益气活血软坚解毒含药血清诱导人肝癌细胞系Bel-7402细胞的凋亡

目的:研究益气活血软坚解毒(YHRJ)含药血清对人肝癌细胞系Bel-7402生长抑制及诱导凋亡作用.方法:将YHRJ含药血清作用于人肝癌细胞系Bel-7402细胞,应用MTT检测对肝癌细胞生长抑制作用,倒置显微镜、荧光显微镜、激光共聚焦扫描显微镜等影像学方法观察细胞形态学变化,以及PI染色单染、AnnexinV-PI双染后,流式细胞术检测对细胞周期影响及诱导细胞凋亡程度.结果:MTT法检测结果显示:YHRJ含药血清具有抑制Bel-7402肿瘤细胞生长作用(其中20%YHRJ等效剂量抑制率49.1%,与NS比,P<0.01);荧光显微镜及激光共聚焦显微镜可观察到典型的凋亡形态学变化.流式细胞术检测结果,细胞周期出现G0/G1期阻滞,并出现典型的凋亡峰,AnnexinV-PI双染法检测到早期及中晚期细胞凋亡.结论:YHRJ含药血清有抑制人肝癌细胞系Bel-7402细胞生长并有诱导细胞凋亡作用.     

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。     

六磷酸肌醇全外对人肝癌7402细胞凋亡的影响

目的观察IP_6对体外培养的肝癌7402细胞凋亡的影响。方法用透射电镜、琼脂糖凝胶电泳和流式细胞仪观察IP_6处理细胞后其生化和形态学指标的改变。结果透射电镜观察7402细胞经IP_6处理6h后,细胞核固缩,形成着边现象,至24h部分细胞脱壁,琼脂糖凝胶电泳呈现梯形条带,流式细胞仪分析显示处理细胞中出现亚二倍体峰,证实处理组细胞发生凋亡。结论 IP_6能诱导肝癌7402细胞凋亡,或许诱导细胞凋亡是IP_6的抗癌机理之一。     

益气活血软坚解毒方含药血清诱导人肝癌细胞系Bel-7402细胞凋亡过程中部分凋亡调控基因变化

目的:研究益气活血软坚解毒方(YHRJ)含药血清对人肝癌细胞系Bel-7402细胞凋亡调控基因Fas,FasL,Bcl-2,Bax,P53,NF-kB表达影响.方法:将细胞分为对照组(NS)组、NS DDP(顺氯氨铂)组、YHRJ等剂量组(YHRJD)、YHRJD DDP组、YHRJ高剂量组(YHRJG)、YHRJG DDP组,应用流式细胞术、免疫组化、原位杂交、RT-PCR等方法对用药24,48h的Bel-7402细胞凋亡的主要调控基因Fas,FasL,Bcl-2,Bax,P53,NF-kBmRNA与蛋白表达进行检测.结果:流式细胞术检测显示:与NS组相比,NS DDP、YHRJD、YHRJD DDP及YHRJG DDP组Fas蛋白表达均明显提高(30.12%±22.94%,10.50%±8.41%,30.35%±22.98%,32.61%±26.87%vs8.77%±6.93%,P<0.01),YHRJG组效果不明显(P>0.05);NS DDP、YHRJD DDP组FasL蛋白表达也升高(16.40%±7.168%,8.41%±6.74%vs4.12%±2.60%,P<0.01),而YHRJG组FasL蛋白表达降低(3.05%±2.53%vs4.12%±2.60%,P<0.01).免疫组化结果显示:除YHRJG DDP组外,其余各组突变型P53蛋白表达明显降低(30.2%,14.6%,19.8%,17.3%vs60.0%,P<0.05);各加药组Bax蛋白表达明显增高(40.7%,40.4%,72.1%,68.9%,42.2%vs30.0%,P<0.05);NS DDP组与YHRJG组Bcl-2蛋白表达明显降低(26.3%,24.4%vs30.5%,P<0.05),而YHRJD、YHRJG DDP、YHRJG DDP组Bcl-2表达明显升高(41.8%,39.3%,45.6%vs30.5%,P<0.05);各加药组NF-kB蛋白表达明显降低(15.9%,13.3%,14.1%,7.8%,14.6%vs24.2%,P<0.05).原位杂交结果显示:各加药组NF-kBmRNA表达明显降低(30.5%,13.3%,21.4%,17.4%,53.2%vs58%,P<0.05).RT-PCR结果显示:YHRJ等效剂组凋亡调控基因Bcl-2mRNA表达明显降低(0.717±0.198vs1.327±0.097,P<0.001);DDP、YHRJD、YHRJG组凋亡调控基因BaxmRNA表达明显增高(46.22±6.22,56.19±7.36,62.32±11.06vs35.22±4.38,P<0.05).结论:YHRJ含药血清诱导人肝癌细胞系Bel-7402细胞凋亡可能的基因调控机制在于通过抑制凋亡信号转导基因FasL基因蛋白表达,促进凋亡调控基因Bax基因蛋白表达,抑制NF-kB基因mRNA及蛋白表达来实现的.     

