9-硝基喜树碱脂质体对肝癌细胞株HepG2的抑制作用及机制

目的 观察9-硝基喜树碱脂质体对HepG2肝癌细胞的抑制作用及其机制.方法 9-硝基喜树碱(9NC)及其脂质体(9NC-LP)处理HepG2细胞后,MTT法测定细胞活性,流式细胞术检测细胞周期及凋亡相关蛋白表达.建立HepG2细胞裸鼠皮下移植瘤模型,随机分为空白对照组,空白脂质体组,DMSO组,9NC低、高剂量组,9NC-LP低、高剂量组,每组10只.尾静脉给药后持续监测各组裸鼠肿瘤体积、体重变化.给药后28 d处死裸鼠,切取肿瘤提取总蛋白后Western Blot检测蛋白表达变化.结果 在体外实验,9NC-LP对肿瘤细胞生长呈时间和剂量依赖性抑制.药物处理后S期和G2/M期细胞明显增多,浓度大于0.1 btmol/L时24 h后细胞完全阻滞于S期;0.1μmol/L 孵育72 h后,超过95%HepG2细胞阻滞于G2/M期.在体内实验,与空白对照组比较,各剂量的9NC及9NC-LP均引起肿瘤体积下降(P<0.05)和裸鼠体重减轻(P<0.05).空白脂质体组与空白对照组、DMSO组比较差异无显著性(P>0.05).肿瘤生长最高抑制率9NC-LP(2.5 mg/kg)组、9NC-LP(1.5 mg/kg)组、9NC(1.5 mg/kg)组,分别为87.02%、51.57%和35.47%.9NC (2.5 mg/kg)组用药14 d过半动物死亡.结论 9NC及9NC-LP可以通过调控细胞周期和诱导凋亡有效抑制肝癌细胞生长.与原料药相比9-硝基喜树碱脂质体在体内对肝癌细胞的抑制增强,副作用减弱.9-硝基喜树碱脂质体是一种极具前景的纳米靶向药物.

Abstract：

Objective To observe the inhibitory effect and mechanism of 9-nitrocamptothecin liposomes on HepG2 liver carcinoma cells. Methods HepG2 cells were incubated with 9-nitrocampto-thecin(9NC) or with 9-nitrocamptothecin liposomes(9NC-LP) for 24 h, 48 h and 72 h. Cell viability was then measured by the MTT assay. Cell cycle and apoptosis were evaluated by flow cytometry.Western Blot was used to determine the expression of cell cycle and apoptosis related proteins. HepG2tumor-bearing mouse models were then established. The HepG2 tumor-bearing mice were randomly divided into control group, free liposomes group, DMSO group, 9NC low dose group, 9NC high dose group, 9NC-LP low dose group and 9NC-LP high dose group. There were 10 mice in each group.Drugs were administered by tail vein and tumor volume and body weight were observed 28 days after administration. Then animals were sacrificed and the expression of proteins from tumor homogenates was analyzed by Western blotting. Results In vitro, HepG2 cell viability was apparently inhibited by 9NC and 9NC-LP, and the inhibitory effect increased in a time-dependent and dose-dependent manner.Both S and G2/M phase arrests were observed after incubation with drugs. HepG2 cells were completely arrested in S phase with 9NC concentration over than 0.1 μmol/L after incubation for 24 h,while more than 95% of cells arrested in G2/M phase when 9NC concentration was 0.1 μmol/L after incubation for 72 h. In vivo, compared with the control group, the average tumor volume was reduced in both the 9NC and 9NC-LP group (P<0.05) , and the average animal body weight also decreased in both the 9NC and 9NC-LP group (P<0.05). There was no significant difference among the control group, free liposomes group, and DMSO group. The lights inhibition rates of tumor growth in the 9NC-LP(2.5 mg/kg),9NC-LP(1.5 mg/kg),and 9NC(1.5 mg/kg)groups were 87.02%, 51.57%and 35.47%, respectively. In the 9NC-LP(2.5 mg/kg)group, >50% of animals died 14 days after drug administration. Conclusion 9NC and 9NC-LP can inhibit HepG2 cell growth via cell cycle arrest and apoptosis induction. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo,which means 9NC-LP is a promising compound for cancer therapy via intravenous administration.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

