Serene Samyesudhas and Dr. Lynn Roy assisted in preparing samples for filming.

1，细胞的卵巢癌细胞株培养和荧光标记<ol><li>制备SKOV3媒体：麦考介质补充有10％胎牛血清（FBS），0.1mM的L-谷氨酰胺，50单位/ ml青霉素和50微克/毫升链霉素。保持OVCA429细胞在最小必需培养基（MEM）中补充有10％FBS，1mM丙酮酸钠，0.1mM的L-谷氨酰胺，50单位/ ml青霉素和50微克/毫升链霉素。 </li><li>在湿润的培养箱中传播的SKOV3和OVCA429细胞系在37℃下用5％的CO 2。细胞生长至40-50％汇合。 </li><li>生成单元，以监测存活在体外和体内表达红色荧光蛋白（RFP）。 <ol><li>以产生SKOV3细胞表达RFP，添加的慢病毒颗粒表达RFP和10微克/毫升的聚凝胺至SKOV3媒体在没有青霉素和链霉素的72小时。 </li><li> 72小时后，选择RFP阳性细胞的荧光激活细胞索尔蒂纳克（FACS），如下所示：Trypsinize RFP和非RFP表达细胞，离心分离机，在125×g离心5分钟，重悬的细胞在含有0.1％BSA的PBS中的10 7个细胞/ ml。在细胞分选仪，使用非RFP的细胞，以确定排序参数。然后收集细胞表达用561 nm激光高的RFP。 </li><li>可选地，如果慢病毒质粒表达的抗生素抗性盒，通过在合适的抗生素培养选择的RFP表达细胞（如10毫克/毫升的杀稻瘟素）。注意：需要使用可以通过分批和由细胞类型而有所不同的慢病毒颗粒的量。合适的慢病毒滴度应为每个小区类型来确定。 </li></ol></li></ol> 2，丰富的肿瘤干细胞<ol><li>治疗SKOV3，SKOV3-RFP或OVCA429细胞系为20μm顺铂72小时。注意：顺铂是有毒的。使用防护服/手套，避免与皮肤，粘膜接触。不要将铂íÑ​​废水。 </li><li>制备CSC媒体：无血清的DMEM / F12培养基补充有5微克/毫升的胰岛素，10毫微克/毫升的人重组表皮生长因子（EGF），10纳克/毫升碱性成纤维细胞生长因子（bFGF），12纳克/毫升白血病抑制因子和0.4％牛血清白蛋白（BSA）。 </li><li> Trypsinize细胞。文化尚存在中信建投的媒体单元。 注：在培养非贴壁球体2天将形成。 </li><li>通过收集介质含有细胞并在125 XG离心5分钟，改变介质每2天。然后重悬在新鲜CSC媒体沉淀。检查细胞活力，每2天计数细胞并进行台盼蓝染色。 </li><li> （137 mM氯化钠，2.7 mM的氯化钾，和11.9 mM磷酸盐缓冲液，pH 7.4的PBS）与然后通过离心129×g离心5分钟，重悬沉淀在含有苯酚胍硫氰酸盐（RNA），磷酸盐缓冲盐水收集的CSCs在进一步的实验0.1％BSA（流式细胞术），或CSC媒体继续培养该细胞。 </li><li>使用光学显微镜在20X和40X的放大倍率监控CSC细胞形态。 </li></ol>通过对干细胞标记基因的表达分析3，中信建投表征<ol><li>收集的CSCs的3制剂通过收集介质含有细胞，在129 XG离心5分钟，重新悬浮沉淀中含有苯酚胍硫氰酸盐的溶液（也可以使用其他方法来提取RNA）。 </li><li>使用TRIzol试剂根据生产商的方案制备的总RNA。利用市售的cDNA反转录试剂盒从0.1-1微克的RNA合成互补DNA（cDNA）。 </li><li>进行定量反转录聚合酶链式反应（QRT-PCR）。 <ol><li>弥补主混合物对于每一组含有QRT-PCR混合物，引物和水至总量每样本8微升引物（引物用不同的荧光团可在同一井被放大）。例如，生成一个主混音的Oct4-FAM，Nanog的六角，和GAPDH-CY5。生成巢和PROM1单独的主混音。加入2μl的样品cDNA模板。 请注意：在本研究中，QRT-PCR使用表1中所列的引物进行。 </li><li>执行所有的荧光定量PCR检测的重复利用实时PCR热循环仪能够检测多种荧光团各样本。包括无模板对照。 </li><li>标准化的干细胞的基因表达，以GAPDH表达为使用ΔΔCT法各样品。 </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0" width="746"><colgroup><col /><col /></colgroup><tbody><tr height="21"><td height="63" rowspan="3" style="height:62px;width:111px;">辛-4-FAM </td><td style="width:636px;">引物1：5&#39;-CCCAAGGAATAGTCTGTAGAAGTG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2：5&#39;-TGCATGAGTCAGTGAACAGG-3&#39;</td></tr><tr height="21"><td height="21" style="height:21px;"> FAM探针：5&#39;-CTTCCAAGC / ZEN / TG​​CCCACCTAACTTCT / 3IABkFQ -3&#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> NANOG-HEX </td><td>引物1：5&#39;-CCTTCTGCGTCACACCATT-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2：5&#39;-AACTCTCCAACATCCTGAACC-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> HEX探针：5&#39;-CTGCCACCT / ZEN.CTTAGATTTCATTCTCTGGT / 3IABkFQ-3&#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> GAPDH-CY5 </td><td>引物1：5&#39;-TGTAGTTGAGGTCAATGAAGGG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2：5&#39;-ACATCGCTCAGACACCATG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> CY5探针：5&#39;-AAGGTCGGAGTCAACGGATTTGGTC / 3IABRQSP-3&#39; </td><html></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;">巢蛋白FAM </td><td>引物1：5&#39;-AGGACCTGAGCGATCTGG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2：5&#39;-CGTTGGAACAGAGGTTGGAG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> FAM探针：5&#39;-AACTTTTCA / ZEN / GTAGCCCGCAGCC / 3IABkFQ -3&#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> CD133-HEX </td><td>引物1：5&#39;-ACTCTCTCCAACAATCCATTCC-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;">引物2：5&#39;-AAACAATTCACCAGCAACGAG-3&#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> HEX探针：5&#39;-ACAATCACT / ZEN / GAGCACTCTATACCAAAGCG / 3IABkFQ-3 </td></tr></tbody></table> 表1引物用于CSC的表征定量RT-PCR检测。 e_title“&gt; 4。在细胞表面标记CD117和CD133的肿瘤干细胞分析<ol><li>收获的CSCs通过收集介质含有细胞和在125 XG离心5分钟。添加的非蛋白水解细胞剥离溶液来分手单元500微升。离心细胞，在125×g离心5分钟以除去细胞剥离溶液。然后洗涤细胞两次用冷的PBS中。 </li><li>孵育在正常生长培养基中进行1小时标记细胞之前。降速细胞5分钟，在129×g下。重悬的细胞在PBS中的0.1％BSA中与抗CD133-PE和抗-CD117-FITC。 </li><li>在1％多聚甲醛中过夜固定细胞。注意：多聚甲醛是有毒的（疑似致癌物）。请在通风橱中，并在一周内使用。 </li><li>流式细胞仪进行流量分析CD117（用抗-CD117 FITC偶联）和CD133（用抗-CD133的缀合PE）上的流式细胞仪的细胞表面表达。收集细胞在FL1（为FITC）和FL3（体育）频道的数据。产生门，用于细胞同时表达FITC和PE。生成的点积与4个象限（FITC-和PE-，FITC + PE-，FITC-PE +，和FITC +和PE +），并判断在每个象限中的细胞数。 </li></ol> 5，分析肿瘤干细胞的治疗反应<ol><li>板5000细胞每孔（SKOV3，SKOV3-RFP，SKOV3 CSC和SKOV3-RFP的CSCs）在96孔板过夜。 </li><li>治疗与顺铂（0，1，10，100，和1000μM）或紫杉醇（0，0.01，0.1，1，10，和100μM）为96小时。 </li><li>进行MTT（3（4,5二甲基-2 - 基）-2,5 - 二苯基溴化）细胞活性分析。 <ol><li>每孔100μl板5000细胞在96孔板（板的细胞，一式两份，并且包括媒体只对照孔）。 </li><li> 24小时后，加入10微升的10毫克/毫升的MTT。在37℃下进行2-4小时（直至形成沉淀）。 </li><li>加入100微升的MTT溶剂4mM的盐酸，0.1％NP-40的异丙醇。孵育在黑暗中15分钟。 </li><li>阅读板块，在570 nm处对酶标仪中进行eader。 </li></ol></li></ol>

