结直肠癌干细胞微球体的培养及耐药机制

Establishment of colorectal cancer stem cell-derived tumorspheres and its multi-drug resistance

目的 :建立结直肠癌HT29细胞微球体耐药模型,并初步探讨微球体对化疗耐药的分子作用机制。方法 :人结肠癌HT29细胞以含表皮生长因子(epidermal growth factor,EGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bF GF)的无血清培养液为介质,通过体外悬浮培养法进行微球体的培养;采用蛋白质印迹法检测微球体中干细胞相关核心蛋白和耐药蛋白的表达水平;以亲本HT29细胞为对照,相差显微镜下观察微球体在化疗药物作用下的形态学变化;通过特异性针对三磷酸腺苷结合转运蛋白G超家族成员2(ATP-binding cassette subfamily G member 2,ABCG2)基因的si RNA沉默ABCG2的表达,并观察ABCG2基因沉默后微球体对化疗药物敏感性的影响。结果 :HT29细胞能在含多种生长因子的无血清培养液中形成稳定传代的微球体,并有自我更新和分化的潜能;微球体中高表达干细胞核心蛋白CD133、C-myc和BMI-1以及耐药蛋白ABCG2和多药耐药1(multidrug resistance 1,MDR1)基因编码的P-糖蛋白(P-glycoprotein,P-gp)(P值均<0.05),并对化疗药物[5-氟尿嘧啶(5-fluorouracil,5-FU)和奥沙利铂)]及靶向药物(西妥昔单抗)均具有一定的抵抗性;沉默ABCG2表达能够逆转微球体对化疗药物的耐药性。结论 :无血清培养能够形成微球体,其具有肿瘤干细胞的分子特征和耐药特点,靶向抑制ABCG2表达能提高结直肠癌微球体对化疗药物的敏感性。

Objective： To establish colorectal cancer HT29 stem cell-derived drug-resistant spheres, and to explore the possible mechanism of chemotherapy resistance.Methods： Serum-free medium supplemented with epidermal growthfactor（EGF） and basic fibroblast growth factor（b FGF） was used to isolate colorectal cancer HT29 stem cell-derived spheres, and the core molecular features of the tumorspheres were investigated by Western blotting. Then the tumorspheres were exposed to chemotherapeutic drugs and observed by the inverted-phase contrast microscope as compared with parent HT29 cells. The effects of ATPbinding cassette subfamily G member 2（ABCG 2） gene silencing on tumorsphere formation and chemosensitivity were observed under the inverted-phase contrast microscope.Results： Tumorspheres could be generated from HT29 cells in serumfree medium and sustained potential of self-differentiation, with high expressions of ＂stemness＂ genes（CD133, C-myc and BMI-1） and multidrug-resistance-related proteins including ABCG2 and P- g l y c o p r o t e i n（ P- g p）, a s c o m p a r e d w i t h p a r e n t H T 2 9 c e l l s（all P 0.05）. The tumorspheres from colorectal cancer HT29 cells were resistant to chemotherapeutic drugs [5-fluorouracil（5-FU） and oxaliplatin] and targeted drug（cetuximab）. Silencing ABCG 2 gene may reverse the multidrug resistance in tumorspheres.Conclusion： Tumorspheres can be generated from HT29 cells by culture with serum-free medium, which are endowed with stem celllike property including expression of ＂stemness＂ genes and multidrug resistance capacity, and ABCG 2 silencing can enhance the chemosensitivity in tumorspheres, thus representing a valid approach to sensitizing colorectal cancer stem cells to conventional treatment.