奥沙利铂长循环脂质体的制备及其对人结直肠癌SW480细胞活性的影响

目的 观察奥沙利铂长循环脂质体的制备以及对结直肠癌SW480细胞活性的影响.方法 通过逆向旋转蒸发法制备奥沙利铂长循环脂质体,检测脂质体的粒径、电位、包封率及外观形态;分析奥沙利铂长循环脂质体对细胞活性的影响.结果 脂质体粒径、电位分别为(151.56±15.57)nm,(-23.68±2.35)mV;包封率为(42.96±6.45)%;细胞对脂质体的摄入在2 h可观察到,平均荧光强度在2 12、24 h分别为198、443、642;2.60 mmol/L的空载脂质体、28.0 mg/L游离奥沙利铂及2.60 mmol/L的奥沙利铂脂质体(含28.00 mg/L奥沙利铂)分别与细胞作用12 h,细胞的活力分别是(84.73±1.73)%、(70.72±2.66)%和(57.69±3.33)%(F=104.428,P＜0.01),同时细胞集落数量减少.结论 奥沙利铂长循环脂质体具有增强对结直肠癌SW480细胞毒性作用.

Abstract：

Objective To investigate the preparation of oxaliplatin long-circulating liposomes and its effect on activity of human colorecal cancer SW480 cells. Methods The rverse-phase evaporation vesicles (REV) method was used to prepare the oxaliplatin long-circulating liposomes. The particle size, electric potential, entrapment efficiency and face shape were analyzed, and the effects of liposomes on activity of cells were also analyzed. Results The particle size and electric potential were ( 151.56 ± 15. 57 ) nmand ( - 23.68 ± 2. 35) mV resepctively. Entrapment efficiency was (42. 96 ± 6. 45 ) %. At 2 h, the uptake of liposomes by cells was observed. At 2, 12, 24 h, immunofluorescent intensity was 198,443 and642 respectively. After cells were treated with 2. 60 mmol/L blank liposomes, 28.00 mg/L free oxaliplatin and 2. 60 mmol/L liposomal oxaliplatin (containing 28 mg/L oxaliplatin), activity of cells was (84. 73 ±1.73 ) %, (70. 72 ± 2, 66 ) % and (57. 69 ± 3. 33 ) % respectively ( F = 104. 428, P ＜ 0. 01 ). Additionally, the number of cell colonies was reduced. Conclusion Oxaliplatin long-circulating liposomes were successfully prepared, and could enhance cytotoxic effects against colorecal cancer SW480 cells.     

卡铂碳包铁纳米笼壳聚糖微球对人肝癌细胞株HepG2的影响

目的探讨卡铂碳包铁纳米笼壳聚糖微球（C-Fe@CN-CN）对人肝癌细胞株HepG2增殖和细胞周期的影响。方法应用反相微乳法制备C-Fe@CN-CN，采用噻唑蓝（MTT）法检测不同药物浓度（20、40、80、160mg／L）的C-Fe@CN-CN对HepG2细胞作用24、48、72h的生长抑制效应；流式细胞仪分析药物浓度为8、16mg／L，作用48h时细胞周期的改变。结果C-Fe@CN—CN对人肝癌细胞增殖有明显抑制作用，并呈剂量和时间依赖性。随剂量增加，24h抑制率由18．8％增至53．2％，随时间延长，抑制率由53．2％增至98．7％。微球作用24h时抑制效应低于原药。作用48h和72h时抑制效应等同于原药。C-Fe@CN-CN或原药作用48h后，肝癌细胞周期阻滞于S期（47．2％），随药物浓度增加，S期阻滞（59．3％）更加明显。空白微球对肝癌细胞增殖和细胞周期分布无影响。结论C-Fe@CN—CN在体外能有效地抑制肝癌细胞增殖，机制与原药相同。均为诱导S期阻滞。     

