hTERT、Bcl-2在宫颈鳞癌中的表达及冬凌草甲素抑制人宫颈癌SiHa细胞增殖作用研究
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摘要
目的:探讨hTERT及Bcl-2在宫颈鳞癌中的表达及其临床意义;研究冬凌草甲素(Oridonin,Ori)对人宫颈癌SiHa细胞增殖的抑制作用及其机制。
方法:应用免疫组化SABC法检测hTERT及Bcl-2在89例宫颈鳞癌及20例正常宫颈组织中的表达。采用MTT法测定Ori对Siha细胞增殖的抑制作用;Wright- Giemasa染色、Hoechst 33258荧光染色、透射电镜观察Ori处理SiHa细胞后的细胞形态学改变;采用流式细胞仪检测Ori对SiHa细胞凋亡的影响;应用RT-PCR检测Ori处理后SiHa细胞中hTERT、Bcl-2及c-myc mRNA的表达。
结果:在正常宫颈组织和宫颈鳞癌组织中,hTERT表达的阳性率分别为20.0%、76.4%,差异显著(P<0.01);Bcl-2的表达阳性率分别为35.0%、79.8%,有显著性差异(P<0.01);且在宫颈鳞癌组织中hTERT的阳性表达与Bcl-2的阳性表达呈正相关性(P<0.01,r=0.332)。MTT法检测显示Ori能显著抑制SiHa细胞的生长,且具有浓度和时间依赖趋势。流式细胞仪检测显示,15μM Ori作用SiHa细胞48 h后,其凋亡率为14.0%,并使细胞周期阻滞在S期, 30μM Ori组凋亡率较15μM组下降。Wright-Giemasa和Hoechst染色可见SiHa细胞变圆,胞浆减少,核染色质边集。电镜可见SiHa细胞线粒体肿胀,内质网囊泡状扩张,核染色质边集、浓缩,细胞膜完整且边界清楚,部分细胞坏死成碎片状。RT-PCR分析显示随着Ori浓度的增加,hTERT、Bcl-2及c-myc mRNA的表达均呈下降趋势。
结论:hTERT、Bcl-2蛋白在宫颈鳞癌组织中阳性表达率显著高于正常宫颈组织,二者共同参与并协同促进宫颈鳞癌的发生与发展。hTERT随着宫颈鳞癌恶性级别增加呈升高表达的特性,使其有望成为判断宫颈鳞癌恶性程度的参考指标之一。Ori能显著抑制SiHa细胞增殖,低剂量可诱导SiHa细胞凋亡,高剂量可诱导SiHa细胞凋亡并致细胞坏死。冬凌草甲素诱导SiHa细胞凋亡可能与其抑制hTERT、Bcl-2和c-myc mRNA表达有关。
Objective: To investigate the expressions of hTERT and Bcl-2 in cervical squamous cell carcinoma and effect of oridonin on human cervical cancer SiHa cell.
Methods: The expressions of hTERT and Bcl-2 were examined in 89 cervical squamous cell carcinoma and 20 normal cervical specimens using immunohistochem- ical technique. The growth inhibition rates of SiHa cell were measured by MTT assay. Morphological changes of SiHa cell treated by oridonin were determined by Wright- Giemasa staining, Hoechst 33258 staining and transmission electron microscope (TEM). The apoptosis ratio of SiHa cell was defected by flow cytometric analysis (FCM). The expressions of c-myc, Bcl-2 and hTERT mRNA were determined by RT-PCR analysis.
Results: In normal cervical specimens and cervical squamous cell carcinoma, the positive rates of hTERT were 20%, 76.4% respectively(P<0.01), and the positive rates of Bcl-2 were 35%, 79.8% respectively(P<0.01). The overexpression of hTERT was positively correlated with that of Bcl-2 (P<0.01,r=0.332). The proliferation of SiHa cells was significantly inhibited by oridonin in a dose and time dependent manner. Cell apoptosis ratio of 15μM Ori was 14.0% and the cell cycle was arrested at synthesis phase, but the apoptosis ratio of 30μM Ori decreased. Morphological changes of apoptosis of SiHa cell were observed by Wright-Giemasa, Hoechst staining and TEM. The expressions of c-myc, hTERT and Bcl-2 mRNA decreased by RT-PCR.
Conclusion: hTERT and Bcl-2 overexpressed in cervical squamous cell carcinoma with promise well as a diagnostic target, both of them participated and promoted the tumorigenesis. Ori inhibited the proliferation of SiHa cells. Low concentration Ori induced Siha cells apoptosis, and high doze Ori led to apoptosis and necrosis. Apoptosis induced by Ori might be associated with inhibiting the expressions of hTERT,Bcl-2 and c-myc mRNA in SiHa cell.
