人肝癌组织中肝癌干细胞的分离培养与初步鉴定

Isolation,Culture and Identification of Human Hepatic Cancer Stem Cells from Hepatocellular Carcinom Tissues

目的体外分离及培养人肝癌组织中的肝癌干细胞并鉴定其生物学特性。方法取临床肝癌患者术后肝癌组织,用酶消化法分离肝癌细胞,将其接种于无血清培养基中培养,并在超低粘附24孔板中观察肝癌干细胞克隆球生长情况;用MTT比色法检测肝癌干细胞克隆球增殖能力;采用流式细胞仪鉴定并分选CD90+细胞、CD90-细胞,CD133+细胞、CD133-细胞,将4种不同细胞组成CD90+细胞CD133+细胞、CD90-细胞CD133+细胞、CD90+细胞CD133-细胞及CD90-细胞CD133-细胞,分别接种于裸鼠右侧腋窝皮下观察其成瘤能力并记录肿瘤体积;将裸鼠肿瘤组织HE染色观察。结果在无血清培养基中,肝癌干细胞可以形成少量的细胞克隆球;在72h内肝癌干细胞克隆球始终处于持续增殖状态;肝癌干细胞表面标记物CD90、CD133阳性率分别是7.2%、8.8%,并成功分选出CD90+细胞、CD90-细胞、CD133+细胞、CD133-细胞,将重组的4种不同细胞组分别接种于裸鼠皮下,结果显示,CD90+细胞CD133+细胞、CD90-细胞CD133+细胞及CD133+细胞CD90-细胞在裸鼠体内均形成肿瘤,CD90+细胞CD133+细胞接种1个月后开始有裸鼠成瘤,CD90-细胞CD133+细胞与CD133+细胞CD90-细胞接种至第2个月开始有裸鼠成瘤,而CD133-细胞CD90-细胞在整个过程中未见肿瘤形成。CD90+细胞CD133+细胞组肿瘤体积大于CD90-细胞CD133+细胞组与CD90+细胞CD133-细胞组(P<0.05);CD90-细胞CD133+细胞组与CD90+细胞CD133-细胞组比较差异无统计学意义(P>0.05)。裸鼠体内肿瘤HE染色观察与人肝癌组织病理特点相似。结论成功建立了从人肝癌组织中分离培养肝癌干细胞的方法;培养得到的肝癌干细胞能在无血清培养基中形成克隆球,且具有较强的增殖能力;其中表达CD90和/或CD133细胞,均在裸鼠体内形成肿瘤;肝癌组织中CD90+细胞和CD133+细胞具有肝癌干细胞特性,可能为肝癌干细胞。

Objective To isolate and culture human hepatic cancer stem cells from tissues of hepatocellular carcinoma and to identify their biological characteristics. Methods Hepatocellular carcinoma tissues from patients with liver cancer after surgery were collected. Hepatic cancer cells were isolated by digestion and cultured in the serum free medium,and the survival of tumor spheres were observed in ultra low adhesion 24-well plates. Besides,the proliferation of hepatic cancer cells was analysed by MTT assay while hepatic cancer cells were sorted by flow cytometry based on CD90 and CD133,and devided into four groups CD90+CD133-,CD90-CD133+,CD90+CD133+and CD90-CD133-respectively. Then,the four group cells were subcutaneously engrafted into nude mice,and tumor was resected and subjected to HE staining. Results Tumor spheres could be found in the serum free culture condition and the hepatic cancer cells kept in an increased state in continued 72 hours. Moreover,the ratio of expression of CD90 and CD133 was 7. 2%,8. 8% respectively.The CD90+,CD133+,CD90-and CD133-cells were scucessfully sorted by flow cytometry. Cell transplantation assays indicated tumor were detected in CD90+CD133+group one month later,and in CD90+CD133-and CD90-CD133+group two months later,while no tumor were observed in CD90-CD133-group. Tumor volume of CD90+CD133+group was not significantly larger than that in both of CD90+CD133-and CD90-CD133+groups. HE staining showed the pathological features of tumor were similar with that of human.Conclusion We succesfully established the method to isolate and culture human hepatic cancer stem cells,which could formed tumor spheres and have ability of self-renewal. Some of the cells had the ability to form tumor spheres,self-renewal. Some of the cells expressed CD90+or CD133+,and could formed tumors under the skin of nude mice. Importantly,the CD90+CD133+cells possess the properties of stem cells,which would be hepatic cancer cells.