细胞周期素D1反义cDNA治疗肝癌的研究

目的 通过基因反义封闭技术抑制细胞周期素D1（CydinD1）的表达，研究其对肝癌细胞增殖以及成瘤性的影响。方法 以肝癌HepG2细胞株为研究对象，通过转染可表达CyclinD1反义互补脱氧核苷酸（AScDNA）的质粒后，观察CyclinD1反义cDNA对肝癌细胞CyclinD1基因表达、体外增殖活性及裸鼠体内成瘤性的影响。结果 噻唑蓝（MTT）法检测细胞增殖活性显示转染表达反义CydinD1的质粒后，HepG2细胞的增殖受到抑制．抑制作用在48h左右最强；逆转录-聚合酶链反应（RT-PCR）检测显示CyclinD1 mRNA基因的表达明显被抑制；间接免疫荧光检测结果显示CyclinD1蛋白表达显著降低；流式细胞仪检测结果显示G0／G1期的细胞比例增高，G2＋M和S期的细胞比例下降，HepG2细胞周期在G1期被阻滞；裸鼠成瘤试验显示肝癌HepG2细胞的成瘤性受到明显抑制。结论 CyclinD1反义cDNA可以特异性的抑制肝癌HepG2细胞株CyclinD1蛋白的表达，从而调控细胞周期，抑制肝癌细胞增殖及体内成瘤性。CyclinD1反义cDNA对于肝细胞癌的生物治疗具有一定的应用前景。     

氯化钴诱导低氧环境对人肝癌HepG2细胞株的生长影响

目的 建立人肝癌HepG2细胞在氯化钴诱导的低氧环境下的生长模型,观察不同浓度氯化钴(CoCl2)诱导的低氧环境对人肝癌HepG2细胞的增殖、细胞的周期与凋亡即细胞的生长状况影响,探讨氯化钴诱导的低氧对人肝癌HepG2细胞生长的作用机制.方法 将对数期人肝癌细胞株HepG2细胞,分为加入不同浓度(50 μm/L、100 μm/L、150 μm/L、200 μm/L)氯化钴的实验组和加入等体积培养基的对照组,(1)通过MTT法检测HepG2细胞增殖情况计算细胞的抑制率并绘制HepG2细胞生长抑制曲线;(2)划痕实验观察HepG2细胞的迁移能力;(3)通过流式细胞仪的Annexin-VFITC/PI双染法和PI单染法检测细胞的凋亡和周期变化;(4)通过Western Blot法检测HepG2细胞的Bcl-2蛋白的表达.结果 (1)通过MTT法检测结果示在一定的时间和浓度范围内氯化钴抑制人肝癌HepG2细胞的增殖,并呈时间-剂量依赖性关系.(2)划痕损伤实验说明:氯化钴抑制HepG2细胞的迁移及损伤修复能力,呈浓度依赖性关系.(3)流式细胞仪的检测结果显示:对照组、不同浓度(50 μm/L、100 μm/L、200 μm/L、300 μm/L、500 μm/L)的实验组的细胞凋亡率(％)分别是:3.42、7.74、13.07、20.56、28.53、44.45(P ＜0.05),与对照组相比实验组的凋亡率明显增加,并呈浓度依赖性.细胞的周期结果显示:随着浓度增加,氯化钴能明显抑制HepG2细胞周期的变化,阻滞细胞周期于G1期,从而抑制细胞的增殖.(4) Western Blot法检测:与对照组相比,经过不同浓度氯化钴处理后的实验组Bcl-2蛋白的表达明显减少.结论 在一定范围内,氯化钻诱导的低氧环境可以抑制人肝癌HepG2细胞的增殖以及愈合迁移的能力,诱导细胞凋亡并呈时间-剂量的依赖性关系,其作用机制可能与Bcl-2蛋白的表达减少有关.     

