斑蝥素酸镁对人肝癌细胞SMMC-7721裸鼠皮下移植瘤凋亡的影响

建立人肝癌细胞SMMC-7721裸鼠皮下移植瘤模型,实验组每只裸鼠瘤周注射斑蝥素酸镁6.26×10-5mmol,对照组给予相同容积的无菌生理盐水瘤周注射。给药22 d后,观察斑蝥素酸镁对皮下移植瘤增殖的影响,并在此基础上,利用HE染色观察药物对人肝癌裸鼠皮下移植瘤组织形态学特征的影响。实验发现斑蝥素酸镁组移植瘤细胞体积变小、胞浆固缩、嗜酸性变,细胞核固缩、碎裂。透射电镜观察人肝癌裸鼠皮下移植瘤组织超微结构的改变,镜下见斑蝥素酸镁组移植瘤细胞核膜基本消失、核染色质聚集成团等改变。免疫组化二步法检测人肝癌裸鼠皮下移植瘤组织中bcl-2、bax的表达水平,结果显示斑蝥素酸镁组瘤组织中bcl-2的表达低于生理盐水组,而bax的表达高于生理盐水组(P0.05)。本实验提示斑蝥素酸镁能明显抑制人肝癌裸鼠皮下移植瘤的增殖,并能诱导移植瘤细胞发生凋亡,其机制可能与上调bax和下调bcl-2表达有关。     

壳寡糖抑制肝癌细胞SMMC-7721的增殖及其机制探讨

探讨壳寡糖抑制人肝癌细胞株SMMC-7721的增殖作用及其分子机制.采用噻唑蓝(MTT)法检测壳寡糖的细胞增殖抑制作用;利用流式细胞仪检测肝癌细胞的凋亡情况;用RT-PCR和Western blot方法研究壳寡糖引起凋亡的原因.结果显示,0.8 mg/mL的壳寡糖能够抑制SMMC-7721细胞增殖,并且促进 SMMC-7721细胞的凋亡,其原因是壳寡糖能够上调促凋亡蛋白Bax的表达.     

蚕蛹复合氨基酸对人肝癌细胞SMMC-7721的抑制作用

本实验探讨蚕蛹复合氨基酸对人肝癌细胞株SMMC-7721的抑制作用.实验经MTT法检测药物对人正常肝脏细胞株QSG-7701的毒性后,计算药物安全浓度.将不同浓度的蚕蛹复合氨基酸与人肝癌细胞SMMC-7721共培养,采用MTT法测定OD值,评定蚕蛹蛋白复合氨基酸对肝癌细胞株的增殖抑制作用;经Hoechst33258染色和倒置显微镜进行形态学观察以检测细胞凋亡率;流式细胞法测定细胞周期和Annexin V/PI双染色法检测细胞凋亡.细胞毒性实验表明:蚕蛹蛋白复合氨基酸的最大无毒浓度为10 mg/mL.不同浓度的蚕蛹蛋白复合氨基酸对SMMC-7721细胞均有抑制作用,各组与对照组相比差异有显著性(P＜0.05),并呈剂量和时间依赖性.Hoechst33258染色和流式细胞术结果亦证实,蚕蛹复合氨基酸能显著促进SMMC-7721细胞的凋亡.     

甘草乙醇浸提液诱导肝癌SMMC-7721细胞凋亡的研究

探讨甘草乙醇浸提液对肝癌细胞SMMC-7721凋亡的影响。经MTT法测定表明甘草乙醇浸提液对肝癌细胞SMMC-7721的半数致死浓度（IC50）为1．246mg／mL,细胞生长抑制率与甘草乙醇浸提液浓度呈正相关。作用后的癌细胞在Hoechst33342荧光染料作用下呈现草蓝色荧光,在形态上具有明显的细胞凋亡现象。提取其DNA进行琼脂糖凝胶电泳,出现明显的梯状条带。因此,甘草乙醇浸提液对肝癌细胞具有显著的抗增殖作用,它能够诱导肝癌细胞的凋亡。     

紫草素对肝癌细胞SMMC-7721凋亡的影响及其机制

目的:探讨紫草素对肝癌SMMC-772细胞的作用及分子机制.方法:SMMC-7721细胞分别经0、5、20、80、320 ng/ml的紫草素作用0h、24 h、48 h和72 h后,适时采用CCK8法观察该细胞增殖的活性,hoechst染色分析细胞的核型变化,流式细胞仪检测细胞凋亡水平,Western blot证实细胞...     