<ol>
	<li>Pennington, K., Pulaski, H., Pennington, M., Liu, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+Much+of+a+Good+Thing:+Suicide+Prevention+Promotes+Chemoresistance+in+Ovarian+Carcinoma.">Too Much of a Good Thing: Suicide Prevention Promotes Chemoresistance in Ovarian Carcinoma.</a> Curr Cancer Drug Targets. 10, 575-583 (2010).</li><li>Thigpen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rational+approach+to+the+management+of+recurrent+or+persistent+ovarian+carcinoma.">A rational approach to the management of recurrent or persistent ovarian carcinoma.</a> Clin Obstet Gynecol. 55, 114-130 (2012).</li><li>Burgos-Ojeda, D., Rueda, B. R., Buckanovich, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+stem+cell+markers:+prognostic+and+therapeutic+implications.">Ovarian cancer stem cell markers: prognostic and therapeutic implications.</a> Cancer letters. 322, 1-7 (2012).</li><li>Ma, L., Lai, D., Liu, T., Cheng, W., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem-like+cells+can+be+isolated+with+drug+selection+in+human+ovarian+cancer+cell+line+SKOV3.">Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3.</a> Acta Biochim Biophys Sin (Shanghai). 42, 593-602 (2010).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nature Medicine. 3, 730-737 (1997).</li><li>Alison, M. R., Lim, S. M., Nicholson, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+problems+for+therapy.">Cancer stem cells: problems for therapy.</a> The Journal of pathology. 223, 147-161 (2011).</li><li>Luo, X., Dong, Z., Chen, Y., Yang, L., Lai, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+ovarian+cancer+stem-like+cells+is+associated+with+epithelial+to+mesenchymal+transition+through+an+miRNA-activated+AKT+pathway.">Enrichment of ovarian cancer stem-like cells is associated with epithelial to mesenchymal transition through an miRNA-activated AKT pathway.</a> Cell proliferation. 46, 436-446 (2013).</li><li>Wang, L., Mezencev, R., Bowen, N. J., Matyunina, L. V., McDonald, J. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+stem-like+cells+show+induced+translineage-differentiation+capacity+and+are+suppressed+by+alkaline+phosphatase+inhibitor.">Ovarian cancer stem-like cells show induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor.</a> Oncotarget. 4 (12), 2366-2382 (2013).</li><li>Liu, T., Cheng, W., Lai, D., Huang, Y., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+ovarian+cancer+cells+in+different+culture+systems.">Characterization of primary ovarian cancer cells in different culture systems.</a> Oncology reports. 23, 1277-1284 (2010).</li></ol>

The authors declare they have no competing financial interest.

肿瘤干细胞有抗药性的治疗可能是治疗原发肿瘤后交代复发。肿瘤干细胞的特性可能导致更好的治疗卵巢癌。在建立使用上述协议化疗耐药的CSCs的临界参数的时序治疗与化疗的长度和化疗的浓度。当使用在Ma 等人的协议。人们发现，在7天后用顺铂和紫杉醇治疗的，无活细胞保持4。通过还原处理，以3天（以20μM的顺铂而不是40μM的顺铂和10μM紫杉醇），可行化疗耐药细胞和肿瘤干细?...

