CAJ | 학술논문

目的对一株抗胰腺癌干细胞单克隆抗体(简称:单抗)进行初步鉴定和体外功能研究,为靶向胰腺癌干细胞治疗胰腺癌提供候选单抗药物.方法应用无血清悬浮培养及PKH26染色确定胰腺癌细胞系PANC-1中肿瘤干细胞的存在.流式细胞术检测PANC-1细胞中有干细胞标志物CD44+CD24+的细胞比例.双色细胞免疫荧光检测CD24和单抗15D2识别的抗原蛋白在PANC-1细胞中的表达情况.无血清悬浮培养法观察单抗15D2对PANC-1成球细胞自我更新的影响.CCK-8法检测单抗15D2对PANC-1细胞增殖和耐药的影响.免疫组织化学检测单抗15D2识别的靶抗原在人胰腺癌、癌旁组织中的表达情况.结果 PANC-1细胞能在无血清培养液中存活、增殖并形成细胞球,成球率为(2.5±0.5)％.PANC-1球体细胞中CD44+ CD24+细胞的比例较亲本细胞提高了11.4倍,其中CD44+ CD24+细胞占CD24+细胞的97％,在此体系中用CD24作为PANC-1细胞干细胞标志物.细胞免疫荧光结果显示单抗15D2识别的抗原分子在细胞膜上表达,并能与CD24在PANC-1细胞共定位.抗体体外功能研究发现,单抗15D2能显著抑制PANC-1细胞在无血清培养液中成球,抑制率达到22％.同时,单抗15D2联合吉西他滨能显著抑制PANC-1球体细胞的增殖,联合组和对照组IC50分别为0.10、0.39 μmol/L.免疫组织化学检测结果显示,单抗15D2所识别的抗原蛋白在76.9％(11/13)的人胰腺癌组织中表达阳性,而在癌旁组织中表达阳性率仅为10.0％(1/10),差异有统计学意义(P＜0.05).结论抗胰腺癌干细胞单抗15D2体外能够显著抑制胰腺癌干细胞的自我更新和耐药能力,为胰腺癌干细胞的靶向治疗提供有应用价值的候选抗体药物.
목적 대일주항이선암간세포단극륭항체(간칭:단항)진행초보감정화체외공능연구,위파향이선암간세포치료이선암제공후선단항약물.방법 응용무혈청현부배양급PKH26염색학정이선암세포계PANC-1중종류간세포적존재.류식세포술검측PANC-1세포중유간세포표지물CD44+CD24+적세포비례.쌍색세포면역형광검측CD24화단항15D2식별적항원단백재PANC-1세포중적표체정황.무혈청현부배양법관찰단항15D2대PANC-1성구세포자아경신적영향.CCK-8법검측단항15D2대PANC-1세포증식화내약적영향.면역조직화학검측단항15D2식별적파항원재인이선암、암방조직중적표체정황.결과 PANC-1세포능재무혈청배양액중존활、증식병형성세포구,성구솔위(2.5±0.5)％.PANC-1구체세포중CD44+ CD24+세포적비례교친본세포제고료11.4배,기중CD44+ CD24+세포점CD24+세포적97％,재차체계중용CD24작위PANC-1세포간세포표지물.세포면역형광결과현시단항15D2식별적항원분자재세포막상표체,병능여CD24재PANC-1세포공정위.항체체외공능연구발현,단항15D2능현저억제PANC-1세포재무혈청배양액중성구,억제솔체도22％.동시,단항15D2연합길서타빈능현저억제PANC-1구체세포적증식,연합조화대조조IC50분별위0.10、0.39 μmol/L.면역조직화학검측결과현시,단항15D2소식별적항원단백재76.9％(11/13)적인이선암조직중표체양성,이재암방조직중표체양성솔부위10.0％(1/10),차이유통계학의의(P＜0.05).결론 항이선암간세포단항15D2체외능구현저억제이선암간세포적자아경신화내약능력,위이선암간세포적파향치료제공유응용개치적후선항체약물.
Objective To identify-and study a monoclonal antibody (McAb) against pancreatic cancer stem cell in vitro,as well as to provide candidate antibody-drug for cancer stem cell-targeted therapy of pancreatic cancer.Methods Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cell in PANC-1 cell line.Flow cytometry was used to detect the expression of CD24 and CD44 in PANC-1 cells and sphere cells,Immunofluorescence was used to detect the expression of CD24 and antigen recognized by 15D2.The effects of 15D2 on self-renewal,proliferation and chemosensitivity to gemcitabine of PANC-1 parent or sphere cells were identified by serum-free suspension culture and CCK-8 assay,Immunohistochemistry was applied to detect the level of antigen recognized by 15D2 in cancer and adjacent tissues.Results PANC-1 cells could survive,proliferate and form sphere cells in serum-free medium.The sphere-forming rate was (2.5±0.5) ％.The percentage of CD44+ CD24+ cells population in sphere cells increased by 11.4 folds compared to PANC-1 cells,in which single nearly 97 ％ CD24+ cells was CD44+ CD24+ cells.Therefore,CD24+ was selected for cancer stem cell marker in PANC-1 in this study.The two-color immunofluorescence assay showed that 15D2 could recognize cells which was also stained by CD24.In vitro functional experiments demonstrated that 15D2 significantly suppressed the sphere formation of PANC-1 cells,with the inhibitory rate being 30.4 ％.Meanwhile,the combination of 15D2 and gemcitabine can significantly attenuate the growth of PANC-1 sphere cells.The IC50 was 0.10 μmol/L in 15D2+gemeitabine group,and 0.39 μmol/L in mlgM+gemcitabine group,Immunohistochemical results showed that the antigen recognized by 15D2 was greatly expressed in about 76.9 ％ (11/13) human pancreatic cancer tissues and hardly detected in adjacent normal tissues (10.0 ％,1/10).Conclusion McAb 15D2 can inhibit self-renewal and drug-resistance of pancreatic cancer stem cell in PANC-1 cell line,and it might become a candidate drug for target pancreatic cancer stem cell treatment.