基于ALDH1与CD44+/CD24-肿瘤干细胞亚群差异探讨化痰散结方对乳腺癌干细胞生物行为的影响

According to cancer stem cells (CSCs) theory, tumors originated from a small group of cancer cells. From strict sense,the identification of CSCs is very difficult due to the rareness of them. At present, CSCs are separated and identified from tumor tissues through specific markers of CSCs. The CSCs isolated through different methods, however, are not same. CSCs with specific biological characteristics are in a degree depend on the corresponding method used to obtain CSCs.Studies have confirmed: breast cancer stem cells subpopulations, which are positively expressed through CD44 + / CD24- and ALDH1 method, just have a small portion of expression overlap, on the contrary, show a big difference at biological characteristics of the tumors between each other. This experiment, which is on the preliminary basis of mechanism research about breast cancer stem cells apoptosis induced by anti-cancer traditional Chinese medicine of the dissipating phlegm and resolving masses prescription, is designed as follows:1.Enrich the cancer stem cells MCF-7/ADR.2. To identify and isolate the breast cancer stem cells respectivly by using the methods of CD44 + CD24-and ALDH1 +.3.The comparison on two groups of breast cancer stem cells about the differences of growth inhibition by the dissipating phlegm and resolving masses prescription.This study will explicate the potential and derection for anti-tumor traditional chinese medicine from the CSC level,and attempts to reveal the characteristics and advantages of traditional Chinese medicine how to confront the tumor from the biological behavior by the observe ,which use the diffirence due to the dissipating phlegm and resolving masses prescription to intervent the different cancer stem cells separated by two different identification methods.

肿瘤干细胞（CSC）学说认为肿瘤起源于一小群肿瘤干细胞，而严格意义上CSC的鉴定十分困难，目前我们只能通过CSC的固有属性将其从肿瘤组织中进行鉴定分离，但不同的鉴定方法分选的CSC是不完全相同的。且其生物学特性更大程度取决于相应的鉴定方法。研究证实：CD44+CD24-与ALDH1阳性的乳腺癌干细胞亚群仅有小部分重叠，并存在较大的肿瘤生物学特征差异。 本实验在抗乳腺癌中药协定方化痰散结方诱导乳腺癌干细胞凋亡机制研究的前期基础上，设计如下：1.肿瘤干细胞MCF-7/ADR的富集2.CD44+CD24-法与ALDH1+法分别鉴定和分离乳腺癌干细胞3.化痰散结方对两组乳腺癌干细胞亚群生物学行为影响的差异性比较。本研究拟通过化痰散结方对两组不同鉴定方法分离出的乳腺癌干细胞的干预作用差异性的观察，以期从CSC层面阐发中药抗肿瘤的潜力和方向，尝试从肿瘤生物学行为角度揭示中药抗癌的优势和特点。

耐药性是导致恶性肿瘤难治、复发的关键因素之一。大量研究表明，肿瘤耐药根源于肿瘤干细胞的耐药性，而凋亡耐药是肿瘤干细胞耐药的重要机制。前期实验研究表明化痰散结类中药可导致耐药乳腺癌细胞株凋亡，在前期工作的基础上，我们设计化痰散结方分别对CD44+/CD44 -以及 ALDH1+乳腺癌干细胞亚群影响的研究进一步阐发干细胞为靶点的抗肿瘤中药的机制特点。首先进行肿瘤干细胞MCF-7/ADR的富集；继而以CD44+CD24-法与ALDH1+法分别鉴定和分离乳腺癌干细胞；最后进行化痰散结方对其生物学行为影响进行比较。从肿瘤干细胞的角度探索中药抗肿瘤的特点和潜力具有相当的挑战性和广泛前景。本课题通过以上研究从CSC层面阐发了化痰散结方逆转凋亡耐药的内在机制，从肿瘤生物学行为角度揭示了中药抗癌的优势和特点。对深入阐发中药抗癌机制具有理论探索和实践指导意义。. 我们分离的SP 细胞中CD44+/CD24-以及ALDH1+比例明显高于非SP细胞。化痰散结方能够逆转人乳腺癌多柔比星/阿霉素耐药，其耐药相关蛋白质相互作用网络复杂，其中PI3K－Akt－mTOR很可能是其中的关键调控点，而化痰散结方抑制MCF－7/ADR细胞增殖的作用机制之一是抑制 PI3K －Akt－mTOR通路的表达，细胞划痕、凋亡等实验也显示出化痰散结方良好的剂量依赖型干预作用。不仅如此，我们还进一步研究了对乳腺癌其他分型(MDA-MB-231)细胞的干预作用。相关研究结果如下：（1）MTT法检测结果显示化痰散结方能够抑制乳腺癌细胞的增殖，且随药物浓度的增加和作用时间的延长，对乳腺癌细胞的抑制作用越强。3个给药组48、72h抑制作用优于24h（P<0.05，P<0.01）；与空白对照组（A组）比较，差异均有统计学意义（P值均＜0.05）。（2）Western blot结果显示，随着化痰散结方含药血清浓度的增加，乳腺癌细胞提取蛋白中的Gli1蛋白的表达逐渐降低，与空白对照组（A组）比较，各实验组Glil蛋白表达均显著降低（P<0.05，P<0.01）。