小干扰RNA沉默垂体瘤转化基因抑制人泌乳素型垂体瘤细胞的生长

Small interfering RNA silencing pituitary tumor transforming gene inhibits growth of human prolactin pituitary tumor cells

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.

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.