视网膜母细胞瘤中Survivin和c-Myc的表达及对凋亡的影响
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摘要
目的:研究survivin、c-Myc蛋白在视网膜母细胞瘤(retinoblastoma, Rb)组织中的表达,探讨Rb中survivin与c-Myc蛋白的表达及与瘤细胞凋亡的关系;体外观察曲古菌素A(trichostatin A,TSA)诱导人Rb细胞株Hxo-rb44的细胞周期阻滞和凋亡的作用,以及TSA对凋亡抑制蛋白survivin和原癌基因c-Myc表达变化的影响;构建针对survivin蛋白的shRNA(short hairpin RNA,短发夹RNA)表达质粒,观察其对人Rb细胞株Hxo-rb44细胞的survivin及c-Myc蛋白表达的抑制作用,以及对细胞凋亡的影响,为探讨shRNA-survivin应用于Rb的基因治疗提供理论基础。
方法:(1)华中科技大学同济医学院附属协和医院眼科1994年12月至2005年8月期间的接受眼球摘除术的Rb病人23例23只眼,用免疫组织化学的Sp法(streptavidin-peroxidase conjunct method,链霉亲和素-过氧化物酶连接法)检测survivin、c-Myc蛋白在这23例Rb组织中的表达;用TUNEL技术(terminal deoxynucleo -tidyl transferase-mediate deoxyuridinetriphosphate nick end labeling, TdT-mediated d-UTP nick end labeling,末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记技术)检测Rb组织中的瘤细胞凋亡情况。(2)对人Rb细胞株Hxo-rb44进行培养传代,用流式细胞仪检测不同浓度的TSA作用不同时间后对Hxo-rb44细胞株之细胞周期和细胞凋亡的影响,用Western blot(蛋白质免疫印迹)法检测凋亡过程中survivin和c-Myc蛋白表达的动态变化。(3)设计合成一对针对人survivin的mRNA寡核苷酸序列,退火后将其连入载体,对重组质粒pSIREN进行酶切鉴定;用脂质体介导的方法将干扰质粒pSIREN转染Hxo-rb44细胞,用Western blot法检测转染对survivin、c-Myc蛋白表达的影响,用Hoechst33258染色法检测转染前后的Hxo-rb44细胞诱导凋亡率,并绘制出细胞生长曲线。
结果:(1)在23例23只眼Rb组织切片中,survivin蛋白阳性表达率为60.9%(14/23),可见阳性部位位于胞浆;c-Myc蛋白阳性表达率为73.9%(17/23),可见阳性部位位于胞核。平均凋亡指数(apoptosis index, AI)为0.52%。survivin蛋白表达与Rb瘤细胞凋亡指数明显呈负相关关系,与c-Myc蛋白表达呈正相关关系(p<0.05)。(2)TSA可明显诱导Rb细胞周期阻滞在G2期并诱导凋亡的发生,而且与药物浓度呈依赖关系;用Western blot法可以观察到survivin和c-Myc蛋白表达水平随着TSA作用时间的延长而显著下降。(3)酶切证实目的寡核苷酸片段已经被克隆到pSIREN,转染pSIREN质粒可以显著抑制瘤细胞的survivin蛋白表达;细胞凋亡率由3.5±1.29%上升到36.1±19.66%,c-Myc蛋白表达也显著下降,瘤细胞生长受抑制。转染对照质粒对细胞survivin、c-Myc蛋白及凋亡均无明显影响。
结论:(1)survivin和c-Myc蛋白在Rb协同高表达的现象,可能在Rb的发生发展过程中发挥抗凋亡促增殖的重要作用。(2)TSA可以有效诱导人Rb细胞株Hxo-rb44的细胞周期阻滞和细胞凋亡,其作用机制与抑制survivin和c-Myc蛋白表达有关。(3)成功构建了人survivin基因的短发夹RNA表达质粒,其对Rb的survivin蛋白表达有显著抑制作用,并伴有瘤细胞凋亡率的增加。提示survivin蛋白表达受抑制及同时引起的c-Myc蛋白表达受抑制可能参与了其导致瘤细胞凋亡的机制。
Objective:To observe the expression of survivin and c-Myc protein and their relationship with apoptosis in 23 retinoblastoma(Rb) samples. And to clarify the induction of cell cycle arrest and apoptosis of human retinoblastoma cell line Hxo-rb44 in vitro induced by trichostatin A(TSA) and the changes of the expression of survivin and c-Myc protein in apoptosis. The plasmid containing short hairpin RNA(shRNA) of survivin protein was costructed in order to be observed the expression of survivin gene in human retinblastoma cell line Hxo-rb44 in vitro. And to explore its effect on the expression of survivin and c-Myc protein and apoptosis.