NF-κB、Caspase-3在腺苷诱导人HepG2细胞凋亡中的作用

目的 研究NF-κB、Caspase-3在腺苷(ADO)体外诱导人肝癌HepG2细胞凋亡中的作用.方法 将不同浓度的ADO(0.1～5 mmol/L)作用于HepG2细胞,采用MTT法测定ADO抑制细胞增殖的时间效应和剂量效应.2.0 mmol/L ADO单用或联合NF-κB抑制剂吡咯烷二硫氨基甲酸(PDTC,100 μmol/L)作用HepG2细胞12 h、24 h,采用Hoechst 33342荧光染色法及流式细胞术(FCM)观察细胞凋亡及细胞周期,Western blot技术检测NF-κB蛋白表达;荧光比色法测定Caspase-3酶活性.结果 ADO对HepG2细胞生长具有显著抑制作用,并呈一定的量效和时效关系.药物作用24 h、48 h的IC50分别为2.52 mmol/L和1.89 mmol/L.ADO单独处理HepG2细胞12 h和24 h或联合PDTC处理后,细胞凋亡率分别为8.30%、22.32%;20.18%、30.89%,均显著高于对照组(0.81%,P<0.001);ADO作用HepG2细胞后荧光显微镜观察到细胞凋亡的形态学改变,FCM分析药物处理组显示典型特征性的亚二倍体凋亡峰(sub-G1),细胞生长周期阻滞于G0/G1期;同时伴有Caspase-3活性显著升高(P<0.05).ADO处理后显著增加了NF-κB蛋白表达(P<0.05);PDTC有效抑制了NF-κB表达,同时增加了 Caspase-3活性及ADO的细胞毒作用(P<0.05).结论 ADO诱导了HepG2细胞凋亡并活化Caspase-3.抑制NF-κB活性可通过Caspase-3途径增强ADO的细胞毒作用.     

MIF和Cyclin D1在肝细胞癌中的表达

目的 探讨巨噬细胞游走抑制因子(macrophage migration inhibitory factor,MIF)和细胞周期蛋白D1(cyclin D1)在原发性肝细胞癌(hepatocellular carcinoma,HCC)组织中的表达及两者在肝癌发病机制及细胞周期调控中的关系.方法 应用定量PCR及Westem blot技术检测MIF和Cyclin D1在肝癌组织和癌旁组织的表达.化学合成MIF siRNA,将PLC细胞和HepG2细胞分成对照组、MIF siRNA 50 nmol/L干预组、MIF siRNA 1130 nmol/L干预组,脂质体法转染肝癌细胞PLC和HepG2,定量PCR技术和Western blot检测MIF siRNA干扰后MIF和Cyclin D1 mRNA和蛋白的表达变化.结果 MIF和Cyclin D1蛋白在肝癌组织中的相对表达量为0.825±0.13和0.843±0.104;MIF和Cyclin D1 mRNA在肝癌组织中的相对表达量为癌旁组织的(7.31±1.85)倍和(4.27±1.05)倍,与癌旁组织相比,差异均具有统计学意义(FMIF=15.5,P<0.01;fCyclin D1=87.5,P<0.01).应用小RNA干扰技术转染PLC和HepG2肝癌细胞后MIF mRNA分别下调71.2%±7.2%、87.4%±2.9%、74.3%±8.9%、88.4%±4.6%(FPLC=315.5,P<0.01;FHHepG2=201.2,P<0.01);MIF蛋白分别下调为0.33±0.03、0.11±0.02、0.81±0.08、0.36±0.02,并呈剂量依赖关系(FPLC=43.9,P<0.01;FHepG2:133.4,P<0.01).伴随MIF mRNA和蛋白的表达F调Cyclin D1 mRNA分别下调68.2%±3%、78.1%±1.4%、65.8%±4.7%、77.3%±2.6%(FPLC=1569,P<0.01;FHepG2=480.4,P<0.01);Cyclin D1蛋白下调0.28±0.06、0.15±0.03、0.44±0.04、0.13±0.02,亦呈剂量依赖关系,与对照组相比差异有统计学意义(P<0.01),两干预组相比差异有统计学意义(FPLC=35.5,P<0.01;FHepG2=114.7,P<0.01).结论 MIF和Cyclin D1在肝细胞癌中过表达,MIF可能在转录水平调控Cyclin D1的表达,并促使肿瘤细胞通过细胞周期检测点,继续增值和分化,导致肿瘤的形成.     

