CAJ | 학술논문

目的观察小干扰RNA(siRNA)沉默垂体瘤转化基因(PTTG)对原代培养的人泌乳素型垂体瘤细胞(HPAs)生长的影响.方法原代培养人泌乳素型垂体腺瘤细胞,稳定传代后分为3组:空白对照组、阴性对照组、siRNA-PTTG转染组,转染48 h后细胞计数试剂盒(CCK-8)法检测各组细胞增殖能力;流式细胞仪检测细胞凋亡率;逆转录聚合酶链反应(RT-PCR)和Western blot法测定PTTG mRNA、蛋白的表达;半胱氨酸天冬氨酸蛋白酶(Caspase)试剂盒检测Caspase-3的活性;放射免疫法测定各组细胞上清中泌乳素的水平.结果与空白对照组及阴性对照组比较,siRNA-PlTG转染组HPAs细胞PTTG mRNA和蛋白表达明显下调,细胞增殖受到抑制[阴性对照组、转染组抑制率分别为(3.02±1.77)％、(25.38±0.11)％,P＜0.05],凋亡明显增多[3组凋亡率分别为:(6.21±2.36)％、(7.23±3.56)％、(32.33±5.69)％,P＜0.05],Caspase-3活性增加(P＜0.05);放射免疫法显示siRNA-PTTG转染组上清中泌乳素分泌水平下降,为空白对照组的32.9％ (P ＜0.05).结论下调PTTG基因可抑制人泌乳素型垂体瘤细胞生长,促进其凋亡,从而降低泌乳素分泌水平.
목적 관찰소간우RNA(siRNA)침묵수체류전화기인(PTTG)대원대배양적인비유소형수체류세포(HPAs)생장적영향.방법 원대배양인비유소형수체선류세포,은정전대후분위3조:공백대조조、음성대조조、siRNA-PTTG전염조,전염48 h후세포계수시제합(CCK-8)법검측각조세포증식능력;류식세포의검측세포조망솔;역전록취합매련반응(RT-PCR)화Western blot법측정PTTG mRNA、단백적표체;반광안산천동안산단백매(Caspase)시제합검측Caspase-3적활성;방사면역법측정각조세포상청중비유소적수평.결과 여공백대조조급음성대조조비교,siRNA-PlTG전염조HPAs세포PTTG mRNA화단백표체명현하조,세포증식수도억제[음성대조조、전염조억제솔분별위(3.02±1.77)％、(25.38±0.11)％,P＜0.05],조망명현증다[3조조망솔분별위:(6.21±2.36)％、(7.23±3.56)％、(32.33±5.69)％,P＜0.05],Caspase-3활성증가(P＜0.05);방사면역법현시siRNA-PTTG전염조상청중비유소분비수평하강,위공백대조조적32.9％ (P ＜0.05).결론 하조PTTG기인가억제인비유소형수체류세포생장,촉진기조망,종이강저비유소분비수평.
Objective To observe the effect of small interfering RNA (siRNA) silencing pituitary tumor transforming gene (PTTG) on the growth of primary cultured human prolactin pituitary adenoma cells (HPAs).Methods After the HPAs were primary cultured and steadily subcultured,the cells were divided into three groups:the blank control group,the negative control group and the siRNA-PTTG transfection group.After 48 h of infection,proliferation rate of HPAs was measured by using cell counting kit-8 (CCK-8).Transfection efficiency and the apoptosis rate were tested by using flow cytometry.The expression of PTTG mRNA and protein were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively.Caspase-3 activity was measured by Caspase activity assay kit.Prolactin changes in cell supernatants were measured by radioimmunoassay.Results Compared to the blank cotnrol group and the negative control group,the transcription of PTTG mRNA and the expression of PTTG protein were significantly down-regulated in the siRNA-PTTG transfection group,and the proliferation was suppressed [the inhibition rate in the negative control group and the siRNA-PTTG transfection group was (3.02 ± 1.77) ％ and (25.38 ± 0.11) ％ respectively (P ＜ 0.05),while the apoptosis was significantly increased [the apoptosis rate in the blank control group,negative control group and siRNA-PTTG transfection group was (6.21 ± 2.36) ％,(7.23 ± 3.56) ％ and (32.33 ± 5.69) ％ respectively (P ＜ 0.05).The activity of Caspase-3 was increased (P ＜ 0.05),and the radioimmunoassay revealed that the prolactin level in the cell supernatants was significantly decreased in the siRNA-PTTG transfection group,32.9％ of the blank control group (P ＜ 0.05).Conclusion Down-regulation of PTTG expression can suppress the proliferation and induce apoptosis of HPAs,which results in the reduction of prolactin secretion.