Methods : (1) The cohort consisted of 23 patients diagnosed with primary retinoblastoma from December 1994 to August 2005 in the Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology. The Sp method (streptavidin-peroxidase conjunct method) of immunohistochemical technique(IHC) was used to detect the expression of survivin and c-Myc protein in all paraffin embedded retinoblastoma samples and evidence of apoptosis cells was examined via the TUNEL (terminal deoxynucleotidyl transferase-mediate deoxyuridinetriphosphate nick end labeling, TdT-mediate d-UTP nick end labeling) technique. (2) The retinoblastoma cell line Hxo-rb44 was chosen in this experiment. The cell cycle arrest effects and apoptosis of Hxo-rb44 cell line were assayed with Flow cytometer(FCM). Expression of survivin and c-Myc protein was detected by Western blot. (3) 64 bp reverse repeated motifs of survivin target sequence were synthesized and inserted into plasmid to construct the plasmid shRNA-survivin and the plasmid was transfected into retinblastoma cell line Hxo-rb44. The exprsession of survivin protein was detected by Western blot. The apoptosis ratio of transfected retinoblastoma cell was evaluated by Hoechst33258 stain. And the growth curve was drawn.
Results: (1) Results showed that survivin protein was expressed in nearly 60.9% (14/23) of samples while the c-Myc protein was positively expressed in 17/23 samples (73.9%). Average apoptosis index (AI) is 0.52%. Expression of survivin protein was found to be significantly related with c-Myc protein (p<0.05). Survivin protein expression level was negatively correlated with apoptosis index. (2) Trichostatin A obviously induced cell cycle arrest and apoptosis with dose and time dependent manner. Occurrence of apoptosis was detected by Anexin V-PI staining also in dose dependent manner. The expression survivin and c-Myc protein markedly decreased with increasing incubation time with trichostatin A. (3) The recombinant plasmid shRNA-survivin was successfully constructed and the recombinant plasmid depress the survivin protein expression by 66% and c-Myc by 53% in retinoblastoma cell. After transfection the apoptosis ration was promoted to 36.1±19.66% compared to 3.5±1.29%.
Conclusions: (1)Survivin and c-Myc protein might play synergetic role in the carcinogenesis of retinoblastoma by inhibiting tumor cell apoptosis and promoting proliferation. (2)Trichostatin A can effectively induce cell cycle arrest and apoptosis in retinoblastoma cell line Hxo-rb44 and the inhibition of survivin and c-Myc gene expression may play a critical role in the retinoblastoma cell apoptosis induced by trichostatin A. (3)The short hairpin RNA of survivin can efficiently reduce its expression in retinoblastoma cell and induce apoptosis. The inhibition of the expression of survivin and the reduced c-Myc protein may also contribute to the promoted apoptosis ratio.
方法:(1)华中科技大学同济医学院附属协和医院眼科1994年12月至2005年8月期间的接受眼球摘除术的Rb病人23例23只眼,用免疫组织化学的Sp法(streptavidin-peroxidase conjunct method,链霉亲和素-过氧化物酶连接法)检测survivin、c-Myc蛋白在这23例Rb组织中的表达;用TUNEL技术(terminal deoxynucleo -tidyl transferase-mediate deoxyuridinetriphosphate nick end labeling, TdT-mediated d-UTP nick end labeling,末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记技术)检测Rb组织中的瘤细胞凋亡情况。(2)对人Rb细胞株Hxo-rb44进行培养传代,用流式细胞仪检测不同浓度的TSA作用不同时间后对Hxo-rb44细胞株之细胞周期和细胞凋亡的影响,用Western blot(蛋白质免疫印迹)法检测凋亡过程中survivin和c-Myc蛋白表达的动态变化。(3)设计合成一对针对人survivin的mRNA寡核苷酸序列,退火后将其连入载体,对重组质粒pSIREN进行酶切鉴定;用脂质体介导的方法将干扰质粒pSIREN转染Hxo-rb44细胞,用Western blot法检测转染对survivin、c-Myc蛋白表达的影响,用Hoechst33258染色法检测转染前后的Hxo-rb44细胞诱导凋亡率,并绘制出细胞生长曲线。
结果:(1)在23例23只眼Rb组织切片中,survivin蛋白阳性表达率为60.9%(14/23),可见阳性部位位于胞浆;c-Myc蛋白阳性表达率为73.9%(17/23),可见阳性部位位于胞核。平均凋亡指数(apoptosis index, AI)为0.52%。survivin蛋白表达与Rb瘤细胞凋亡指数明显呈负相关关系,与c-Myc蛋白表达呈正相关关系(p<0.05)。(2)TSA可明显诱导Rb细胞周期阻滞在G2期并诱导凋亡的发生,而且与药物浓度呈依赖关系;用Western blot法可以观察到survivin和c-Myc蛋白表达水平随着TSA作用时间的延长而显著下降。(3)酶切证实目的寡核苷酸片段已经被克隆到pSIREN,转染pSIREN质粒可以显著抑制瘤细胞的survivin蛋白表达;细胞凋亡率由3.5±1.29%上升到36.1±19.66%,c-Myc蛋白表达也显著下降,瘤细胞生长受抑制。转染对照质粒对细胞survivin、c-Myc蛋白及凋亡均无明显影响。