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。     

肝泰煎剂含药血清诱导人肝癌细胞系Bel-7402细胞的凋亡

目的:观察肝泰煎剂(GTJJ)含药血清诱导人肝癌细胞系Bel-7402细胞凋亡现象。方法:将GTJJ含药血清作用于人肝癌细胞系Bel-7402细胞,应用MTT检测对肝癌细胞生长抑制作用,倒置显微镜、荧光显微镜、激光共聚焦扫描显微镜等影像学方法观察细胞形态学变化,以及PI染色单染、AnnexinV-PI双染后,流式细胞术检测对细胞周期影响及诱导细胞凋亡程度。结果:MTT法检测结果显示:GTJJ含药血清具有抑制Bel-7402肿瘤细胞生长作用[其中20%GTJJ等效剂量抑制率50.09%,与生理盐水(NS)组比,P<0.01];荧光显微镜及激光共聚焦显微镜可观察到典型的凋亡形态学变化;流式细胞术检测显示,细胞周期出现G0/G1期阻滞,并出现典型的凋亡峰;AnnexinV-PI双染法检测到早期及中晚期细胞凋亡。结论:GTJJ含药血清有诱导细胞凋亡作用。     

扶正抗癌方药物血清对人肝癌细胞增殖的影响

目的:阐明扶正抗癌方的抗癌机制。方法:以不同剂量灌胃给药后,不同时间采集大鼠血清,处理体外培养的人肝癌细胞SMMC_(7721),用MTT法和氚标胸腺嘧啶(~3H-TdR)掺入法观察细胞增殖能力。结果:扶正抗癌方(1.6g/kg,8.0g/kg)2次给药后1小时和2小时药物血清处理细胞,MTT转化率和~3H-TdR掺入率明显下降。结论:扶正抗癌方具有明显的抑制人肝癌细胞增殖作用。     

苯乙酸钠对人肝癌细胞凋亡的研究

为了观察苯乙酸钠对人肝癌细胞的分化诱导作用，应用流式细胞仪（ＦＣＭ）、台盼蓝染色计数法和＾３Ｈ－ＴｄＲ掺入法观察茉乙酸钠对人肝癌细胞株的细胞周期动力学变化，另用喉癌细胞株（Ｈｅｐ－２）做对照细胞，结果显示苯乙内对人肝癌细胞呈剂量依赖性抑制，细胞周期的Ｇ１期下降，Ｓ期相对升高，但对喉癌抑制作用不明显。表明苯乙酸钠可抑制人肝癌细胞株增殖，主要是抑制细胞周期的Ｇ１期。     

刺五加皂甙对肝癌细胞凋亡的影响

探讨刺五加皂甙（ＡＳＳ）对体外培养肝癌细胞凋亡的影响。采用透射电镜及琼脂糖凝胶电泳观察ＡＳＳ作用肝癌凋亡的形态学改变和ＤＮＡ电泳。电镜观察ＡＳＳ作用肝癌细胞出现异染色体边集,线粒体肿胀等凋亡的改变。ＡＳＳ作用肝癌细胞ＤＮＡ出现典型梯状改变。提示ＡＳＳ促进体外培养肝癌细胞凋亡。且随着时间的增加和剂量加大,诱发凋亡程度增高。     

Effects of Pinus massoniana bark extract on cell proliferation and apoptosis of human hepatoma BEL-7402 cells

AIM:To study the effects of Pinus massoniana bark extract (PMBE) on cell proliferation and apoptosis of human hepatoma BEL-7402 cells and to elucidate its molecular mechanism. METHODS:BEL-7402 cells were incubated with various concentrations (20-200 ug/mL) of PMBE for different periods of time. After 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was evaluated by morphological observation, agarose gel electrophoresis, and flow cytometry analysis. Possible molecular mechanisms were primarily explored through immunohistochemical staining. RESULTS:PMBE (20-200 ug/mL) significantly suppressed BEL-7402 cell proliferation in a time-and dose-dependent manner. After treatment of BEL-7402 cells with 160 ug/mL PMBE for 24, 48, or 72 h, a typical apoptotic "DNA ladder"was observed using agarose gel electrophoresis. Nuclear condensation and boundary aggregation or split, apoptotic bodies were seen by fluorescence and electron microscopy. Sub-G_1 curves were displayed by flow cytometry analysis. PMBE decreased the expression levels of Bcl-2 protein in a time-dependent manner after treatment of cells with 160 ug/mL PMBE. CONCLUSION:PMBE suppresses proliferation of BEL-7402 cells in a time-and dose-dependent manner and induces cell apoptosis by possibly downregulating the expression of the bcl-2 gene.     

