Delta样蛋白1在垂体腺瘤细胞株中的表达及生物学功能研究  

Expression and biological function of Delta-like protein 1 gene in pituitary adenoma cell lines

作　　者：董伟 陈一元[1] 李振业 张亚卓[1] 高华[1] Dong Wei;Chen Yiyuan;Li Zhenye;Zhang Yazhuo;Gao Hua(Beijing Neurosurgical Institute,Capital Medical University,Beijing 100070,China;Department of Neurosurgery,Tangshan People′s Hospital,Tangshan 063001,China)

机构地区：[1]首都医科大学,北京市神经外科研究所,北京100070 [2]唐山市人民医院神经外科,唐山063001

出　　处：《中华神经外科杂志》2023年第3期282-287,共6页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金(82103048);河北省自然科学基金精准医学联合基金细化项目(H2020105017);中央引导地方科技发展资金(226Z7704G)。

摘　　要：目的探讨Delta样蛋白1(DLK1)在垂体腺瘤细胞株中的表达水平及生物学功能。方法体外培养垂体腺瘤GH3、MMQ和ATT20细胞,分为实验组和对照组,实验组细胞分别给予抗DLK1抗体1μg/ml和5μg/ml,对照组细胞给予等体积的二甲基亚砜,共培养24、48及72 h。采用蛋白质免疫印迹实验检测各个细胞株DLK1的水平和谱系来源,细胞增殖实验检测细胞的活力,酶联免疫吸附(ELISA)法检测细胞的分泌能力[GH3细胞:GH和胰岛素样生长因子(IGF-1),MMQ细胞:催乳素(PRL),ATT2细胞:促肾上腺皮质激素(ACTH)],细胞免疫荧光实验和蛋白质免疫印迹实验检测抗DLK1抗体对GH3细胞表达PIT1蛋白水平的影响。结果蛋白质免疫印迹实验结果证实,GH3和MMQ细胞为POU结构域转录因子1(POU1F1)谱系细胞,DLK1表达水平高;ATT20细胞为T盒转录因子19(TBX19)谱系细胞,DLK1表达水平低。与抗DLK1抗体共培养48 h,仅GH3细胞5μg/ml实验组的细胞活力高于对照组(P=0.017);共培养72 h,GH3细胞1μg/ml、5μg/ml实验组(均P<0.05)及MMQ细胞5μg/ml实验组(P=0.041)的细胞活力高于对照组;ATT20细胞的实验组与对照组所有时间点的细胞活力差异均无统计学意义(均P>0.05)。与抗DLK1抗体培养24、48、72 h,GH3细胞对照组培养上清中GH含量分别为(8.8±0.6)、(10.4±0.8)及(13.2±0.9)ng/ml,5μg/ml实验组分别为(6.7±0.7)、(6.1±0.4)及(4.7±0.4)ng/ml,各时间点两组比较差异均有统计学意义(均P<0.001);GH3细胞对照组3个时间点培养上清中IGF-1的含量分别为(12.5±0.7)、(14.6±1.0)及(15.9±1.1)ng/ml,5μg/ml实验组分别为(10.9±0.7)、(11.8±0.8)及(8.3±0.7)ng/ml,48 h和72 h时间点两组的差异均有统计学意义(均P<0.05);与对照组相比,ATT20细胞5μg/ml实验组各时间点分泌ACTH的量及MMQ细胞分泌PRL的量差异均无统计学意义(均P>0.05)。细胞免疫荧光实验和蛋白质免疫印迹实验均显示,与抗DLK1抗体共培养24 h,GH3细胞5μg/ml实验组的DLK1和PIT1Objective To study the expression levels and biology function of Delta-like homolog 1(DLK1)in pituitary adenoma cell lines.Methods Pituitary adenoma cell lines including ATT20,GH3 and MMQ were cultured ex vivo and cells were divided into experimental groups(anti-DLK1 antibody:1μg/ml and 5μg/ml)and control group(equal volume of dimethylsulphoxide)for 24 h,48 h and 72 h.Cell lineages and DLK expression levels of ATT20,GH3 and MMQ were validated by Western blot;and cell proliferation was assessed using cell viability;and enzyme linked immunosorbent assay(ELISA)was used to measure the hormone secretion[GH3 cell:growth hormone(GH)and insulin-like growth factor 1(IGF-1),MMQ:prolactin(PRL),ATT20 cell:adrenocorticotrophic hormone(ACTH)];and immunofluorescence and Western blot were used to measure the levels of POU domain transcription factor 1(POU1F1)-encoded protein,PIT1,in GH3 cells after anti-DLK1 antibody treatment.Results Western blotting experiment verified that GH3 cell line and MMQ cell line were originally in the POU1F1 lineage and DLK1 was highly expressed,and ATT20 cell line was originally in the T-Box Transcription Factor 19(TBX19)lineage and DLK1 was poorly expressed.After 48 h co-culturing with anti-DLK1 antibody,only the cell viability of GH3 experimental group was higher compared with control group(P=0.017);after 72 h co-culturing,the cell viability of GH3 experimental groups(P<0.01)and MMQ experimental group(5μg/ml)were higher compared with controls(P=0.041);and there was no statistical difference among ATT20 experimental groups and control in any timepoint(all P>0.05).After 24 h,48 h and 72 h co-culturing with anti-DLK1 antibody,the GH level in cell culture supernatants of control GH3 cell was 8.8±0.6 ng/ml,10.4±0.8 ng/ml and 13.2±0.9 ng/ml respectively,and that in the experimental group(5μg/ml)was 6.7±0.7 ng/ml,6.1±0.4 ng/ml and 4.7±0.4 ng/ml respectively,and the differences between control and experimental groups were significant at all 3 timepoints(all P<0.001).After 24 h,48 h and 72 h co-c

关 键 词：垂体肿瘤 细胞 体外培养 Delta样蛋白1 POU结构域转录因子1 

分 类 号：R736.4[医药卫生—肿瘤]