ALDH^(hi)CD44^+细胞在乳腺癌组织中的表达及其与相关细胞信号传导通路之关系研究

Study on the expression of ALDHhiCD44^+ cells in breast cancer tissues and the association between ALDHhiCD44^+ cells and cell signal pathways

目的:①探讨具有干细胞标记特性的ALDHhiCD44+细胞在乳腺癌组织及癌旁组织中的表达情况;②探讨乳腺癌MDA-MB-231细胞系中是否存在乳腺癌ALDHhiCD44+细胞亚群,并分析Wnt/β-catenin与Notch细胞信号通路中的重要基殷β-catenin和Notch1,在ALDHhiCD44+细胞亚群中的表达情况。方法:①试验组为30例乳腺癌患者的术中新鲜癌组织,另取19例癌旁2 cm组织标本作为对照;②机械-酶方法消化组织、收集细胞悬液,并行肿瘤细胞原代培养,再经流式细胞仪检测ALDH-hiCD44+细胞的含量;③流式细胞技术检测乳腺癌细胞系MDA-MB-231细胞中ALDHhiCD44+细胞的表达含量,并分选出AL-DHhiCD44+细胞,利用荧光定量RT-PCR技术检测ALDHhiCD44+细胞亚群和ALDHlow CD44+细胞亚群中β-catenin和Notch1的扩增情况。结果:①30例浸润性乳腺癌组织中有28例原代培养成功,19例癌旁组织中有15例能够培养出原代细胞。ALDH-hiCD44+细胞在癌组织原代培养的细胞中的含量为(3.85±2.59)%(0.86～8.00%),在癌旁组织原代细胞中含量为(0.13±0.07)%(0.05～0.19%),两值相比差异有统计学意义(P=0.035,P<0.05);②乳腺癌细胞系MDA-MB-231中,ALDHhiCD44+细胞含量为(0.82±0.067)%,且与ALDHlowCD44+细胞相比,ALDHhiCD44+细胞中β-catenin和Notch1的表达量是增高的(P<0.05)。结论:通过肿瘤组织原代细胞培养、检测新途径,进一步证实ALDHhiCD44+乳腺癌干细胞的存在,且其在癌灶的表达量高于癌旁组织。结合细胞系MDA-MB-231中的ALDHhiCD44+细胞检测,其中的β-catenin和Notch1表达增高,表明ALDH-hiCD44+细胞中Wnt/β-catenin和Notch细胞信号通路处于活化状态。

Objective （ 1 ） To explore the expression of stem cell markers characteristic ALDHhiCD44 ＋ cells in breast cancer tissues and cancer adjacent tissues ; （ 2 ） To explore whether there was breast cancer stem cells ALDHhiCD44 ~ in breast ceneer cell line MDA - MB -231 and to analyze the gene amplification levels of the important genes 13 - catenin and Notchl in Wnt/β - eatenin and Notch signal pathways from ALDHhiCD44 ＊ cells. Method （ I ） We collected the 30 eases of fresh cancer tissues during surgery as the experimental group and 19 cases of cancer adjacent tissue samples as control group; （2）We used the method of machinery - enzymes to digest the tis- sues for primary culture, and then the content of ALDHhiCD44 ＋ cells were deteeted by flow eytometry; （ 3 ） ALDHhiCD44 ＋ cells were studied in MDA- MB -231 cells by flow eytometry,the ALDHhiCD44＋ cells and ALDHt~＇CD44 ~ cells were separated by fluorescence - activated cell sorting, and the gene amplification of β- catenin and Notchl in Wnt and Notch signal pathways of ALDHhiCDd-4＋ and AL- DHI~＇CD44 ~ were also studied by real time polymerase chain reaction（ real time PCR）. Results （1） 28 out of 30 cases of invasive breast cancer tissues and 15 out of 19 cases of cancer adjacent tissues had been primary cultured successfully ,the average content of ALDHhiCD44 ＋ cells in cancer tissues were （3.85 ± 2. 59） % （0. 86 ± 8.00% ） , in eancer adjacent tissues were（ 0. 13 ±0. 07 ）% （0.05± O. 19% ）. The expression of ALDHhiCD44 ＋ cells in cancer tissues was higher than the adjacent tissues （P =0. 035 ,P 〈0. 05）. （2）The content of AL- DHhiCD44 ＋ ceils was approximately（ 0. 82± 0. 067 ）% in MDA - MB -231 cells, the mRNA levels of 13 -eatenin and Noteh l were high- er in ALDHhiCD44 ＋ cells than ALDHlowCD44＋ cells（ P 〈0. 05）. Condusion We have uesd the method of the culture and detection of primary cells from breast cancer and cancer adjacent tissues to further confirmed the presence of breast cancer stem cells ALDHhiCD44 ＋ , and the content of ALDHhiCD44 ＋ cells were higher in the breast cancer tissues than cancer adjacent tissues ; the expression of 13 - eatenin and Notchl in ALDHhiCD44 ＋ cells from MDA - MB - 231 cells were higher than ALDHlowCD44＋ cells indieated that Wnt / β- caterfin and Notch cell signaling pathways in the ALDHhiCD44 ＋ cells were activated.