作者：贾焕珍，陈一元，赵斯达，郑淑荣

单位：

1.首都医科大学燕京医学院中心实验室2.首都医科大学附属北京天坛医院神经外科3.首都医科大学北京市神经外科研究所中心实验室

单位（英文）：

1.Central Lab, Yanjing Medical College, Capital Medical University, Beijing 101321, China 2.Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China 3.Central Lab, Beijing Neurosurgical Institute, Capital Medical University, Beijing 100070, China

关键词：

垂体肿瘤; 生长激素; Delta样蛋白1同源物; 细胞增殖; p70核糖体蛋白S6激酶; 起始因子4E结合蛋白1; 雷帕霉素靶蛋白;

关键词（英文）：

Pituitary neoplasms; Growth hormone; Delta-like homolog 1; Cell proliferation; p70S6k; 4EBP1; mTOR

中图分类号：

DOI：

10.12114/j.issn.1008-5971.2023.00.115

基金项目：

国家自然科学基金资助项目（82103048）；

摘要：

目的 分析Delta样蛋白1同源物（DLK1）在生长激素（GH）腺瘤中的表达情况及其临床意义。方法本实验时间为2020年10月至2022年6月。肿瘤标本来自2016—2020年于首都医科大学附属北京天坛医院神经外科行手术切除的34例GH腺瘤患者。采用免疫组织化学染色检测肿瘤标本中DLK1、GH表达情况。取对数生长期的大鼠GH3细胞，将其随机分为A组、B组、C组、D组[B组、C组、D组分别加入1、5、20μg/ml抗DLK1抗体，A组加入等体积的二甲基亚砜（DMSO）]，分别于培养0、24、48、72 h后采用细胞增殖实验检测各组细胞活力。取对数生长期的大鼠GH3细胞，将其随机分为E组、F组（F组加入5μg/ml抗DLK1抗体，E组加入等体积的DMSO），分别于培养0、24、48、72 h后采用ELISA检测各组GH3细胞培养上清液中GH水平。取对数生长期的大鼠GH3细胞，将其随机分为G组、H组、I组、J组（H组、I组、J组分别加入1、5、20μg/ml抗DLK1抗体，G组加入等体积的DMSO），采用Western blot法检测各组GH3细胞中磷酸化p70核糖体蛋白S6激酶（p-p70S6K）、磷酸化起始因子4E结合蛋白1(p-4EBP1)、磷酸化雷帕霉素靶蛋白（p-mTOR）水平。结果 DLK1主要位于稀疏颗粒型肿瘤标本的细胞核及致密颗粒型肿瘤标本的细胞质，GH主要位于疏松颗粒型、致密颗粒型肿瘤标本的细胞质。Pearson相关分析结果显示，肿瘤标本的DLK1评分与GH评分呈正相关（r=0.550,P<0.001）。根据DLK1评分中位数将肿瘤标本分为高DLK1评分组（≥110分，n=17）和低DLK1评分组（<110分，n=17）。高DLK1评分组患者血清GH及肿瘤标本GH评分、临床表型为致密颗粒型者占比高于低DLK1评分组（P<0.05）。B组培养48、72 h后细胞活力高于A组，C组、D组培养24、48、72 h后细胞活力高于A组（P<0.05）;C组培养48 h后细胞活力高于B组，D组培养24、48、72 h后细胞活力高于B组（P<0.05）;D组培养48、72 h后细胞活力高于C组（P<0.05）。A组、B组、C组、D组培养24、48、72 h后细胞活力分别高于本组培养0 h后，培养48、72 h后细胞活力分别高于本组培养24 h后，培养72 h后细胞活力分别高于本组培养48 h后（P<0.05）。F组培养48、72 h后GH3细胞培养上清液中GH水平低于E组（P<0.05）。E组培养48、72 h后GH3细胞培养上清液中GH水平高于本组培养0 h后，F组培养48、72 h后GH3细胞培养上清液中GH水平低于本组培养0 h后（P<0.05）;E组培养72 h后GH3细胞培养上清液中GH水平高于本组培养24 h后，F组培养72 h后GH3细胞培养上清液中GH水平低于本组培养24 h后（P<0.05）。H组、I组、J组GH3细胞中p-p70S6K水平高于G组，J组GH3细胞中p-p70S6K水平低于H组、I组（P<0.05）;I组、J组GH3细胞中p-4EBP1水平低于G组、H组（P<0.05）;H组、I组、J组GH3细胞中p-mTOR水平高于G组，I组、J组GH3细胞中p-mTOR水平高于H组，J组GH3细胞中p-mTOR水平高于I组（P<0.05）。结论 DLK1主要在致密颗粒型GH腺瘤中表达；DLK1可抑制GH3细胞增殖，增加血清GH水平，其机制可能与DLK1可抑制GH3细胞中p70核糖体蛋白S6激酶、雷帕霉素靶蛋白的磷酸化及促进起始因子4E结合蛋白1的磷酸化有关。

