Apr-1基因对胆管癌细胞QBC939细胞周期基因表达的影响

目的探讨Apr-1基因调控QBC939胆管癌细胞周期的机制。方法运用脂质体介导法将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)。采用细胞周期基因芯片,观察转染Apr-1基因前后胆管癌细胞细胞周期相关基因表达的变化。结果转染Apr-1基因的QBC939-Apr-1细胞出现Apr-1 mRNA的表达,成功建立了稳定表达Apr-1基因的细胞模型。基因芯片检测结果发现:Skp2及UBE基因的表达水平明显上调,而CDC6,Cyclin H等25种基因的表达下降,其中MRE11A,CKS2,CDK8及CDC45等下调在3倍以上。结论过表达的Apr-1基因可诱导QBC939细胞多种细胞周期相关基因表达发生变化,为深入认识Apr-1基因参与调节细胞周期的机制提供了新的思路。     

干扰Livin基因对人胆管癌QBC939细胞增殖影响的研究

目的 研究转染Livin反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)对人胆管癌细胞株QBC939细胞Livin mRNA及Livin蛋白表达以及细胞增殖的影响.方法 设计合成全硫代磷酸化修饰并5'端FITC荧光标记的Livin ASODN.用脂质体介导Livin ASODN转染QBC939细胞,荧光显微镜观察24 h Livin ASODN转染人细胞的情况,并计算转染率.MTT法检测Livin反义寡核苷酸对QBC939细胞增殖的抑制作用.RT-PCR和细胞免疫荧光化学分别检测转染后48 h Livin mRNA表达及Livin蛋白的变化.结果 500 mol/L ASODN转染后24 h可达到最佳的转染效果;转染后60 h,能明显抑制胆管癌细胞的增殖.转染60 h后,RT-PCR及细胞免疫荧光化学检测分别显示Livin mRNA及Livin蛋白表达水平明显低于对照组(P<0.05).结论 脂质体介导转染Livin ASODN能特异性地抑制QBC939细胞中的Livin基因及Livin蛋白表达,抑制胆管癌QBC939细胞的增殖,降低癌细胞活力.

Abstract：

Objective To study the effect of transfection of livin antisense oligodeoxynucleotide (Livin ASODN) on Livin mRNA and Livin protein expression and proliferation of QBC939 cells.Methods Livin ASODN was transfected into cell line QBC939 by LipofectamineTM 2000. Fluorescence microscopy was used to observe the ASODN transfected cells and to calculate the rate of transfection.to measure Livin mRNA and Livin protein expression by RT-PCR and immunohistochemistry and con-focal laser scanning microscopy after the transfection. Changes in cell proliferation were detected by MTT. Results The highest efficiency was at 24 hours after 500 nmol/L Livin ASODN transfection.The results of MTT showed that the inhibition of cell proliferation of QBC939 cells was most obvious at 60 hours after Livin ASODN transfection (P<0. 05). The level of Livin mRNA and Livin protein expression in the ASODN group was obviously lower than that in the control group (P<0. 05).Conclusion The transfection of Livin ASODN inhibited Livin gene and Livin protein expression, and obviously inhibited the proliferation and depressed the vitality of QBC939 cells.     

丁酸钠对人胰腺癌细胞ASPC-1生长的影响及其机制的研究

目的 探讨丁酸钠对人胰腺癌ASPC-1细胞生长的影响并探讨其作用机制.方法 使用不同浓度丁酸钠处理ASPC-1细胞.MTT法观察肿瘤细胞的增殖;流式细胞仪检测细胞凋亡和细胞周期的变化;Western印迹法检测细胞内p21、p53、bcl-2、bax蛋白表达的变化,实时定量PCR检测p21和bcl-2的表达.结果 丁酸钠能明显抑制ASPC-1细胞的增殖,其作用具有明显的时间和剂量依赖性.丁酸钠处理24 h后ASPC-1细胞凋亡率明显提高(P＜0.05),S期细胞比例显著降低(P＜0.05),G0/G1期细胞比例显著升高(P＜0.05).p21、bax蛋白表达明显上调(P＜0.05);bcl-2蛋白的表达显著降低(P＜0.05);p53蛋白表达无明显变化(P＞0.05).结论 丁酸钠可以通过诱导胰腺癌细胞的凋亡和周期阻滞而抑制癌细胞生长;其发生机制可能与下调抗凋亡基因bcl-2、上调促凋亡基因bax和肿瘤抑制基因p21的表达有关.

