生长抑素受体5抑制垂体催乳素腺瘤激素的异常分泌

Activation of somatostatin receptor 5 suppresses prolactin synthesis in prolactinoma cells

目的探讨生长抑素受体5(SSTR5)活化在垂体催乳素腺瘤中激素分泌中的抑制作用。方法使用大鼠GH3细胞(美国细胞中心,ATCC)作为垂体催乳素腺瘤的体外细胞模型,外源性转染SSTR5质粒构建SSTR5过表达模型,使用流式细胞仪分选技术分选转染阳性细胞,并用蛋白质印迹法(Western blot)加以验证,荧光素酶报告基因检测PRL-luc启动子活性,CellTiter Glo试剂盒及酶联免疫吸附试验(ELISA)试剂盒检测细胞活性及细胞上清PRL的浓度。使用SSTR5激动剂BIM23052进行细胞刺激,检测空白组和转染组细胞上清PRL浓度及细胞活性。应用Student’s t检验和单向方差分析进行组间比较。结果蛋白印迹法检测流式分选SSTR5过表达质粒转染细胞GH3SSTR5可发现SSTR5蛋白高表达,单纯过表达SSTR5显著降低PRL-luc报告基因的活性[(71.07±4.38)%比(99.96±0.88)%,t=6.47,P<0.01],抑制细胞上清PRL的分泌[(65.24±10.71)%比(100.00±4.44)%,t=2.99,P<0.05];使用不同浓度的BIM23052刺激GH3SSTR5细胞,PRL-luc报告基因活性呈现U型剂量反应曲线,BIM23052浓度在10 nmol/L时达到最大抑制效应[(45.23±1.21)%对(99.96±1.24)%,t=31.62,P<0.01],而BIM23052并不影响SSTR5空载质粒mock转染组GH3mock细胞的PRL-luc报告基因活性;BIM23052并不影响GH3mock细胞上清PRL的浓度,但BIM23052可显著抑制GH3SSTR5细胞上清PRL的浓度[(57.10±6.41)%比(96.14±6.82)%,t=4.16,P<0.01]。BIM23052并不改变GH3mock和GH3SSTR5细胞的活性。结论SSTR5活化可以通过抑制PRL mRNA的转录抑制垂体催乳素腺瘤激素的异常分泌。

Objective To investigate the inhibitory role of somatostatin receptor 5(SSTR5)activation in hormone secretion in pituitary prolactinomas.Methods Rat GH3 cells were used as an in vitro cell model of pituitary prolactin adenoma,and SSTR5 overexpression model was constructed by exogenous transfection of SSTR5 plasmid.The luciferase reporter gene assay was used to detect PRL-luc promoter activity.CellTiter Glo kit and enzyme linked immunosorbent assay(ELISA)kit were used to detect cell activity and the concentration of PRL in cell supernatant.Cell stimulation was performed using the SSTR5 agonist BIM23052,and the supernatant PRL concentration and cell activity of the blank group and transfection group were detected.Statistical analysis was performed using GraphPad Prism(V.7).Student’s t-test and one-way analysis of variance were used for comparison between groups.Results SSTR5 protein was highly expressed in GH3SSTR5 cells transfected with SSTR5-overexpressing plasmid detecting by Western blotting.Overexpression of SSTR5 alone significantly reduced the activity of the PRL-luc reporter gene[(71.07±4.38)%vs.(99.96±0.88)%,t=6.47,P<0.01],inhibited the secretion of PRL in the cell supernatant[(65.24±10.71)%vs.(100.00±4.44)%,t=2.99,P<0.05].The GH3SSTR5 cells were stimulated with different concentrations of BIM23052,PRL-luc reporter gene activity presented a specific U-shaped dose-response curve,and the BIM23052 concentration reached the maximum inhibitory effect at 10 nmol/L[(45.23±1.21)%vs.(99.96±1.24)%,t=31.62,P<0.01],while BIM23052 did not affect PRL-luc reporter gene activity of GH3mock cells in mock transfection group.BIM23052 did not affect the concentration of PRL in the supernatant of GH3mock cells,but BIM23052 significantly inhibited the concentration of PRL in the supernatant of GH3SSTR5 cells[(57.10±6.41)%vs.(96.14±6.82)%,t=4.16,P<0.01].BIM23052 did not alter the activity of GH3mock and GH3SSTR5 cells.Conclusion SSTR5 activation can inhibit the abnormal secretion of pituitary prolactinomas hormone by inhibiting the transcription of PRL mRNA,which provides a theoretical basis for the second-generation SSA pasireotide in the treatment of dopamine receptor agonist-resistant pituitary prolactinomas.