永生化人甲状旁腺细胞系的建立及功能鉴定  

Establishment and functional identification of an immortalized human parathyroid cell line

作　　者：蒋楠 李博厚 邓淑婷 袁也 吴琼[2] 温似春 陈柏锡 赵泽文 彭思琪 夏郁彬 陶一鸣 刘双信[1,2] JIANG Nan;LI Bo-hou;DENG Shu-ting;YUAN Ye;WU Qiong;WEN Si-chun;CHEN Bai-xi;ZHAO Zeweng;PENG Si-qi;XIA Yu-bing;TAOYi-ming;LIU Shuang-xin(School of Medicine,South China University of Technology,Guangzhou 510006,China;Department of Nephrology,Guangdong Provincial People’s Hospital(Guangdong Academy of Medical Sciences),Southern Medical University,Guangzhou 510080,China)

机构地区：[1]华南理工大学医学院,广州510006 [2]南方医科大学附属广东省人民医院(广东省医学科学院)肾内科,广州510080

出　　处：《中国血液净化》2023年第5期359-363,共5页Chinese Journal of Blood Purification

基　　金：国家自然基金项目(81870508,81873616,82170730);广东省登峰计划项目(DFJH201901);广东省自然科学基金项目(2022A1515012374,2023A1515010024)。

摘　　要：目的 建立永生化的人甲状旁腺细胞系并进行功能鉴定。方法 采用慢病毒感染的方式将SV40(simian vacuolating virus 40,SV40)大T抗原导入细胞,转染后用嘌呤霉素筛选转染成功的细胞。通过细胞形态学、实时荧光定量聚合酶链式反应(real time quantitative polymerase chain reaction,RT-qPCR)及免疫荧光检测转染后传代甲状旁腺细胞表达甲状旁腺激素(parathyroid hormone,PTH)、钙敏感受体(calcium sensitive receptor,CaSR)和神经胶质细胞缺失因子2(glial cells missing homolog 2,GCM2)mRNA及蛋白的表达情况。通过细胞增殖实验检测转染的细胞对Ca~(2+)的反应性。结果 转染成功的甲状旁腺细胞传代后大部分呈梭形,细胞核椭圆形,胞浆均质透明;RT-qPCR结果显示甲状旁腺细胞表达CaSR、GCM2以及PTH mRNA;免疫荧光显示永生化的甲状旁腺细胞系表达甲状旁腺相关蛋白CaSR、GCM2以及PTH。细胞增殖实验显示低钙能够促进甲状旁腺细胞系增殖。结论 本研究成功建立了永生化的甲状旁腺细胞系,为进一步探讨继发性甲状旁腺功能亢进的发病机制及治疗提供了实验基础。Objective To establish an immortalized human parathyroid cell line and to identify its func-tions.Methods SV40(Simian vacuolating virus 40,SV40)large antigen was introduced into the parathyroid cells by lentivirus infection,and the transfected cells were screened by puromycin.The expressions of parathy-roid hormone(PTH),calcium sensitive receptor(CaSR)and glial cells missing homolog 2,(GCM2)mRNAs and proteins in the parathyroid cells were detected by cell morphology,RT-qPCR and immunofluorescence.The reactivity of transfected cells to Ca2+was detected by cell proliferation assay.Results Most of the trans-fected parathyroid cells were fusiform,with oval nuclei and transparent cytoplasm.RT-qPCR showed that the parathyroid cells expressed CaSR,GCM2 and PTH mRNAs.Immunofluorescence showed that the immortal-ized parathyroid cell line expressed parathyroid associated proteins of CaSR,GCM2 and PTH.Cell prolifera-tion experiments showed that low calcium could promote the proliferation of parathyroid cells.Conclusion An immortalized parathyroid cell line was successfully established in this study,which provides an experimen-tal basis for further exploring the pathogenesis and treatment of secondary hyperparathyroidism.

关 键 词：继发性甲状旁腺功能亢进 甲状旁腺细胞 永生化 

分 类 号：R363[医药卫生—病理学]