结论:(1)survivin和c-Myc蛋白在Rb协同高表达的现象,可能在Rb的发生发展过程中发挥抗凋亡促增殖的重要作用。(2)TSA可以有效诱导人Rb细胞株Hxo-rb44的细胞周期阻滞和细胞凋亡,其作用机制与抑制survivin和c-Myc蛋白表达有关。(3)成功构建了人survivin基因的短发夹RNA表达质粒,其对Rb的survivin蛋白表达有显著抑制作用,并伴有瘤细胞凋亡率的增加。提示survivin蛋白表达受抑制及同时引起的c-Myc蛋白表达受抑制可能参与了其导致瘤细胞凋亡的机制。
Objective:To observe the expression of survivin and c-Myc protein and their relationship with apoptosis in 23 retinoblastoma(Rb) samples. And to clarify the induction of cell cycle arrest and apoptosis of human retinoblastoma cell line Hxo-rb44 in vitro induced by trichostatin A(TSA) and the changes of the expression of survivin and c-Myc protein in apoptosis. The plasmid containing short hairpin RNA(shRNA) of survivin protein was costructed in order to be observed the expression of survivin gene in human retinblastoma cell line Hxo-rb44 in vitro. And to explore its effect on the expression of survivin and c-Myc protein and apoptosis.
Methods : (1) The cohort consisted of 23 patients diagnosed with primary retinoblastoma from December 1994 to August 2005 in the Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology. The Sp method (streptavidin-peroxidase conjunct method) of immunohistochemical technique(IHC) was used to detect the expression of survivin and c-Myc protein in all paraffin embedded retinoblastoma samples and evidence of apoptosis cells was examined via the TUNEL (terminal deoxynucleotidyl transferase-mediate deoxyuridinetriphosphate nick end labeling, TdT-mediate d-UTP nick end labeling) technique. (2) The retinoblastoma cell line Hxo-rb44 was chosen in this experiment. The cell cycle arrest effects and apoptosis of Hxo-rb44 cell line were assayed with Flow cytometer(FCM). Expression of survivin and c-Myc protein was detected by Western blot. (3) 64 bp reverse repeated motifs of survivin target sequence were synthesized and inserted into plasmid to construct the plasmid shRNA-survivin and the plasmid was transfected into retinblastoma cell line Hxo-rb44. The exprsession of survivin protein was detected by Western blot. The apoptosis ratio of transfected retinoblastoma cell was evaluated by Hoechst33258 stain. And the growth curve was drawn.
Results: (1) Results showed that survivin protein was expressed in nearly 60.9% (14/23) of samples while the c-Myc protein was positively expressed in 17/23 samples (73.9%). Average apoptosis index (AI) is 0.52%. Expression of survivin protein was found to be significantly related with c-Myc protein (p<0.05). Survivin protein expression level was negatively correlated with apoptosis index. (2) Trichostatin A obviously induced cell cycle arrest and apoptosis with dose and time dependent manner. Occurrence of apoptosis was detected by Anexin V-PI staining also in dose dependent manner. The expression survivin and c-Myc protein markedly decreased with increasing incubation time with trichostatin A. (3) The recombinant plasmid shRNA-survivin was successfully constructed and the recombinant plasmid depress the survivin protein expression by 66% and c-Myc by 53% in retinoblastoma cell. After transfection the apoptosis ration was promoted to 36.1±19.66% compared to 3.5±1.29%.
Conclusions: (1)Survivin and c-Myc protein might play synergetic role in the carcinogenesis of retinoblastoma by inhibiting tumor cell apoptosis and promoting proliferation. (2)Trichostatin A can effectively induce cell cycle arrest and apoptosis in retinoblastoma cell line Hxo-rb44 and the inhibition of survivin and c-Myc gene expression may play a critical role in the retinoblastoma cell apoptosis induced by trichostatin A. (3)The short hairpin RNA of survivin can efficiently reduce its expression in retinoblastoma cell and induce apoptosis. The inhibition of the expression of survivin and the reduced c-Myc protein may also contribute to the promoted apoptosis ratio.
引文
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