姜黄素通过PPAR-γ途径促进人肾癌细胞的调亡

目的观察姜黄素对人肾癌细胞株A498细胞增殖和凋亡的影响,并探讨PPAR-γ途径在其中的作用。方法姜黄素(10、50、100μmol/L)处理A498细胞12、24、48h。采用MTT法测定细胞活力,并计算肿瘤细胞抑制率,流式细胞术测定细胞凋亡。实时定量PCR和Western Blot分别检测Bcl-2、Bax及PPAR-γmRNA和蛋白的表达。观察PPAR-γ阻断剂GW9662对姜黄素作用的影响。结果姜黄素(10、50、100μmol/L)分别处理人肾癌细胞株A498细胞24、48、72h后,以浓度和时间依赖的方式抑制细胞增殖促进细胞凋亡,同时以浓度依赖的方式下调了A498细胞中Bcl-2的表达,增加了Bax和PPAR-γ的表达。GW9662部分地消除了姜黄素对A498细胞增殖和凋亡的影响。结论姜黄素抑制人肾癌细胞增殖促进其凋亡,其机制涉及到PPAR-γ途径。     

雄激素对MCF-7乳腺癌细胞株增殖凋亡的影响

目的 观察睾丸酮对MCF-7乳腺癌细胞株增殖、凋亡的影响,并探讨其机制.方法 将1×10~(-5)、1×10~(-7)、1×10~(-9)、1×10~(-11) mol/L的睾丸酮分别作用于乳腺癌MCF-7细胞24、48、72 h,噻唑蓝(MTT)比色法检测细胞生长,流式细胞术检测不同浓度睾丸酮作用48 h时MCF-7细胞的周期分布和凋亡以及该细胞株中细胞周期素D1蛋白和雄激素受体的表达.结果 高浓度睾丸酮抑制MCF-7乳腺癌细胞株的增殖,1×10~(-5) mol/L睾丸酮作用48 h时,细胞生长抑制率为22.21%,细胞凋亡率为(58.60±5.41)%,但可使细胞周期由G_1 期进入S期,并可使细胞周期素D1蛋白表达量增加,雄激素受体蛋白表达量下降.低浓度睾丸酮(1×10~(-9) mol/L)作用后细胞周期素D1蛋白表达量无明显变化,雄激素受体蛋白表达量升高,在短时间内(24 h)可促进MCF-7细胞增殖.结论 高浓度睾丸酮在体外可使MCF-7乳腺癌细胞株细胞周期素D1蛋白表达增加,使细胞周期由G_1进入S期,而同时促使细胞凋亡,表现出抗肿瘤作用.低浓度睾丸酮有短暂的促进MCF-7乳腺癌细胞增殖的作用.     

五羟黄酮调节肝癌HepG2细胞bax基因表达诱导细胞凋亡的实验研究

目的:探讨三磷酸肌醇( IP3) 和bax基因表达变化在五羟黄酮(Quercetin)诱导肝癌细胞凋亡中的作用。方法:以肝癌HepG2 细胞培养72 h为对照,以20，40，60，80μmol/L Quercetin作用于 HepG2 细胞72 h 时和60μmol/L Quercetin 作用于 HepG2 细胞 6，12，24，48，72 h，应用同位素试剂盒检测细胞IP3含量,RT-PCR分析bax mRNA表达，Western blotting 分析细胞bax蛋白表达,流式细胞仪检测细胞凋亡率。结果:各浓度的Quercetin作用于肝癌HepG2 细胞72 h，IP3含量显著低于对照组(P<0.01)，bax mRNA和bax蛋白表达显著高于对照组，细胞凋亡率显著高于对照组(P<0.01); 60 μmol/L Quercetin 作用于肝癌HepG2 细胞6，12，24，48，72 h,各时相IP3含量显著低于对照组(P<0.01）;12 h后bax mRNA和bax蛋白表达显著高于对照组，24 h后各时相细胞凋亡率为显著高于对照组（P<0.01）。结论:Quercetin能减少IP3生成,上调bax基因表达,诱导肝癌细胞凋亡。