p53促进化疗抵抗肺癌干细胞不对称自我更新的机制研究

Cancer stem cells(CSCs) are estimated as the culprit of cancer recurrence. Two types of self-renewal have been identified in CSCs-asymmetric and symmetric self-renewal. Symmetric division expands the 'stem pool',thus promotes tumor recurrence. We presume that drug resistant lung cancer stem cells divide symmetricly. We have previously enriched a population of stem-like cells from lung cancer cell line A549 using cisplatin treatment, and defined them as Drug-resistant lung cancer stem cells(Dr-LCSCs). Dr-LCSCs divided into two daughters cells symmetriclly and inherited stemness, whereas asymmetriclly when p53 was upregulated, resulting in one daughter cell inherited stemness and the other started to differentiate. The cell fate determinate Numb decide the self-renewal division mode. We proposed that the self-renewal division mode of Dr-LCSCs is tightly associated with the recurrence of lung cancer by controlling'stem cell pool'. Thus, we plan to enriched Dr-LCSCs from more lung cancer cell lines, investigate the self-renewal division mode of Dr-LCSCs by PKH26 staining and BrdU-labled detain assy, and find out the mechanism that p53 regulate division mode along with Numb using Co-immunoprecipitaion(Co-IP) and kinase assay. The results is hoping to provide molecular targets that to prevent lung cancer recurrence.

肿瘤干细胞（CSCs）理论认为 CSCs是肿瘤复发的根源。CSCs具有两种自我更新模式-对称分裂和不对称分裂，CSCs对称分裂使"干细胞池"迅速扩增，促进肿瘤复发。探讨化疗后残余肺癌干细胞分裂自我更新模式及其调控机制有利于理解肺癌化疗后复发。前期我们从A549细胞中富集到一群化疗抵抗肺癌干细胞（Dr-LCSCs），观察到Dr-LCSCs/A549通过对称分裂完成自我更新，上调p53能逆转Dr-LCSCs/A549对称分裂，促进不对称分裂。为了进一步明确Dr-LCSCs的自我更新模式及其调控机制，本研究拟从多株肺癌细胞系中富集Dr-LCSCs，利用PKH26标记、BrdU滞留实验等方法探明Dr-LCSCs自我更新模式，采用质粒转染、免疫共沉淀、激酶活性测定等手段探讨p53调控Dr-LCSCs自我更新模式的具体途径。本研究结果将为防治肺癌复发提供理论基础和分子靶点。

肿瘤干细胞（CSCs）具有对称分裂和不对称分裂两种分裂模式，对称分裂使“干细胞池”迅速扩增，促进肿瘤复发。探讨化疗后残余肺癌干细胞分裂自我更新模式及其调控有利于理解肺癌化疗后复发机制。本研究利用顺铂体外诱导肺癌细胞系A549、H460、H1299和H661细胞，体外富集耐药肿瘤干细胞样细胞（Dr-LCSCs），探讨Dr-LCSCs 自我更新模式的具体机制。研究结果表明：（1）通过体外顺铂诱导的方式建立了稳定的顺铂耐药的非小细胞肺癌细胞系A549/CisR、H460/CisR、H1299/CisR、H661/CisR；（2）相对亲代细胞，顺铂耐药A549/CisR、H460/CisR、H1299/CisR、H661/CisR细胞存在着标志物ABCG2、CD90、CD44及CD133的过表达（3）采用流式细胞术分离耐药A549/CisR、H460/CisR、H1299/CisR及H661/CisR细胞中CD44+/CD90+双阳性细胞，并采用体外成球实验、自我更新实验验证其具有干细胞自我更新能力，定义为耐药肺癌干细胞样细胞（Dr-LCSCs）。（4）野生型P53背景的A549/CisR(CD44+/CD90+)、H460/CisR(CD44+/CD90+)和p53突变背景的H661/CisR (CD44+/CD90+)、以及p53缺失背景的H1299/CisR (CD44+/CD90+)比较，体外自我更新能力较弱。（5）Dr-LCSCs在体外主要采用对称分裂模式，野生型p53 A549/CisR(CD44+/CD90+)、H460/CisR(CD44+/CD90+)不对称分裂的比例较高，提示野生型p53可能具有促进不对称分裂的作用。（6）外源性野生型p53具有促进H1299/CisR (CD44+/CD90+)不对称分裂的作用；（7）转染野生型 P53或突变型 P53进入 Dr-LCSCs，使用 p53抗体进行免疫共沉淀，未能检测到P53与ASPP2结合（阳性对照有条带）；转染野生型 p53 或突变型 p53 进入 Dr-LCSCs，使用 Par3 抗体，利用免疫共沉淀，未能检测到 Dr-LCSCs内 par3与 aPKC/Par6形成复合物。（8）野生型p53通过促进CD44、CD90分子标志物在子代细胞中不对称分布机制促进Dr-LCSCs不对称分裂。