Apr-1基因对胆管癌细胞QBC939细胞周期的调节机制研究

目的 研究Apr-1基因对胆管癌细胞的细胞周期调节作用及机制.方法 采用脂质体介导法将Apr-1基因转染胆管癌细胞系QBC939,建立稳定表达Apr-1基因的细胞模型(QBC939-Apr-1).运用RT-PCR检测转染前后QBC939细胞系中Apr-1 mRNA的表达;流式细胞分析以及生长曲线等方法观察目的 基因对该细胞系的细胞周期的影响.采用细胞周期基因芯片,观察Apr-1基因对细胞周期相关基因表达的调节作用.结果 Apr-1基因在QBC939细胞系中呈阴性表达,转染Apr-1基因的QBC939-Apr-1细胞出现了Apr-1 mRNA的表达,成功建立了稳定表达Apr-1基因的细胞模型,该细胞模型表现出细胞生长受抑,细胞周期显示G2期细胞由9%增加至13%(P＜0.01).细胞周期抑制;并且细胞周期基因芯片检测结果发现:Skp2、UBE基因的表达水平出现明显上调,而CDC6、Cyclin H等25种基因的表达下降,其中MRE11A、CKS2、CDK8、CDC45下调在3倍以上.结论 Apr-1基因在体外能够使QBC939细胞的细胞周期在G2期延长,出现细胞周期多种调节基因的表达差异.

Abstract：

Objective To investigate the role and the mechanism of Apr-1 gene on cholangiocarcinoma QBC939 cell lines proliferation and cell cycle regulation. Methods Apr-1 gene was transfected into QBC939 cells by using liposomes to establish a QBC939 cell model ( QBC939-Apr-1 ) stably expressing Apr-1 gene. Apr-1 mRNA expression and the changes in cell cycle and cell growth of QBC939 cells were analyzed by RT-PCR, flow cytometry ( FCM ) and growth curve before and after transfection. The regulatory effect of Apr-1 gene on the expression of cell cycle-related genes was investigated in QBC939 cells before and after Apr-1 transfection using cell cycle gene microarrays. Results Significant suppression of cell growth was observed with the cell model stably expressing Apr-1 gene. Apr-1 over-expression caused cell arrest from 9% to 13% (P ＜0. 01 ) increase in G2 population. Cell cycle gene microarrays demonstrated that the expression of Skp2 、UBE1 was up-regulated, while the expression of MRE11A 、CKS2 、CDK8 、CDC45 was down-regulated by more than 3 folds. Conclusions Apr-1 gene suppresses QBC939 cell proliferation in vitro, QBC939 cells presented with differences in the expression of cell cycle-related genes after Apr-1 gene transfection.     

WWOX在胆管癌组织中的表达及其对胆管癌细胞生物学行为的影响

目的 研究抑癌基因WWOX表达对胆管癌RBE细胞生长的影响.方法 采用免疫组化方法检测2005年7月至2010年5月54例胆管癌组织、12例正常胆管组织中WWOX蛋白表达水平.将携有WWOX基因的真核表达载体转染胆管癌细胞系RBE细胞(RBE/WWOX组),筛选稳定转染的细胞并扩增培养,以转染空载质粒(RBE/con组)及未经转染(自然生长组)的RBE细胞作为对照.荧光定量RT-PCR和Western Blot法检测WWOX在RBE细胞中的表达情况;噻唑蓝实验检测转染前后各组细胞增殖活性;FCM法检测各组细胞的凋亡;JC-1染色法检测细胞线粒体膜电位;Transwell小室侵袭实验检测各组肿瘤细胞侵袭力;荧光定量RT-PCR和Western Blot法检测胆管癌细胞bcl-2、bax、FasL、caspase-3表达的变化.结果 WWOX在胆管癌中的表达低于正常胆管组织(P＜0.05),蛋白表达的缺失频率为40.7%.建立稳定表达WWOX基因的RBE/WWOX细胞株,mRNA及蛋白表达明显增加.转染后的RBE细胞噻唑蓝吸光度明显下降(P＜0.05).与自然生长组和RBE/con组比较,FCM显示RBE/WWOX组细胞的凋亡率明显增高(P＜0.01),JC-1显示转染后的线粒体膜电位下降(P＜0.01),侵袭实验显示转移至下室滤膜的细胞数明显减少(P＜0.01).荧光定量RT-PCR结果显示bcl-2 mRNA表达是自然生长组的0.12倍,bax、caspase-3 mRNA分别是自然生长组的4.72和2.57倍,FasL mRNA的表达无明显变化;Western Blot法检测发现bcl-2蛋白的表达降低,bax、caspase-3蛋白表达升高,FasL无明显变化.结论 抑癌基因WWOX通过诱导胆管癌细胞凋亡发挥抗肿瘤增殖的作用.