肝癥口服液含药血清对SMMC-7721肝癌细胞TGFβ1/Smads信号转导通路的影响

目的观察肝癥口服液含药血清对SMMC-7721肝癌细胞TGFβ1/Smads信号转导通路的影响。方法以肝癌患者和健康志愿者服药前后血清培养人SMMC-7721肝癌细胞,应用RT-PCR和WesternBlotting方法观察含药血清对肝癌细胞SmadsmRNA及其蛋白表达的影响。结果肝癌患者肝癥口服液含药血清可增强Smad4mRNA及其蛋白表达,而抑制Smad7的过强表达,具有显著性差异;健康志愿者肝癥口服液含药血清可明显抑制肝癌细胞Smad7mRNA表达。结论解除TGFβ1/Smads信号转导通路过度受抑状态,可能是肝癥口服液抗癌的机制之一。     

斑蝥素酸镁与斑蝥酸钠对喉癌Hep-2细胞抑制活性比较的初步研究

目的:探讨自主研发产物--斑蝥素酸镁对人喉癌上皮细胞的增殖抑制活性是否好于临床抗癌药物斑蝥酸钠。方法:采用磺酰罗丹明染色法(SRB法)、细胞集落形成实验分别考察斑蝥素酸镁和斑蝥酸钠对人喉癌上皮细胞Hep-2的影响。结果:与斑蝥酸钠相比,斑蝥素酸镁作用喉癌Hep-2细胞的效果更加显著。其IC50仅为2.19μmol·L-1,远低于斑蝥酸钠的IC50(15.75μmol·L-1)。细胞集落形成实验显示,与斑蝥酸钠相比,斑蝥素酸镁明显阻止细胞集落的形成,最低有效浓度为1.75μmol·L-1,远低于斑蝥酸钠的最低有效浓度(3.50μmol·L-1)。结论:与斑蝥酸钠相比,斑蝥素酸镁对喉癌Hep-2细胞具有更加显著的抑制效果,可进行更加深入的研究。     

腺病毒介导IL-24-E1A双基因载体的构建及其体外抑制SMMC-7721肝癌细胞生长作用初探

构建IL-24和E1A双基因腺病毒载体,获得Ad-IL-24-E1A重组腺病毒子,分析其体外抑瘤作用。PCR及BglⅡ和SalⅠ酶切获得IL-24,与空载体构建成pAdTrack-IL-24-IRES。XhoI和EcoRV酶切获得E1A片段,与pAdTrack-IL-24-IRES连接后成功构建pAdTrack-IL-24-IRES-E1A,同源重组、包装和扩增获得Ad-IL-24-E1A重组病毒子。用50MOI重组腺病毒感染SMMC-7721肝癌细胞,MTT法测定Ad-IL-24-E1A的细胞生长抑制作用;流式细胞仪分析细胞凋亡情况。本研究成功获得Ad-IL-24-E1A重组病毒子。与其他组相比,Ad-IL-24-E1A明显抑制肿瘤细胞生长,感染48h后凋亡率达52%,而抑制正常细胞作用不明显。研究提示Ad-IL-24-E1A双基因重组载体明显抑制SMMC-7721肝癌细胞生长。     

胡桃醌不同给药途径对人肝癌细胞BEL-7402裸鼠皮下移植瘤生长的影响

目的采用裸鼠皮下移植瘤模型,通过不同给药途径对胡桃醌抗肿瘤活性和毒性进行评价。方法建立人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,通过腹腔注射和局部注射两个给药途径观察胡桃醌抑制肿瘤生长的效果。结果①以600、300和150μg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,发现该剂量胡桃醌对肿瘤生长没有明显的影响;NK细胞活性检测发现,600、300μg/kg胡桃醌对裸鼠免疫功能有影响(P均<0.01),150μg/kg胡桃醌则没有影响(P>0.05);与阳性对照组(5-Fu)相比,600μg/kg胡桃醌组NK细胞活性差异无显著性(P>0.05),300和150μg/kg胡桃醌组NK细胞活性差异有显著性(P<0.05,P<0.01),结果提示胡桃醌对小鼠免疫系统有一定的损伤作用。②以4.5、3和1.5 mg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,抑瘤率分别为为78.24%、66.57%、48.94%;4.5、3 mg/kg胡桃醌的抑瘤作用可与阳性对照组比拟(P均>0.05)。但4.5 mg/kg胡桃醌组裸鼠出现明显的皮下脂肪减少、消瘦,并有死亡现象。③以pH 7.4和pH 4.0的600、300和150μg/kg胡桃醌人肝癌BEL-7402细胞裸鼠皮下移植瘤模型局部给药,结果发现不同pH(pH 7.4或4.0)600、300μg/kg的胡桃醌局部注射抑瘤作用与阳性对照组(5-Fu)组差异无显著性(P>0.05),而不同pH的150μg/kg胡桃醌抑瘤作用不明显。同一浓度不同pH药物的抑瘤作用差异无显著性(P均>0.05),但pH 4.0的胡桃醌组肿瘤细胞肝转移较少。结论胡桃醌不同给药途径均可抑制人肝癌BEL-7402细胞裸鼠皮下移植瘤的生长,但有一定的毒副作用,药物安全范围较小。     

