protease activity.     

当归补血汤对体外造血微环境中小鼠肌卫星细胞增殖及c-kit表达的影响

目的观察当归补血汤对体外造血微环境中小鼠肌卫星细胞增殖及c-kit表达的影响。方法分离培养小鼠卫星细胞并鉴定。制备含有不同剂量当归补血汤的大鼠载药血清及对应剂量的骨髓基质细胞条件培养基。将肌卫星细胞随机分为8组:正常大鼠血清组、当归补血汤载药血清1～3组、正常大鼠血清条件培养基组、条件培养基1～3组,MTT法检测细胞增殖活性,免疫组化法检测细胞c-kit的表达情况,荧光实时定量PCR检测细胞c-kit mRNA的表达。结果培养的肌卫星细胞呈Desmin免疫阳性;与正常大鼠血清组相比,载药血清各组及条件培养基各组细胞增殖显著,载药血清3组、条件培养基各组阳性细胞c-kit蛋白及mRNA表达量有显著性差异,随当归补血汤载药血清浓度增大c-kit表达量增多,条件培养基也呈同样的变化趋势。结论当归补血汤载药血清及含载药血清的条件培养基可促进肌卫星细胞增殖及c-kit的表达。     

Analysis of malignancy in human cells: malignant and transformed phenotypes are under separate genetic control.

Human cell hybrids derived from malignant HeLa and normal fibroblast parental cells expressed many of the transformed properties of the HeLa parent but their tumor-producing capability was suppressed. Hybrids derived from HeLa/HeLa fusions retained both their transformed and malignant phenotypes. Thus, an apparent separation of the control of the transformed versus malignant phenotype is indicated. Furthermore, several transformed properties--including lack of density-dependent inhibition of growth, lectin agglutination, lowered requirement for serum growth factors, and anchorage independence--are expressed coordinately in the nontumorigenic hybrids. This finding suggests that none of these properties by themselves, or in concert, endows a cell with tumorigenic potential.     

格林巴利综合征相关及单纯腹泻相关空肠弯曲菌细胞毒作用分析

目的分析格林-巴利综合征(GBS)相关空肠弯曲菌细胞毒素(CDT)作用。方法9株GBS相关空肠弯曲菌以及4株腹泻患者分离菌株的全菌蛋白分别以0.001μg～5μg/mL的浓度作用于HeLa细胞,通过观察细胞形态的改变,获得不同菌株对细胞作用的差异。结果不同菌株间产生细胞毒作用的浓度范围从0.01μg/mL到5μg/mL。结论GBS相关菌株对HeLa细胞的细胞毒素作用与腹泻患者分离株相比没有显著差异。不同空肠弯曲菌菌株对HeLa细胞的细胞毒素作用具有菌株特异性。