英文摘要：

Objective To analyze the expression and clinical significance of Delta-like homolog 1 (DLK1) in growth hormone (GH) adenomas. Methods This experiment was conducted from October 2020 to June 2022. Tumor specimens were obtained from 34 patients with GH adenomas who underwent surgical resection at Beijing Tiantan Hospital, Capital Medical University from 2016 to 2020. Immunohistochemical staining was used to detect the expression of DLK1 and GH in tumor specimens. Rat GH3 cells at logarithmic growth stage were randomly divided into groups A, B, C and D [groups B, C and D were added with 1, 5 and 20 μg/ml anti-DLK1 antibody, respectively, and group A was added with equal volume of dimethyl sulfoxide (DMSO) ] . Cell proliferation assay was used to detect cell viability in each group after 0, 24, 48, and 72 hours of culture. Rat GH3 cells at logarithmic growth stage were randomly divided into groups E and F (group F was added with 5 μg/ml anti-DLK1 antibody and group E was added with equal volume of DMSO) . After 0, 24, 48, and 72 hours of culture, the level of GH in the supernatant of GH3 cell culture in each group was detected by ELISA. Rat GH3 cells at logarithmic growth stage were randomly divided into groups G, H, I and J (groups H, I and J were added with 1, 5 and 20 μg/ml anti-DLK1 antibody, respectively, and group G was added with equal volume of DMSO) . The levels of phosphorylated p70 ribosomal protein S6 kinase (p-p70S6K) , phosphorylated eukaryotic initiation factor 4E-binding protein1 (p-4EBP1) and phosphorylated mechanistic target of rapamycin (p-mTOR) in GH3 cells of each group were detected by Western blot. Results DLK1 was mainly located in the nucleus of loose granular tumor specimens and in the cytoplasm of dense granular tumor specimens, while GH was mainly located in the cytoplasm of loose granular and dense granular tumor specimens. Pearson correlation analysis showed that the DLK1 score of tumor specimens was positively correlated with the GH score (r=0.550, P < 0.001) . According to the median DLK1 score, tumor specimens were divided into high DLK1 score group ( ≥ 110 points, n=17) and low DLK1 score group ( < 110 points, n=17) . The serum GH of patients, GH score and proportion of dense granular phenotype of tumor specimen in the high DLK1 score group were higher than those in the low DLK1 score group (P < 0.05) . The cell viability of group B was higher than that of group A after 48 and 72 hours of culture, and that of groups C and D was higher than that of group A after 24, 48 and 72 hours of culture (P < 0.05) . The cell viability of group C after 48 hours of culture was higher than that of group B, and that of group D after 24, 48, and 72 hours of culture was higher than that of group B (P < 0.05) . After 48 and 72 hours of culture, the cell viability of group D was higher than that of group C (P < 0.05) . In groups A, B, C and D, the cell viability after 24, 48, and 72 hours of culture was higher than that after 0 hour of culture, the cell viability after 48 and 72 hours of culture was higher than that after 24 hours of culture, the cell viability after 72 hours of culture was higher than that after 48 hours of culture, respectively (P < 0.05) . After 48 and 72 hours of culture, the level of GH in the supernatant of GH3 cells in group F was lower than that in group E (P < 0.05) . The level of GH in the supernatant of GH3 cell culture in group E after 48 and 72 hours of culture was higher than that after 0 hour of culture, the level of GH in the supernatant of GH3 cell culture in group F after 48 and 72 hours of culture was lower than that after 0 hour of culture (P < 0.05) . The GH level in the supernatant of GH3 cell culture in group E after 72 hours of culture was higher than that after 24 hours of culture, the level of GH in the supernatant of GH3 cell culture in group F after 72 hours of culture was lower than that after 24 hours of culture (P < 0.05) . The level of p-p70S6K in GH3 cells in groups H, I, and J was higher than that in group G, while the level of p-p70S6K in GH3 cells in group J was lower than that in groups H and I (P < 0.05) . The level of p-4EBP1 in GH3 cells in groups I and J was lower than that in groups G and H (P < 0.05) . The level of p-mTOR in GH3 cells in groups H, I, and J was higher than that in group G, the level of p-mTOR in GH3 cells in groups I and J was higher than that in group H, the level of p-mTOR in GH3 cells in group J was higher than that in group I (P < 0.05) . Conclusion DLK1 is mainly expressed in dense granular GH adenoma. DLK1 can inhibit the proliferation of GH3 cells and increase the level of serum GH, and the mechanism may be related to DLK1 inhibiting the phosphorylation of p70 ribosomal protein S6 kinase and mechanistic target of rapamycin in GH3 cells and promoting the phosphorylation of eukaryotic initiation factor 4E-binding protein1.

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