PTPRZ 1单克隆抗体对胶质瘤干细胞自我更新和侵袭的抑制作用  

Inhibiting and invasive self-renewal effect of PTPRZ 1 monoclonal antibody on glioma stem cells

作　　者：刘庆 杨莹 安乐乐 张晓宁 罗敏 李学刚 李飞 许森林 时雨 LIU Qing;YANG Ying;AN Lele;ZHANG Xiaoning;LUO Min;LI Xuegang;LI Fei;XU Senlin;SHI Yu(Department of Pathology,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Laboratory and Pathology,No.953 Hospitalof PLA Army,Shigatse,Xizang Autonomous Region,857000;Department of Neurosurgery,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Yu-Yue Pathology Scientific Research Center,Jinfeng Laboratory,Chongqing,400039,China)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院病理科,重庆400038 [2]陆军第九五三医院检验病理科,西藏自治区日喀则857000 [3]陆军军医大学(第三军医大学)第一附属医院神经外科,重庆400038 [4]金凤实验室,渝粤病理科学研究中心,重庆400039

出　　处：《陆军军医大学学报》2023年第24期2530-2536,共7页Journal of Army Medical University

基　　金：国家重点研发计划干细胞专项青年科学家项目(2021YFA1103000);重庆市自然科学基金杰出青年基金项目(2023NSCQ-JQX0049);国家自然科学基金面上项目(82172854)。

摘　　要：目的制备靶向蛋白酪氨酸磷酸酶受体Z 1型(protein tyrosine phosphatase receptor-type Z polypeptide 1,PTPRZ 1)单克隆抗体,明确其能否阻断多效生长因子(pleiotrophin,PTN)-PTPRZ 1旁分泌轴,阐明其对胶质瘤干细胞自我更新、迁移和侵袭的抑制效应。方法以PTPRZ 1胞外段(26~300 aa)结构域作为抗原,对8~10周龄雌性BALB/c小鼠进行免疫,2周后通过尾静脉采血、离心收集上清并用ELISA检测血清效价,再进行B细胞融合及阳性杂交瘤细胞筛选,从而获得小鼠源性PTPRZ 1单克隆抗体;通过抗体亲和力检测实验,筛选亲和力最强的抗体株。用sg-PTPRZ1慢病毒感染胶质瘤干细胞进行PTPRZ 1基因沉默,RT-qPCR检测PTPRZ1基因敲除效率。通过Western blot检测抗体对PTPRZ 1抗原的特异性识别能力和胶质瘤干细胞内PTPRZ 1下游通路表达。胶质瘤干细胞功能和机制实验共分为4组:对照组、人重组PTN蛋白处理组、鼠抗PTPRZ 1抗体处理组、人重组PTN蛋白+鼠抗PTPRZ 1抗体处理组。通过克隆形成实验、迁移和侵袭实验检测胶质瘤干细胞自我更新、迁移和侵袭能力的影响。结果制备并成功获得5株小鼠抗人PTPRZ 1单克隆抗体,其中克隆株2F10的抗体亲和力最强,该株抗体能用于检测PTPRZ 1蛋白表达。与对照组比较,人重组PTN蛋白刺激能显著促进胶质瘤干细胞内PTPRZ 1下游ERK磷酸化激活,促进胶质瘤干细胞自我更新(P<0.01)、迁移(P<0.01)和侵袭(P<0.05)。鼠抗PTPRZ 1抗体处理能阻断PTN介导的ERK磷酸化激活,抑制胶质瘤干细胞自我更新(P<0.01)、迁移(P<0.01)和侵袭(P<0.05)能力。结论克隆株2F10的鼠抗PTPRZ 1单克隆抗体能高效识别胶质瘤干细胞PTPRZ 1蛋白,并阻断PTN介导的胶质瘤干细胞自我更新、迁移和侵袭。ObjectiveTo prepare a monoclonal antibody targeting protein tyrosine phosphatase receptor-type Z polypeptide1(PTPRZ 1),to investigate the potential to disrupt the pleiotrophin(PTN)-PTPRZ 1 paracrine signaling axis,and to evaluate the inhibitory effects on the self-renewal,migration,and invasion of glioma stem cells.MethodsThe extracellular domain of PTPRZ 1(amino acids 26-300)served as the antigen to immunize female BALB/c mice aged 8 to 10 weeks.Following a 2-week immunization period,serum samples were collected via tail vein puncture,centrifuged to separate,and the antibody titer was quantified by ELISA.Subsequently,B cell fusion and positive hybridoma cell selection were conducted to isolate murine monoclonal antibodies targeting PTPRZ 1.Antibody clones with the highest affinity were identified through specialized affinity assays.Human glioma stem cells were infected with sgRNA-PTPRZ 1 lentivirus for PTPRZ 1 gene silencing,and the changes in the expression of PTPRZ 1 at mRNA level were analyzed using RT-qPCR.Western blotting was employed to confirm the antibodies’specificity for PTPRZ 1.The experiments on the functions and mechanisms of glioma stem cells were divided into 4 groups:the control group,the human recombinant PTN-treated group,the mouse anti-PTPRZ 1 antibody(clone 2F10)-treated group,and the combination treatment of human recombinant PTN and mouse anti-PTPRZ 1 antibody(clone 2F10)group.The expression of PTPRZ 1 downstream signaling pathways in glioma stem cells was examined via Western blotting.Clonogenic,migration,and invasion assays were utilized to determine the effects on self-renewal,migration,and invasion capabilities of glioma stem cells.ResultsFive murine monoclonal antibodies targeting PTPRZ 1 were successfully generated,with clone 2F10 displaying the highest binding affinity that was found to be effective for detecting PTPRZ 1 protein expression.Compared with the control group,treatment with human recombinant PTN significantly increased ERK phosphorylation downstream of PTPRZ 1 in glioma

关 键 词：胶质瘤 肿瘤干细胞 单克隆抗体 蛋白酪氨酸磷酸酶受体Z1型 

分 类 号：R392.11[医药卫生—免疫学] R73-354[医药卫生—基础医学] R730.264