方法:应用免疫组化SABC法检测hTERT及Bcl-2在89例宫颈鳞癌及20例正常宫颈组织中的表达。采用MTT法测定Ori对Siha细胞增殖的抑制作用;Wright- Giemasa染色、Hoechst 33258荧光染色、透射电镜观察Ori处理SiHa细胞后的细胞形态学改变;采用流式细胞仪检测Ori对SiHa细胞凋亡的影响;应用RT-PCR检测Ori处理后SiHa细胞中hTERT、Bcl-2及c-myc mRNA的表达。
结果:在正常宫颈组织和宫颈鳞癌组织中,hTERT表达的阳性率分别为20.0%、76.4%,差异显著(P<0.01);Bcl-2的表达阳性率分别为35.0%、79.8%,有显著性差异(P<0.01);且在宫颈鳞癌组织中hTERT的阳性表达与Bcl-2的阳性表达呈正相关性(P<0.01,r=0.332)。MTT法检测显示Ori能显著抑制SiHa细胞的生长,且具有浓度和时间依赖趋势。流式细胞仪检测显示,15μM Ori作用SiHa细胞48 h后,其凋亡率为14.0%,并使细胞周期阻滞在S期, 30μM Ori组凋亡率较15μM组下降。Wright-Giemasa和Hoechst染色可见SiHa细胞变圆,胞浆减少,核染色质边集。电镜可见SiHa细胞线粒体肿胀,内质网囊泡状扩张,核染色质边集、浓缩,细胞膜完整且边界清楚,部分细胞坏死成碎片状。RT-PCR分析显示随着Ori浓度的增加,hTERT、Bcl-2及c-myc mRNA的表达均呈下降趋势。
结论:hTERT、Bcl-2蛋白在宫颈鳞癌组织中阳性表达率显著高于正常宫颈组织,二者共同参与并协同促进宫颈鳞癌的发生与发展。hTERT随着宫颈鳞癌恶性级别增加呈升高表达的特性,使其有望成为判断宫颈鳞癌恶性程度的参考指标之一。Ori能显著抑制SiHa细胞增殖,低剂量可诱导SiHa细胞凋亡,高剂量可诱导SiHa细胞凋亡并致细胞坏死。冬凌草甲素诱导SiHa细胞凋亡可能与其抑制hTERT、Bcl-2和c-myc mRNA表达有关。
Objective: To investigate the expressions of hTERT and Bcl-2 in cervical squamous cell carcinoma and effect of oridonin on human cervical cancer SiHa cell.
Methods: The expressions of hTERT and Bcl-2 were examined in 89 cervical squamous cell carcinoma and 20 normal cervical specimens using immunohistochem- ical technique. The growth inhibition rates of SiHa cell were measured by MTT assay. Morphological changes of SiHa cell treated by oridonin were determined by Wright- Giemasa staining, Hoechst 33258 staining and transmission electron microscope (TEM). The apoptosis ratio of SiHa cell was defected by flow cytometric analysis (FCM). The expressions of c-myc, Bcl-2 and hTERT mRNA were determined by RT-PCR analysis.
Results: In normal cervical specimens and cervical squamous cell carcinoma, the positive rates of hTERT were 20%, 76.4% respectively(P<0.01), and the positive rates of Bcl-2 were 35%, 79.8% respectively(P<0.01). The overexpression of hTERT was positively correlated with that of Bcl-2 (P<0.01,r=0.332). The proliferation of SiHa cells was significantly inhibited by oridonin in a dose and time dependent manner. Cell apoptosis ratio of 15μM Ori was 14.0% and the cell cycle was arrested at synthesis phase, but the apoptosis ratio of 30μM Ori decreased. Morphological changes of apoptosis of SiHa cell were observed by Wright-Giemasa, Hoechst staining and TEM. The expressions of c-myc, hTERT and Bcl-2 mRNA decreased by RT-PCR.
Conclusion: hTERT and Bcl-2 overexpressed in cervical squamous cell carcinoma with promise well as a diagnostic target, both of them participated and promoted the tumorigenesis. Ori inhibited the proliferation of SiHa cells. Low concentration Ori induced Siha cells apoptosis, and high doze Ori led to apoptosis and necrosis. Apoptosis induced by Ori might be associated with inhibiting the expressions of hTERT,Bcl-2 and c-myc mRNA in SiHa cell.
引文
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