Abstract：

Objective To study the effects of anti-oncogene WWOX on cell growth of cholangiocarcinoma. Methods The expression of WWOX protein was detected with immunohistochemical method-SP in 54 patients with cholangiocarcinoma from July 2005 to May 2010 and 12 samples of normal bile duct tissues. The recombinant WWOX eukaryotic expression plasmid was introduced into RBE cells by liposome-mediated transfection and positive cell clones were selected and amplified. The mRNA and protein expressions in RBE cells stably transfected with WWOX were investigated by quantitative RT-PCR and Western Blot before and after transfection. Cell proliferation was tested by MTT, cell apoptosis was assessed by FCM, the alteration of mitochondria membrane potential (△Ψm) was detected by JC-1 staining method,cell invasion was determined by Transwell chamber assay. The expression change of bcl-2, bax, FasL,caspase-3 mRNA and protein was detected by quantitative RT-PCR and Western Blot. Results The expression of WWOX protein was significantly lower in cholangiocarcinoma than that in normal bile duct tissues and loss of WWOX protein expression was found in 40. 7% of cholangiocarcinoma specimens ( P ＜0.05). RBE cells with stable transfection of WWOX were established. Quantitative RT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western Blot demonstrated that WWOX protein expression was markedly increased. MTT showed that WWOX gene transfection significantly decreased the proliferation of RBE cells ( P ＜ 0. 05 ). FCM analysis showed that the apoptosis rate after transfection was significantly promoted [( 1.1 ± 0. 6 ) % vs. ( 1.7 ± 0. 5 ) % vs. ( 35.2 ± 4. 4 ) %, P ＜ 0. 01], JC-1 staining method indicated that the experimental group was loss of △Ψm [( 12. 6 ± 1.9 ) % vs. ( 13.6 ± 1.8 ) % vs.(48. 7 ± 2. 9 ) %, P ＜ 0. 01], transwell chamber assay showed that the number of transfected cells that passed the transwell membrane was significantly less than those of control groups ( 77 ± 6 vs. 72 ± 8 vs. 48 ±6, P ＜0. 01 ). Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased and the expression of bax, caspase-3 were significantly increased. There was no significant change in the expression of FasI. Conclusion WWOX exerts its antitumor effect against proliferation through inducing cell apoptosis in cholangiocarcinoma.     

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.     

干扰X连锁凋亡抑制蛋白基因对人胆管癌QBC939细胞增殖及凋亡的作用

目的 观察反义寡核苷酸(ASODN)对人胆管癌QBC939细胞株X连锁凋亡抑制蛋白(XIAP)基因的抑制作用及对癌细胞增殖及凋亡的影响.方法 脂质体法将ASODN转染入QBC939细胞中,转染12、24、48 h后荧光显微镜观察转染后细胞状态及转染率,应用噻唑蓝(MTT)比色法、逆转录-聚合酶链反应(RT-PCR)法和流式细胞仪法测定1.11μmol/L时细胞生长抑制率、XIAP mRNA表达和细胞周期及凋亡率.结果 转染效率可达95%;与对照组比较,1.11μumol/L细胞抑制率为(27.63±1.15)%(24 h),(42.95±1.07)%(48 h);mRNA下调23%(P<0.05);G_0/G_1期细胞、凋亡率高于对照组约27.34%、9.94%(P<0.05).结论 ASODN能有效下调QBC939细胞XIAP基因表达,抑制细胞增殖并诱导凋亡.     

Bcl-2反义寡核苷酸对VP-16抑制人胆管癌细胞株增殖和诱导凋亡的影响

目的:探讨bcl-2 反义寡核苷酸（ASODN）对VP-16抑制人胆管癌QBC939细胞生长和诱导细胞凋亡的影响。方法:合成特异性靶向Bcl-2 ASODN，并将其转染QBC939细胞;四甲基噻唑蓝（MTT）试验检测Bcl-2 ASODN对VP-16抑制QBC939细胞增殖的影响;流式细胞术观察Bcl-2 ASODN对VP-16诱导QBC939细胞凋亡的影响。结果:ASODN组细胞的存活率显著低于无义寡核苷酸（NSODN）组(P＜0.05);ASODN+VP-16组细胞存活率显著低于ASODN组及VP-16组(P＜0.05);ASODN组、ASODN+VP-16组和VP-16组细胞凋亡率显著高于对照组和NSODN组（P＜0.05);ASODN+VP-16组细胞凋亡率显著高于ASODN组及VP-16组(P＜0.05)。结论:Bcl-2 ASODN对VP-16抑制人胆管癌QBC939细胞生长和诱导细胞凋亡有促进作用。     

