玄丹清金颗粒通过Hh-Ptch/Smo-Gli调节轴抑制肿瘤干细胞特性逆转奥希替尼耐药  

Xuandan Qingjin Granule Reversing Osimertinib Resistance by Inhibiting Tumor Stem Cells Through Hh-Ptch/Smo-Gli Regulatory Axis

作　　者：李洋 刘磊 林家茂 Li Yang;Liu Lei;Lin Jiamao(Shandong Provincial Key Laboratory of Precision Oncology,the Affiliated Cancer Hospital of Shandong First Medical University,Jinan 250117,China;College of Integrated Traditional Chinese and Western Medicine,Jining Medical University of Shandong Province,Jinan 250117,China;Department of Medical Oncology,the Affiliated Cancer Hospital of Shandong First Medical University,Jining 272067,China)

机构地区：[1]山东第一医科大学附属肿瘤医院山东省精准肿瘤学重点实验室,济南250117 [2]山东省济宁医学院中西医结合学院,济南250117 [3]山东第一医科大学附属肿瘤医院肿瘤内科,济宁272067

出　　处：《华中科技大学学报(医学版)》2025年第1期52-59,共8页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81904186);山东省自然科学基金创新发展联合基金项目(No.ZR2023LZY007);山东省中医药重点学科建设项目(No.鲁卫中医药科教字[2022]4号)。

摘　　要：目的探讨玄丹清金颗粒对奥希替尼耐药的人肺腺癌PC9/OR细胞的逆转作用及机制。方法构建奥希替尼耐药的人肺腺癌细胞PC9/OR,CCK-8检测不同浓度奥希替尼对PC9和PC9/OR的增殖能力的影响及不同梯度浓度玄丹清金颗粒含药血清对PC9/OR增殖的影响,Western blot和qRT-PCR鉴定PC9/OR耐药性及肿瘤干细胞特性,软琼脂克隆形成实验检测细胞成球能力,侧群分选测定肿瘤干细胞比例,应用转录组测序筛选肿瘤干细胞相关调控通路并利用Western blot反向验证。结果与PC9相比,PC9/OR的奥希替尼IC_(50)值显著升高,奥希替尼对p-EGFR、p-ERK和p-RPS6的抑制被明显削弱,与干细胞特性相关的调控分子亦呈不同程度上调。CCK-8检测提示以培养液稀释的40%及以下浓度含药血清可排除大鼠血清的细胞毒性干扰。CCK-8及克隆形成实验提示奥希替尼与玄丹清金颗粒含药血清的联合组PC9/OR细胞存活率及成球能力显著下降。侧群分选证实联合组干细胞比例显著下调。RNA-seq发现与肿瘤干细胞调节相关的PTCH2、SMO、GLI1、GLI2、GLI3发生显著变化。反证实验发现联合组加入激活剂(purmorphamine,PM)后,克隆形成及细胞存活率升高,OCT4、NANOG、SOX2、SHH、SMO、GLI1、GLI2上调,PTCH2下调。结论玄丹清金颗粒能够通过抑制肿瘤干细胞特性逆转PC9/OR耐药,具体机制与调控Hh-Ptch/Smo-Gli调节轴相关。Objective To explore the reversal effect of Xuandan Qingjin(XDQJ)granule on human lung adenocarcinoma PC9/OR osimertinib-resistant cells,and its mechanism.Methods The human lung cancer cell line PC9 was cultivated,and PC9/OR osimertinib-resistant cells were established.CCK-8 was employed to detect the proliferation of PC9 and PC9/OR treated with different concentrations of Osimertinib,and the effect of different gradient concentrations of XDQJ granule-containing serum on the proliferation of PC9/OR.Western blot and qRT-PCR were utilized to identify the resistance of PC9/OR and the characteristics of tumor stem cells.The soft agar cloning assay was adopted to detect the cell viability.Hoechst33342 and PI double staining were used to determine the proportion of tumor stem cells.Transcriptome sequencing was utilized to screen the related regulatory pathways of tumor stem cells.Western blot was applied to verify the combination of purmorphamine(PM)for reverse validation of tumor stem cell-related regulatory pathways.Results The IC_(50)of PC9/OR was 45.51μmol/mL,which was significantly higher than that of PC9(0.07μmol/mL).The expression levels of p-EGFR,p-ERK,and p-RPS6 of PC9 were significantly decreased after oisimertinib treatment.Compared with PC9,the expression levels of PC9/OR and regulatory molecules related to stem cell characteristics were upregulated to varying extents.CCK-8 assay indicated that diluted drug-containing serum with concentration of 40%or less could eliminate cytotoxic interference in rat serum.By means of CCK-8 and cloning experiments,it was discovered that the combination of XDQJ granules and Osimertinib could reverse the drug resistance of PC9/OR.The proportion of stem cells in the combination group was significantly reduced by side group sorting.RNA-seq found significant changes in PTCH2,SMO,GLI1,GLI2,and GLI3,which are related to tumor stem cell regulation.In the combination group,the survival rate of clonogenic cells increased,OCT4,NANOG,SOX2,SHH,SMO,GLI1 and GLI2 were upregulated,and P

关 键 词：肿瘤干细胞 奥希替尼 耐药 玄丹清金颗粒 

分 类 号：R734.2[医药卫生—肿瘤]