Resistance to chemotherapy is a major impediment to the treatment and cure of cancer. Ovarian cancer is the 5th leading cause of cancer-related deaths among women in the United States (American Cancer Society Facts and Figures 2013). Patients initially respond well to chemotherapy, but most patients relapse1. After relapse patients are treated with a variety of additional chemotherapy agents with very little benefit2. General properties of CSCs include unlimited proliferative capabilities, retention of an undifferentiated state, resistance to drug treatment, efficient DNA repair, and ability to generate malignant tumor cells with different phenotypes3. CSCs frequently exhibit expression of stem cell genes such as Nanog, Oct4, Sox2, Nestin, CD133, and CD117. CSCs often express elevated levels of ABCG2, and ALDH genes that may contribute to drug efflux and metabolism3,4. 
The first definitive evidence for CSCs was demonstrated by isolating acute myeloid leukemia-initiating cells that were capable of self-renewal and tumor generation5. These leukemic stem cells expressed surface CD34 and generated leukemia in NOD/SCID (immunocompromised) mice5. Since then CSCs have been identified in many cancer types including leukemias/lymphomas, breast, bladder, colorectal, endometrial, sarcomas, hepatocellular carcinoma, melanomas, gliomas, ovarian, pancreatic, prostate, squamous cell carcinoma, and lung6. Therefore, being able to study this subtype of cancer cell is advantageous.
The goal of this study is to create a protocol for the selection and isolation of chemoresistant CSCs. Several methods have been reported for the isolation of CSCs from ovarian cancer cell lines. Non-adherent spheroids isolated from OVCAR-3, SKOV3, or HO8910 cultures demonstrate stem-like properties7,8. Isolation of CD133+ cells from OVCAR-3 cultures also yields CSCs. CSCs have also been selected in culture by treatment with chemotherapeutic agents. Treating tumor cell lines (OVCA433, Hey, and SKOV3 cells) with cisplatin and paclitaxel allows for the expansion and isolation of CSCs4,9. While culture of some cell types in CSC media leads to isolation of CSCs, SKOV3 cells did not survive culture in serum-free media or form sphere cells4. Therefore, treatment of cells with cisplatin and paclitaxel aided the expansion or isolation of this population4.
Using a modification of the procedure presented by Ma and colleagues4 we developed a method to isolate CSCs from the ovarian cancer cell lines. Our protocol is advantageous as it yields more viable cells while using less toxic chemotherapeutic agents. Cells are treated with cisplatin and subsequently grown in serum-free media supplemented with growth factors (stem cell media). We isolate the resulting non-adherent sphere cells and assay them for their expression of stem cell markers. This model enables the study of CSC properties and response to drug therapy.

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enrichment for chemoresistant ovarian cancer stem cells from human cell lines

癌症干细胞（CSCs的）被定义为慢骑自行车和未分化细胞的不对称分裂，产生高度增殖，浸润和化疗耐药的肿瘤细胞的一个子集。因此，肿瘤干细胞是细胞的一个有吸引力的人群为目标的治疗。 CSCs的被预测为向多种类型的恶性肿瘤，包括那些在血液，脑，肺，胃肠道，前列腺和卵巢的。分离和富集肿瘤细胞群的肿瘤干细胞将使研究人员能够研究性质，遗传学和肿瘤干细胞治疗的反应。我们产生的一种协议，它可再现丰富的卵巢癌细胞株（SKOV3和OVCA429）卵巢癌肿瘤干细胞。细胞系是用20μM的顺铂处理3天。存活细胞的分离及培养在含细胞因子和生长因子的无血清干细胞培养基。我们通过分析分离出的细胞对于k证明这些肿瘤干细胞纯化的浓缩nown干细胞标记物的Oct4，Nanog和PROM1（CD133）和CD177和CD133的细胞表面表达。在社区体育会显示增加的耐药。这种方法对于肿瘤干细胞的分离，是研究耐药和肿瘤复发的肿瘤干细胞的作用，一个有用的工具。

表明我们用顺铂治疗中分离的CSC从卵巢上皮癌的细胞系，我们首先在治疗之前和选择后获得的细胞系中的图像。我们使用光学显微镜拍摄附着的（未处理的）的SKOV3和OVCA429细胞和SKOV3和OVCA429的CSCs（ 图1）的图像。 CSCs的出现圆形和未附着到组织培养板中（ 图1和2）。我们还表明，这表明该细胞是可行的（ 图2B）中的CSCs的分离后SKOV3细胞转导的RFP保...

Watch this Scientific Journal Video about Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines at JoVE.com

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

肿瘤干细胞（肿瘤干细胞）有牵连的肿瘤复发，由于化疗。我们已经进行了优化的协议选择和扩展，从卵巢癌细胞系的CSCs的。通过处理细胞与化疗顺铂和在干细胞培养促进我们丰富的非粘附CSC培养介质。

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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13.4K 次观看.  Indiana University School of Medicine. 肿瘤干细胞（肿瘤干细胞）有牵连的肿瘤复发，由于化疗。我们已经进行了优化的协议选择和扩展，从卵巢癌细胞系的CSCs的。通过处理细胞与化疗顺铂和在干细胞培养促进我们丰富的非粘附CSC培养介质。 