Differential regulation of the expression of aquaporins 3 and 9 by Auphen and dbcAMP in the SMMC-7721 hepatocellular carcinoma cell line

Aquaglycero-aquaporins (agAQPs) are the structural foundation of rapid water transport and they appear to participate in cancer proliferation and malignancy. AQP3 expression is increased and AQP9 expression is decreased in hepatocellular carcinoma (HCC) compared to normal liver, which suggests their possible use as targets for cancer treatment. AQP-based modifiers, such as Auphen and dibutyryladenosine 3′, 5′-cyclic monophosphate (dbcAMP), might be used to treat several diseases and as chemical tools for assessing the functions of AQPs in biological systems. We investigated the effects of both Auphen on AQP3 and dbcAMP on AQP9 in SMMC-7721 cells. We used western blotting, real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry to evaluate changes in AQP3 and AQP9 expression in SMMC-7721 cells after culturing with Auphen and dbcAMP, respectively. We also determined the proliferation of SMMC-7721 cells. We found that compared to HL-7702 (L02) liver cells, Auphen increased AQP3 expression in tumor cells, whereas dbcAMP decreased expression of AQP9 in these cells. Also, high concentrations of Auphen and dbcAMP inhibited proliferation of SMMC-7721 cells in vitro. Auphen and dbcAMP may inhibit HCC development and could be considered targets for HCC diagnosis and therapy.     

80MeV/u20Ne10+离子对SMMC-7721细胞DNA损伤及细胞周期的影响

从辐照剂量和修复时间两个角度研究了重离子辐照对肿瘤细胞DNA损伤及细胞周期的影响,为重离子治癌的临床应用积累基础数据。不同剂量的80MeV／u^20Ne^10 辐照SMMC—7721细胞样品,利用单细胞凝胶电泳技术(Single Cell Gel Electrophoresis,SCGE)对细胞DNA损伤进行了检测,利用流式细胞技术(Flow Cytometry Methods,FCM)对细胞周期变化进行了分析。80MeV／u^20Ne^10 辐照后4小时内,SMMC—7721细胞的DNA损伤与辐照剂量呈线性关系,在0小时组其线性相关因子r为0．9621,4小时组为0．914;随着修复时间的增加,DNA损伤与辐照剂量不再线性相关,但0．5Gy,1Gy和2Gy三个剂量点的DNA损伤程度极为相近。另外,重离子辐照后SMMC—7721细胞发生S期和G2／M期阻滞现象,其随剂量变化及时间变化的规律不同于X、γ等低LET(Linear Energy Transfer)射线辐照。     

壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响

目的检测壳寡糖对人肝癌SMMC-7721细胞的抑制效果及对凋亡调控蛋白Bcl-2和Caspase-3的影响。方法采用噻唑蓝（MTT）法检测不同浓度壳寡糖对肝癌细胞SMMC-7721细胞增殖的抑制作用,并利用荧光Hoechst33258染色法检测细胞凋亡状况。最后通过免疫细胞化学方法研究壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响。结果壳寡糖能够抑制SMMC-7721细胞增殖,并且促进SMMC-7721细胞的凋亡,并且壳寡糖能够上调促凋亡蛋白Caspase-3的表达和降低抑制凋亡蛋白Bcl-2的表达。结论壳寡糖对人肝癌SMMC-7721细胞的增殖有抑制作用,此作用可能是通过促进Caspase-3的表达和抑制Bcl-2的表达来实现的。     

四逆散对人肝癌HepG2细胞增殖、凋亡的影响及其机制

目的:探讨含四逆散药液血清对人肝癌HepG2细胞增殖、凋亡的影响及机制。方法:将人肝癌HepG2细胞分为5组,每组3个复孔。实验组细胞用五氟尿嘧啶(5-FU)或不同浓度的含四逆散药液血清处理48 h后,用倒置显微镜观察含四逆散药液血清处理后人肝癌HepG2细胞形态的变化; MTT法检测含四逆散药液血清对HepG2细胞生长的抑制作用;荧光染色和流式细胞术分别分析含四逆散药液血清对HepG2细胞凋亡的影响。Rho123染色法检测线粒体膜电位变化,Western blot检测细胞凋亡相关蛋白的表达。结果:与对照组比较,含四逆散药液血清处理人肝癌HepG2细胞后,细胞数量显著减少(P<0.01),形态发生改变,呈现典型的凋亡细胞形态; G1期细胞数明显增加,而G2期细胞数量显著减少(P<0.05); Bax、Caspase-3、-9和Cyt-c的表达显著升高,而Bcl-2的表达显著降低(P<0.05);随着含四逆散药液血清浓度增大,HepG2细胞线粒体膜电位显著下降(P<0.05)。结论:四逆散可以抑制HepG2细胞增殖,并通过线粒体途径诱导细胞凋亡。     

siRNA blocking the RAS signalling pathway and inhibits the growth of oesophageal squamous cell carcinoma in nude mice

