抗胰腺癌干细胞单克隆抗体的筛选和鉴定

目的：从抗胰腺癌干细胞单抗库中筛选、鉴定识别胰腺癌干细胞的功能性单抗，为胰腺癌干细胞靶向治疗提供候选抗体药物。方法：无血清悬浮培养及PKH26染色确定胰腺癌HPAC细胞系中肿瘤干细胞的存在。流式细胞术检测HPAC的干细胞标志物CD133在球体细胞中的阳性比例，检测20株杂交瘤单抗在HPAC亲本和球体细胞中的阳性表达。双色荧光标记流式细胞术检测CD133和单抗在HPAC亲本和球体细胞中的共表达比例；无血清悬浮培养法观察单抗15E9对HPAC成球细胞自我更新的影响。CCK一8法检测单抗15E9对HPAC细胞增殖和耐药的影响。结果：HPAC细胞能在无血清培养基中存活、增殖并形成细胞球，成球率为4．8％±0．6％。HPAC球体细胞中CD133’细胞的比例较亲本细胞提高至11．6倍。20株候选杂交瘤单抗中有3株单抗能识别HPAC球体细胞中CD133’细胞，其中单抗15E9共染比例为3．5％，并能显著抑制HPAC细胞的成球，抑制率达到30．4％。单抗15E9联合吉西他滨能显著抑制HPAC球体细胞的增殖，联合组和对照组Ic50分别为30．8nmol／L和58．1nmol／L。结论：本研究成功筛选出1株杂交瘤单抗可以识别胰腺癌干细胞，并且可识别CD133＋的胰腺癌干细胞；体外功能显示该抗体具有抑制HPAC干细胞的自我更新能力，抗体干预后显著降低HPAC耐药能力，可能是潜在的胰腺癌干细胞的靶向治疗抗体药物。

Screen and identification of monoclonal antibody against pancreatic cancer stem cells

Objective：To prepare functional monoclonal antibodies （mcAbs） against pancreatic cancer stem cell, so as to provide candidate antibody drugs for stem cell targeted therapy of pancreatic cancer. Methods ： HPAC ceils were cultured in serum free medium. Flow cytometry was used to detect the expression of CD133 in HPAC cells and sphere cells. The effects of hybridoma meAbs on self renewal, proliferation and chemosensitivity to Gemcitabine of HPAC parent or sphere cells were identified by serum free suspension culture and CCK 8 assay. Results： HPAC cells could survive,proliferate and form sphere cells in serum free medium. The sphere forming rate was 4.8% ＋ 0.6%. The percentage of CD133 ~ cells population in sphere cells increased by 11.6 folds compared to HPAC cells. The positive rate of CD133 in HPAC sphere cells was significantly higher than that in HPAC parent cells. Among the 20 candidate mcAbs, 3 mcAbs can recognize CD 133 ~ cells in HPAC sphere cells. The positive rate of 15E9 was 3.5%. Further investigation showed that mcAbs 15E9 significantly suppressed the sphere formation ability of HPAC cells ,with the inhibitory rates being 30.4%. The IC50 was 30.8nmol/L in 15E9 ＋ Gemcitabine group, and 58. lnmol/ L in mlgM ＋ Gemcitabine group. Conclusion： We have successfully screened and identified mcAbs 15E9 from large mcAb library against pancreatic cancer stem cell, which can specifically recognize CD133 ＋ HPAC cells and inhibit self renewal, mcAb 15E9 could be the candidate antibody drug targeting cancer stem cell.