维甲酸对人肝癌细胞凋亡的影响及临床意义

目的：观察全反式维甲酸（ATRA）对肝癌细胞凋亡的作用并探讨ATR的作用机制。方法：在肝癌细胞株SMMC－7721细胞加入ATRA,使终浓度为0．5μg/ml,对照组加等剂量的0．02％DMSO,培养4d,ATRA对SMMC－7721细胞的影响通过苏木精－伊红染色,在光镜下进行细胞形态学观察;通过细胞计数绘制生长曲线及计算细胞生长抑制率,流式细胞术分析不同DNA含量的细胞分布,计算细胞凋亡百分率,凋亡相关基因Fas,p53,Bcl-2蛋白表达的检测亦采用流式细胞仪检测。结果：苏木精－伊红染色可见较多细胞核碎裂,胞浆浓缩,染色质深染,聚集于核膜下,部分细胞膜突起呈小泡样,小泡脱落形成凋亡小体,而对照组无明显的形态学改变,作用4d生细胞生长抑制率为47．5％,与对照组比较,差异显著（P＜0．05）,流式细胞仪分析在G1期前出现亚二倍体凋亡峰;凋亡细胞百分比为16．0％,与对照的6．9％比较有显著性差异（P＜0．01）。观察发现,ATRA对SMMC－771细胞Fax,P53的表达明显增加,并下调Bcl-2的表达,结论：0．5μg/ml的ATRA对肝癌细胞的生长有显著抑制效果,并可见癌细胞凋亡形态改变,ATRA促进SMMC－721肝癌细胞凋亡的机制之一可能是通过调控Fas,P53及Bcl-2的表达。     

脂质体介导姜黄素抑制Bel-7402细胞增殖和诱导其凋亡的作用

目的:研究提高姜黄素治疗肝癌生物学效应的新技术和新方法.方法:对比观察脂质体-姜黄素水溶制剂对人肝癌细胞(Bel-7402)凋亡及凋亡相关调控基因Bax、Bcl-2的影响.MTT(四甲基偶氮唑蓝)法观察脂质体-姜黄素对Bel-7402细胞增殖的抑制作用.原位末端标记法(TUNEL技术)观察脂质体-姜黄素诱导Bel-7402细胞凋亡的作用.免疫组织化学染色(SABC)法检测脂质体-姜黄素对Bax和Bcl-2基因表达的影响.结果:10μg/ml、5μg/ml、2.5μg/ml脂质体-姜黄素对Bel-7402细胞增殖有显著抑制作用,P＜0.01,其抑制率分别为38.67%、26.67%和17.33%,与同浓度的姜黄素的抑制率(29.33%、20.00%、5.33%)比较,差异有显著性意义(P＜0.05);增殖抑制作用随药物浓度增高而有加强趋势,3个有效浓度组间差异均有显著性意义(P＜0.05).10μg/ml、5μg/ml、2.5μg/ml脂质体-姜黄素处理组Bel-7402细胞凋亡率分别为68.9%、43.4%、26.9%,与同浓度姜黄素组的Bel-7402细胞凋亡率(53.3%、30.2%、14.9%)比较,差异有显著性意义(P＜0.05).脂质体-姜黄素对细胞凋亡相关基因Bax、Bcl-2表达的影响,与对照组比较差异无显著性意义.结论:脂质体能显著提高姜黄素对Bel-7402细胞增殖的抑制和诱导其凋亡的作用.脂质体-姜黄素对Bax、Bcl-2的表达无显著影响.     

Prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay

AIM: To investigate the prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay. METHODS: Clonogenic assay, flow cytometry, and CB micronuclei assay were used to survey the cell survival rate, radiation-induced apoptosis and micronucleus frequency of hepatocarcinoma cell lines SMMC-7721, HL-7702, and HepG2 after being irradiated by X-ray at the dosage ranging 0-8 Gy. RESULTS: After irradiation, there was a dose-effect relationship between micronucleus frequency and radiation dosage among the three cell lines (P<0.05). A positive relationship was observed between apoptosis and radiation dosage among the three cell lines. The HepG2 cells had a significant correlation (P<0.05) but apoptosis incidence had a negative relationship with micronucleus frequency. There was a positive relationship between apoptosis and radiation dosage and the correlation between SMMC-7721 and HL-7702 cell lines had a significant difference (P<0.01). After irradiation, a negative relationship between cell survival rate and radiation dosages was found among the three cell lines (P<0.01). There was a positive relationship between cell survival rate and micronucleus frequency (P<0.01). No correlation was observed between apoptosis and cell survival rate. CONCLUSION: The radiosensitivity of hepatocarcinoma cells can be reflected by apoptosis and micronuclei. Detection of apoptosis and micronuclei could enhance the accuracy for predicting radiosensitivity.     

