不同MEK1/2抑制剂对NF1敲低的施万细胞增殖活性的影响

doi:10.19438/j.cjoms.2020.04.003

中国口腔颌面外科杂志 ›› 2020, Vol. 18 ›› Issue (4): 302-307.doi: 10.19438/j.cjoms.2020.04.003

不同MEK1/2抑制剂对NF1敲低的施万细胞增殖活性的影响

刘嘉靓, 杜仲, 郑家伟, 游元和, 王延安

上海交通大学医学院附属第九人民医院·口腔医学院口腔颌面-头颈肿瘤科,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海 200011

收稿日期:2019-12-12 出版日期:2020-07-20 发布日期:2020-09-10
通讯作者: 王延安,E-mail:wangyan_an@163.com
作者简介:刘嘉靓（1993-）,男,硕士,住院医师,E-mail:573078695@qq.com
基金资助:
国家自然科学基金（81371164,81870780）

Effects of different MEK1/2 inhibitors on proliferation of NF1 knockdown Schwann cells

LIU Jia-liang, DU Zhong, ZHENG Jia-wei, YOU Yuan-he, WANG Yan-an

Department of Oromaxillofacial Head and Neck Oncology,Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology. Shanghai 200011, China;

Received:2019-12-12 Online:2020-07-20 Published:2020-09-10

摘要/Abstract

摘要： 目的: 构建慢病毒干扰载体介导的NF1 敲低RSC96（NF1^kdRSC96)施万细胞株,对比不同MEK1/2抑制剂对该稳转细胞株增殖的影响。方法: 运用慢病毒干扰载体构建NF1^kd的施万细胞模型,采用qRT-PCR及Western 免疫印迹验证慢病毒干扰载体组（NF1^kd组）和空白载体组的NF1表达水平;应用5种MEK1/2抑制剂,包括司美替尼（Selumetinib）、贝美替尼（Binimetinib）、考比替尼（Cobimetinib）、曲美替尼（Trametinib）、PD0325901处理细胞,CCK-8法检测不同MEK1/2抑制剂在时间梯度和浓度梯度下的抑制作用。使用Graphpad Prism 5计算NF1^kd组和空白载体组中5种抑制剂的半抑制浓度（IC₅₀）值,采用SPSS 22.0软件包对数据进行统计学分析。结果: 5种MEK1/2抑制剂均对NF1^kd施万细胞的增殖有抑制作用（P<0.05）。72 h时,PD0325901、Cobimetinib和Trametinib 3种抑制剂的抑制作用显著强于Selumetinib和Binimetinib（P<0.05)。IC₅₀实验中,Cobimetinib抑制NF1^kd组及空白载体组的药物浓度分别为（2.35±0.98）μmol/L和（14.22±1.67） mol/L,组间差异显著（P<0.05);Binimetinib抑制NF1^kd组及空白载体组的药物浓度分别为（18.96±2.37） mol/L和（114.1±5.98） mol/L,组间差异显著（P<0.05）,而Selumetinib、Trametinib和PD0325901抑制NF1^kd组及对照组的IC₅₀值无显著差异（P>0.05）。结论: 成功构建了NF1敲低的施万细胞稳转株。5种MEK1/2抑制剂均能抑制NF1^kdRSC96稳转细胞株的增殖活性,PD0325901、Trametinib和Cobimetinib具有更持久的抑制作用。相同药物浓度下,Cobimetinib和Binimetinib能更特异地抑制NF1^kd施万细胞的增殖活性。Cobimetinib同时具备药效持久性和抑制特异性,可能具有一定的临床应用前景。

关键词: NF1, 慢病毒, 施万细胞, MEK1/2抑制剂, I型神经纤维瘤

Abstract: PURPOSE: To establish a stable RSC96 cell line with knockdown of neurofibromin 1(NF1) gene by lentiviral vectors, and assess the influence of different MEK1/2 inhibitors on proliferation of NF1 knockdown RSC96 Schwann cell. METHODS: Lentivirus was used to construct a Schwann cell model with decreased expression of NF1 gene. qRT-PCR and Western blot (WB) were used to verify the expression level of NF1 gene. Five MEK1/2 inhibitors were included in this experiment: Selumetinib, Binimetinib, Cobimetinib, Trametinib and PD0325901. CCK-8 assay was used to detect the inhibition of different MEK1/2 inhibitors under time gradient and concentration gradient. IC₅₀ values in the blank control group and NF1^kd group of 5 inhibitors were calculated by Graphpad Prism software package. SPSS 22.0 software package was used for statistical analysis. RESULTS: All MEK1/2 inhibitors could inhibit the proliferation of NF1^kd Schwann cells (P<0.05). At 72 h, PD0325901, Cobimetinib and Trametinib had stronger inhibitory effects than Selumetinib and Binimetinib (P<0.05). In IC₅₀ experiment, the drug concentrations of Cobimetinib in NF1^kd group and blank control group were (2.35±0.98) mol/L and (14.22±1.67) mol/L, respectively(P<0.05); the drug concentrations of Binimetinib in NF1^kd group and blank control group were (18.96±2.37) mol/L and (114.1±5.98) mol/L, respectively; and the differences between the groups were significant (P<0.05). There was no significant difference in IC₅₀ values within Selumetinib, Trametinib and PD0325901 in NF1^kd group and control group (P>0.05). CONCLUSIONS: Stable Schwann cell line with knockdown of NF1 gene was successfully constructed. All five MEK inhibitors can inhibit the proliferation of NF1^kd RSC96 cell line; PD0325901, Trametinib and Cobimetinib processed longer lasting inhibiting ability. Cobimetinib and Binimetinib can both kill NF1^kd Schwann cells more specifically than the blank control group at the same drug concentration. Cobimetinib has both long-lasting efficacy and killing specificity, which may have more clinical prospects.

Key words: NF1, Lentivirus, Schwann cells, MEK1/2 inhibitors, Neurofibromatosis type 1

中图分类号:

刘嘉靓, 杜仲, 郑家伟, 游元和, 王延安. 不同MEK1/2抑制剂对NF1敲低的施万细胞增殖活性的影响[J]. 中国口腔颌面外科杂志, 2020, 18(4): 302-307.

LIU Jia-liang, DU Zhong, ZHENG Jia-wei, YOU Yuan-he, WANG Yan-an. Effects of different MEK1/2 inhibitors on proliferation of NF1 knockdown Schwann cells[J]. China J Oral Maxillofac Surg, 2020, 18(4): 302-307.

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不同MEK1/2抑制剂对NF1敲低的施万细胞增殖活性的影响

Effects of different MEK1/2 inhibitors on proliferation of NF1 knockdown Schwann cells

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