The aim of this study was to study RAS‐siRNA blocking RAS pathway and suppressing cell growth in human oesophageal squamous cell carcinoma in nude mice. The methods in this study was to construct RAS‐siRNA expression vector, establish 40 oesophageal squamous cell carcinoma xenograft animal models and divided them into five groups: control group, siRNA control group, RAS‐siRNA group, paclitaxel group and RAS‐siRNA and paclitaxel group. We observed tumour growth in nude mice, studied histology by HE staining, tumour growth inhibition by TUNEL assay and detected the RAS, MAPK and cyclin D1 protein expression by immunohistochemistry and western blot. We have obtained the following results: (i) successfully established animal models; (ii) nude mice in each group after treatment inhibited tumour volume was significantly reduced compared with the control group (p < 0.05); (iii) compared with the control group, the number of apoptotic cells were significantly increased in the siRNA control group and the RAS‐siRNA group, and the number of apoptosis cells in the paclitaxel and RAS‐siRNA group is significantly most than the paclitaxel group and RAS‐siRNA group (p < 0.05); and (iv) after treatment, RAS, MAPK and cyclin D1 expression in five groups was decreasing gradually. After adding paclitaxel, the protein expression in the paclitaxel and RAS‐siRNA group was significantly lower than that of paclitaxel group, negative control and paclitaxel group (p < 0.05). We therefore conclude that RAS‐siRNA can block the RAS signal transduction pathway, reduce the activity of tumour cells, arrest tumour cell cycle, promote apoptosis, inhibit cell proliferation and increase tumour cell sensitivity to chemotherapeutic drugs. Copyright © 2014 John Wiley & Sons, Ltd.     

A new human pancreatic carcinoma cell line developed for adoptive immunotherapy studies with lymphokine-activated killer cells in nude mice

Summary A human tumor cell line designated SU.86 has been established from a moderate-to-poorly differentiated pancreatic carcinoma of ductal origin specifically for adoptive immunotherapy studies. This line was characterized as to its ability to be lysed in vitro by autologous and allogeneic lymphokine-activated killer (LAK) and natural killer cells and to grow in nude mice. SU.86 has been growing continuously in cell culture for more than 100 passages since 22 September 1986. Transplantation orthotopically and heterotopically into athymic Swiss nude mice showed that tumor take was 100% in the orthotopic position when young (4 to 6 wk old) mice were used and 0% when adult (8 wk old) mice were used (P=0.004). In the heterotopic position (subcutaneous), tumor take was 100% in neonate (2 to 3 wk old) and young mice and 50% in adults. The rate of tumor growth was inversely correlated with age (P<0.001). The histologic pattern is similar to that observed in most human pancreatic carcinomas with pseudoglandular structures and frequent mitotic figures. SU.86 has a doubling time of 77 h in vitro and produces carcinoembryonic antigen, 594 ng/10⁶ cells in 3 d. Chromosomal analysis shows heterogeneity with two notable cell subpopulations. The cell line is moderately sensitive to lysis by LAK cells in a standard, 4-h chromium-51 release assay (35.4±4.0%). When grown together with LAK cells in vitro, it is lysed completely in culture in 8 to 15 d, depending on the serum concentration.     

Occurrence and morphology of tumors induced in nude mice transplanted with chrysotile-transformed rat pleural mesothelial cells

Rat pleural mesothelial cells treated in vitro with chrysotile fibers have been successfully transplanted into nude mice. Three cultures (1 untreated, 2 treated) were injected at passage 75; a fourth culture was obtained from a mesothelioma induced in rat by chrysotile fibers. Overall, tumors grew in each series, but the delay between cell injection and tumor formation was 22 wk with untreated cells whereas only 1 or 2 wk were needed with treated cells, and 1 wk with cells from in vivo-induced mesothelioma. Pathological study by light and electron microscopy of tumors is reported here and showed the mesothelial nature of the cells. Comparison between the ultrastructure of the injected cells and tumor cells indicated that the morphology of injected cells was retained in tumors even if the delay in tumor formation was long. These results suggest that this model is useful for investigating mesothelial cell transformation resulting from in vitro or in vivo exposure to certain carcinogens.