Effects of dendritic cells from cord blood CD34^＋ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocartinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11d vs 7 d,P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.     

Effects of a Shuangling Fuzheng anticancer preparation on the proliferation of SGC-7901 cells and immune function in a cyclophosphamide-treated murine model

AIM: To study the inhibitory effects of a Shuangling Fuzheng anticancer preparation (SFAP) on the human gastric cancer cell line SGC-7901 in vitro as well as its immune-modulated effects in a cyclophosphamide-treated murine model. METHODS: MTT experiments and immunocytochemistry ABC experiments were performed for detecting the proliferation of SGC-7901 cells in vitro and protein expression of c-myc. The staphylococcal protein A (SPA) rosette test was utilized for measuring the ratio of T-lymphocyte subsets from peripheral blood in a cyclophosphamide-treated murine model. Enzyme- linked immunosorbant assay (ELISA) was performed for measuring the levels of serum sIL-2R in treated mice, while immunoturbidimetry was used for measuring the levels of immunoglobulins (Ig). RESULTS: SFAP (40-640 mg/L, 48 h) inhibited the proliferation of SGC-7901 cells, and a positive correlation was noted between inhibitory effects and dosage. At a dosage of 160-320 mg/L in cultured cells, the expression of c-myc was decreased. SFAP (50-200 mg/kg) increased the percentage of CD3 and CD4 T-lymphocytes, the ratio of CD4/CD8, and the contents of Ig such as IgM, IgG or IgA, but decreased the levels of serum sIL-2R in peripheral blood from cyclophosphamide-treated mice. CONCLUSION: SFAP can inhibit the proliferation of SGC-7901 cells via the c-myc gene. In addition, SFAP can modulat the cellular and humoral immunity in cyclophosphamide-induced immunosuppressed mice.     

丹皮酚联合5-FU对食管癌EC9706细胞增殖及凋亡的影响

目的:探讨丹皮酚(paeonol,Pae)单独及联合5-Fu对人食管癌EC9706细胞的增殖抑制及凋亡诱导作用.方法:采用6种浓度的Pae(7.81、15.63、31.25、62.50、125.00、250.00 mg/L)、3种浓度的5-FU(12.50、25.00、50.00 mg/L)及Pae(31.25 mg/L)和5-FU(12.50 mg/L)联合分别处理EC9706细胞24、48、72 h.同时设对照组(细胞不做处理),采用MTT法检测各个时间段细胞的增殖情况:采用流式细胞术检测4种浓度的Pae(31-25、62.50、125.00、250.00 mg/L)处理EC9706细胞72 h后细胞周期的变化:倒置显微镜下观察各Pae组细胞各时间段形态学变化,HE染色光镜下观察凋亡细胞:采用免疫细胞化学法检测经Pae(31.25 mg/L)、5-FU(12.50mg/L)单独和联合作用48 h后细胞中凋亡相关蛋白Bcl-2及Bax的表达.结果:Pae、5-FU可明显抑制EC9706细胞增殖,并随着浓度的增加和作用时间的延长而增强(P<0.05),Pae与5-FU联合用药比单用Pae或5.FU抑制效果更明显(P<0.05);Pae作用后EC9706细胞中G0/G1期和G2/M期细胞比例下降、S期细胞比例上升(Pae 125.00 mg/L组:G0/G1期21.18%±2.28% vs 62.17%±5.23%、G2/M期0.76%±0.54% vs 9.92%±3.10%、S期78.06%±2.82% vs 27.91%±2.13%,均P<0.05):HE染色光镜下可见典型的肿瘤细胞凋亡改变:Pae、5-FU可下调EC9706细胞中Bcl-2蛋白表达,同时增强EC9706细胞中Bax蛋白的表达,联合用药组较单药组作用更为明显(2.21±0.14 vs 5.67±0.30,4.22±0.34;8.55±0.33 vs 3.90±0.27,6.28±0.26,均P<0.05).结论:Pae可明显抑制人食管癌EC9706细胞的增殖.促进其凋亡,Pae联合5-FU作用更为明显.