RNA干扰诱导型一氧化氮合酶基因表达对人胆管癌细胞生长的影响

目的:探讨RNA干扰诱导型一氧化氮合酶(iNOS)基因的表达对人胆管癌细胞生长的影响。方法:针对iNOS设计并合成3种iNOS-siRNA序列(siRNA1、siRNA2、siRNA3)及阴性对照siRNA序列后,分别转染人胆管癌细胞QBC939细胞,用荧光显微镜下观察转染效率,通过iNOS mRNA与蛋白的变化分析干扰效果,选择干扰效果最为明显的iNOS-siRNA序列,观察其干扰后QBC939细胞增殖、细胞周期及细胞凋亡的变化。实验以无处理的QBC939细胞为空白对照。结果:合成的3种iNOS-siRNA序列均可有效转染人胆管癌QBC939细胞,且转染后均能明显降低QBC939细胞中iNOS mRNA与蛋白的表达(均P0.05),其中siRNA2对iNOS的抑制作用最为明显,阴性对照siRNA序列对QBC939细胞中iNOS mRNA与蛋白的表达无明显影响(均P0.05)。转染siRNA2后,QBC939细胞增值明显降低、出现明显的G0/G1期阻滞、凋亡率明显增加(均P0.05);转染阴性对照siRNA序列的QBC939细胞无上述改变(均P0.05)。结论:RNA干扰能有效降低iNOS基因在胆管癌细胞中的表达,从而抑制胆管癌细胞的增殖,并促进其凋亡。     

脆性组氨酸三联体基因对胆管癌细胞凋亡的影响

目的 观察脆性组氨酸三联体(FHIT)基因对胆管癌细胞株QBC939凋亡的影响,探讨FHIT基因对细胞周期蛋白Cyclin D1的调控作用.方法 通过构建重组人FHIT真核表达质粒转染QBC939细胞,应用流式细胞仪检测转染前后细胞凋亡的变化,并通过荧光定量逆转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹法(Western blot)检测胆管癌细胞Cyclin D1表达的变化.结果 成功构建出重组人FHIT真核表达质粒并转染入QBC939细胞,转染后QBC939细胞的凋亡率明显增高(25.8%比30.9%比55.4%,P<0.05);并且Cyclin D1 mRNA表达下降至原细胞株的0.03倍,该基因蛋白质的表达也明显下降(0.33±0.02对比0.33±0.01比0.14±0.02,P<0.05).结论 FHIT基因可以促进QBC939细胞凋亡,并能下调Cyclin D1基因的表达.     

反义调控DNA甲基转移酶3b基因对人胆管癌细胞QBC939生长的影响

目的观察转染反义DNMT3b基因真核表达质粒对人胆管癌细胞QBC939生长的影响，初步探讨DNMT3b基因在胆管癌发生中的作用。方法构建反义DNMT3b基因真核表达质粒，用脂质体介导法将其转人人胆管癌细胞株QBC939；Western blot检测转染前后DNMT3b蛋白表达的变化；MTT法和软琼脂克隆形成试验观察细胞的生长增殖能力；流式细胞术观察细胞生长周期及凋亡率的变化。结果转染反义基因能使DNMT3b蛋白表达水平降低；转染反义DNMT3b基因不能抑制QBC939的生长曲线，对其软琼脂克隆形成率亦无影响（P=0．717）；转染反义DNMT3b基因不能改变QBC939的细胞周期和促进细胞凋亡（P=0．089）。结论转染反义DNMT3b基因真核表达质粒可下调DNMT3b在QBC939细胞中的表达水平，但对QBC939的生长和增殖无影响，也不能改变QBC939的细胞周期和促进细胞凋亡的发生。     