Abstract：

Objective To investigate the effects of sodium butyrate(NaBT) on proliferation of human pancreatic cancer cell line ASPC-1 and explore the possible mechanism. Methods The methylthiazolyl tetrazolium assay (MTT) method was used to detect cell proliferation and draw a curve. The cell apoptosis and cell cycle were determined with flow cytometry. Western blot was used to study the effect NaBT on the pancreatic cancer cells and explore its mechansim. Real-time PCR was employed to assess the expression levels of p53, p21, bcl-2 and cell cycle regulation gene p21. Results After incubation with different concentrations of NaBT for 24 to 72 h, ASPC-1 cell proliferation was inhibited dramatically. NaBT induced an increase of G0/G1 phase cells and a significant decrease in the ratio of S phase cells. The expression of p21 and bax was up-regulated at protein and mRNA level. The expression of bcl-2 was down-regulated at protein and mRNA level. There was no significant difference in the expression of p53 at protein and mRNA level. Conclusion TSA-induced growth inhibition is associated with a block in the G0/G1 phase and apoptosis, which may occur through down-regulating the expression of apoptosis gene bcl-2 and up-regulating the expression of cell cycle regulation gene p21and pro-apoptotic gene bax.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.     

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma.     

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。     

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。     

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.     

RhoC基因转染对人胆管癌QBC 939细胞增殖和侵袭性的影响

目的研究RhoC（ras homolog gene family，member C）基因对胆管癌QBC939细胞株增殖和侵袭能力的影响。方法以脂质体将RhoC cDNA真核表达载体转染人胆管癌QBC939细胞。应用克隆形成试验检测细胞增殖活性的变化，流式细胞仪检测转染前后细胞周期的变化，Boyden小室侵袭实验检测侵袭能力的变化。结果RhoC基因能促进胆管癌QBC939细胞增殖，流式细胞仪分析结果显示转染后细胞出现G1期细胞减少，侵袭实验显示转染后细胞侵袭能力较转染前有显著加强。结论RhoC基因能够促进胆管癌QBC939细胞株的体外增殖和侵袭能力。     

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。     

突变型p27kip1过表达对胆管癌细胞增殖及凋亡的影响

目的探讨外源性突变型p27^kip1高表达，对人源性胆管癌细胞增殖及凋亡的影响。方法通过携带人突变p27^kip1基因的重组腺病毒载体Ad-p27mt和重组腺病毒Ad-LacZ，转染人胆管癌细胞系QBC9。逆转录-聚合酶链反应（RT-PCR）和Western印记，检测p27^kip1在胆管癌细胞系中mRNA和蛋白的表达水平；噻唑蓝（MIT）比色法、克隆形成试验及流式细胞术（PCM），检测p27^kip1过表达对胆管癌细胞生长抑制、细胞周期和凋亡的影响。结果Ad-p27mt能显著抑制胆管癌细胞的生长，诱导G0／G1期阻滞和细胞凋亡。结论携带人突变p27^kip1基因的重组腺病毒对QBC939细胞的增殖有明显的抑制作用，并诱导强烈的G1期阻滞和细胞凋亡，是胆管癌基因治疗的新途径。     

反义MBD1基因真核表达载体转染对人胆管癌细胞MBD1基因表达的影响

目的：探讨反义MBD1基因真核表达载体转染对人胆管癌QBC 939细胞中MBD1基因表达的影响，为进一步研究该基因的作用提供依据。方法：构建反义MBD1基因真核表达载体，通过脂质体转染入人胆管癌细胞株QBC 939中，以G418进行筛选得到稳定转染细胞株，用PCR鉴定外源性neoR基因在转染细胞中的表达以确定转染成功。半定量RT PCR观察转染前后MBD1基因mRNA水平的变化，流式细胞术检测转染前后MBD1蛋白的变化。 结果：人胆管癌QBC 939细胞中MBD1基因的mRNA水平从0.912±0.022降低至0.215±0.017，转染前后的差异有显著性(P＜0.01)；MBD1蛋白水平从(80.19±5.05)％降低到(35.11±4.05)％，转染前后的差异有显著性(P＜0.01)。 结论：反义MBD1基因转染能降低人胆管癌QBC 939细胞中MBD1基因的表达水平，提示MBD1基因在胆管癌的发生发展中具有重要作用，反义MBD1基因转染有可能成为胆管癌治疗的一种新方法。     

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。