乙型肝炎病毒X基因转染人胆管癌细胞系及对端粒酶逆转录酶mRNA表达的影响

目的了解乙型肝炎病毒X(HBx)基因转染对胆管癌细胞端粒酶逆转录酶(hTERT)mRNA的表达影响,阐明HBx基因调节hTERT基因转录表达在胆管癌发生中的意义。方法体外培养胆管癌细胞QBC939,应用脂质体介导的基因转染技术,将含HBx基因的真核表达载体转染到胆管癌细胞中;转染36h后以增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)表达判定转染成功率,流式细胞仪检测转染率;收集细胞提取总RNA,逆转录聚合酶链式反应(RT-PCR)分析转染后细胞内hTERT mRNA的表达变化,并通过细胞免疫化学和Western Blotting了解转染后胆管癌细胞内有无HBx蛋白的表达。结果转染含HBx基因表达载体和空载体的QBC939转染率为29．6％;转染HBx基因后hTERT mRNA表达量比未经转染或转染空载体的hTERT mRNA表达量明显增加;细胞免疫组化和Western Blotting也证实只在转染了HBx基因的胆管癌细胞有HBx蛋白表达。结论HBx基因转染能上调胆管癌细胞hTERT mRNA的转录表达。     

p21waf1基因真核表达载体的构建和对人胆管癌细胞生长的影响

目的　探讨p2 1waf1基因过表达在人胆管癌细胞增殖和凋亡中的作用。方法　包含 2 .1kb的 p2 1waf1cDNA片段经阳离子脂质体DOTAP包裹转染人胆管癌细胞QBC939中 ,并得到稳定表达 ,通过聚合酶链反应 (PCR)、逆转录 PCR(RT PCR)、流式细胞仪及免疫组织化学等方法检测p2 1waf1基因表达、细胞生长和细胞凋亡等情况。结果　p2 1waf1基因转染后 ,细胞生长明显受抑制 ,细胞周期停滞于G1期 ,表现为与对照组相比 ,细胞周期G1期比例明显增加 ,S和G2 /M期比例明显减少(G1期 :5 7.32 %→ 73 .6 9% ;S期 :2 1.81%→ 15 .85 % ;G2 /M期 :19.35 %→ 11.76 % ) ,细胞凋亡率增加(3 .47%→ 15 .81% ) ,裸鼠致瘤性降低。结论　p2 1waf1基因能够抑制人胆管癌细胞增殖和诱导细胞凋亡。     

γ-氨基丁酸对人胆管癌细胞株QBC939增殖和侵袭的影响

目的 观察抑制性神经递质γ-氨基丁酸(GABA)对人胆管癌细胞株QBC939生物学行为的影响.方法 采用MTF比色法观察GABA对人系胆管癌细胞QBC939增殖的作用,PCR-ELISA法观察其对QBC939细胞端粒酶活性的影响,Transwell小室分析GABA对胆管癌细胞侵袭能力的影响,逆转录聚合酶链反应(RT-PCR)、明胶酶谱法分析GABA对QBC939细胞分泌的基质金属蛋白酶(matrix metalloproteinase,MMP)mRNA和酶活性的影响,数据采用单因素方差分析和Dunnett法处理.结果 GABA对人胆管癌细胞QBC939的增殖有抑制作用(抑制率由2.6%增至26.8%,P＜0.05);抑制癌细胞中端粒酶的活性(0.82±0.05)vs.(0.56±0.05)(P＜0.05);抑制胆管癌细胞穿透Matrigel胶的能力(在100 μmol/L浓度的GABA作用下,穿透细胞数由(60±10)个降至(43±4)个(P＜0.05),同时胆管癌细胞分泌的基质蛋白酶MMP-2和MMP-9的mRNA和活性下降,这三种效应均成浓度依赖性.结论 GABA抑制胆管癌细胞QBC939的生长并减弱侵袭转移能力,其机制可能与其抑制端粒酶活性和基质蛋白酶MMP-2和MMP-9的分泌和活性有关.     

靶向survivin的shRNA对人胆管癌细胞体外增殖及凋亡的影响

目的：探讨靶向survivin的shRNA对人胆管癌细胞体外增殖及凋亡的影响。方法：合成具有互补序列的能够编码短发卡RNA（shRNA）的双链寡核苷酸并构建pSilencer2．1真核表达质粒,经稳定转染QBC939细胞后对转基因后胆管癌细胞的生物学行为进行观察。结果：neo基因稳定表达于转染阳性质粒及阴性对照质粒的QBC939细胞中。通过RT—PCR及westernblot检测证实shRNA在mRNA及蛋白质水平抑制survivin表达率分别达68固％和613％。细胞计数及平板克隆形成实验显示转染细胞生长速度及细胞克隆明显减缓（P〈O．01）。流式细胞术测定细胞周期显示转染细胞G1期细胞明显增多,S期细胞明显减少。DNAladder、透射电镜观察及流式细胞定量研究显示转染细胞凋亡明显增多。结论：靶向survivin的shRNA能有效抑制目的基因的表达,通过增加细胞凋亡在体外抑制人胆管癌细胞的生长,为胆管癌的基因治疗提供一定的实验基础。