HT29和HCT116结肠癌细胞无血清培养形成肿瘤干细胞球特性的差异性研究

目的比较HT29和HCT116两种结肠癌细胞系中肿瘤干细胞的差异，初步探讨结肠癌干细胞研究模型。方法以无血清培养法培养HT29和HCT116细胞，观察其在不同时间形成肿瘤干细胞球的差异，用限制性稀释法计算两者的成球率；流式细胞术分析HT29和HCT116细胞系中CD44／CD24的表达情况；裸鼠体内成瘤实验鉴定HT29细胞球与HCT116细胞球成瘤能力。结果无血清培养法发现HCT116较HT29更易形成肿瘤干细胞球且所需时间更短，即HT29在无血清培养的第7天开始形成规则的球体，而HCT116则在第5天就已形成规则的球体，HCT116成球率（11．4±1．15）％高于HT29（3．31±0．27）％，且差异有统计学意义（P〈0．05）；HT29和HCT116中CD44±／CD24±各细胞含量有显著差异，结果显示具有干细胞特性的CD44＋／CD24＋结肠癌细胞在HCT116中所占比例（60．33±5．75）％明显高于HT29（9．23±2．15）％，差异有统计学意义（t=13．939，P〈0．05）；体内成瘤实验发现HCT116细胞球在裸鼠体内的成瘤能力明显强于HT29细胞球，HCT116细胞球的成瘤速度及瘤体生长速度都较HT29细胞球快。结论与HT29相比，HCT116结肠癌细胞系更适合作为肿瘤干细胞研究的模型。     

人胰腺癌细胞系SW1990中肿瘤干细胞样侧群细胞的分离及生物学特性研究

目的 从人胰腺癌细胞系SW1990中分离肿瘤干细胞样侧群(side population,SP)细胞,并进一步研究其生物学特性.方法 细胞常规培养后应用Hoechst 33342和PI染色、荧光激发流式细胞仪检测并分选SP细胞,MTT法检测细胞活性,流式细胞仪分析干细胞标志物CD133的表达,克隆平板测定细胞克隆形成能力,Western blot检测细胞ABC超家族成员G2(ABCG2)蛋白表达.结果 人胰腺癌细胞系SW1990中SP细胞比例为2.70%,可完全被维拉帕米阻断.培养9 d后,SP细胞的A492值为2.1,克隆形成率为(38.7±6.8)%,CD133阳性率为69.63%,均显著高于非SP细胞的0.5,(15.5±2.8)%,16.71%;SP细胞ABCG2表达也较非SP细胞强.结论 胰腺癌细胞系SW1990存在SP细胞.     

侧群细胞与消化系恶性肿瘤干细胞研究

侧群(side population,SP)细胞是利用Hoechst33342染料和流式细胞术进行造血干/祖细胞分离时发现的一群特殊细胞,既有干细胞样自我更新和多向分化潜能,而且具有独特的SP表型标志,为干细胞研究提供了新的方向。侧群细胞在正常组织和恶性肿瘤细胞中均有表达,且其有与肿瘤干细胞相似的生物学特性。近年来在消化系肿瘤干细胞的研究中发现侧群细胞对于分选鉴定肿瘤干细胞意义重大。本文就侧群细胞与消化系肿瘤干细胞的关系进行研究。     

羟基喜树碱富集结肠肿瘤干细胞样群体的初步研究

背景:肿瘤干细胞是临床上肿瘤耐药的主要机制,识别并靶向肿瘤干细胞为肿瘤治疗的研究带来了新的思路。目的:探讨在体外以化疗药物富集结肠肿瘤干细胞样群体的可行性。方法:应用前期实验中以药物浓度递增诱导法建立的羟基喜树碱(HCPT)耐药人结肠癌细胞株SW1116/HCPT,免疫荧光标记干细胞表面抗原CD133,Hoechst33342/PI双染流式细胞仪检测、分选侧群(SP)细胞,实时PCR检测干细胞相关和多药耐药相关基因表达。结果:亲代细胞株SW1116中CD133阳性群体和SP群体分别仅占2%和1%,耐药细胞株SW1116/HCPT中则高达7%和61%,多药耐药基因MDR1抑制剂维拉帕米可使两组SP群体均下降至接近0水平。SW1116/HCPT细胞SP群体中干细胞相关基因CD133、Nanog和多药耐药相关基因MDR1、MRP1、MRP2、MRP6 mRNA表达水平较非SP群体显著上调(P0.05)。结论:HCPT药物浓度递增诱导法可成功富集结肠肿瘤干细胞样群体,其SP亚群中存在于细胞相关和多药耐药相关基因高表达。     

干细胞培养基成球培养法筛选结肠癌干细胞的生物学特性

目的探讨干细胞培养基成球培养法筛选结肠癌干细胞的相关蛋白表达及生物学特性。方法用干细胞培养基成球培养法筛选结肠癌干细胞,Western blot检测干细胞相关蛋白的表达,流式细胞仪检测细胞周期;同时检测干细胞增殖、黏附、耐药及侵袭能力,并摸索结肠癌干细胞最佳冻存条件。结果用干细胞培养基成球培养法成功培养出了结肠癌细胞球,检测发现干细胞相关蛋白表达增强,静止期细胞比例明显升高,细胞增殖速度慢,黏附性、耐药性、侵袭性均强于亲本细胞。结论干细胞培养基成球培养法筛选的细胞球中富集了肿瘤干细胞,为结肠癌干细胞的研究提供了良好的细胞模型。     

无血清培养胆囊癌GBC-SD细胞形成肿瘤细胞球的鉴定

目的:分离扩增肿瘤干细胞,并鉴定其生物学性质.方法:用无血清培齐基培养人胆囊癌GBC-SD细胞得到肿瘤细胞球.将肿瘤细胞球传代扩增,并用含血清培养基培养促使其分化;将肿瘤球和普通GBC-SD细胞分别种入96孔板,MTT检测增殖能力,并将肿瘤球和普通GBC-SD细胞分别植入裸鼠皮下,观察移植瘤的形成:流式细胞术检测CD15s和CD24在肿瘤球细胞和普通GBC-SD细胞中的表达,筛选细胞表面标志物.结果:在无血清培养基中,胆囊癌细胞可以形成少量的肿瘤细胞球,并显示很强的自我更新和增殖能力,含血清环境能够诱导其分化而贴壁生长;在动物实验中,肿瘤球细胞较普通GBC-SD细胞显示更强的致瘤能力(80.00%vs 10.00%,P<0.05);标志物CD15s在肿瘤球的表达较普通GBC-SD细胞明显增高(2.56%±0.38%vs 10.77%±0.93%,t=18.25,P<0.05).结论:人胆囊癌细胞GBC-SD的肿瘤干细胞可以通过无血清培养环境来分离和扩增,CD15s可能为其细胞表面标志物.     

胃癌MKN-28肿瘤细胞系SP细胞亚群初步分析

目的:分析胃癌细胞株MKN-28中是否包含肿瘤干细胞相关的SP(side population)细胞亚群.方法:采用免疫荧光方法检测干细胞标记ABcG2在胃癌细胞株MKN-28的表达;制备MKN-28细胞悬液,经Hoechst33342染色,流式细胞仪分析SP亚群.结果:ABCG2在胃癌细胞系MKN-28中有少量表达,ABCG2阳性细胞约占1.7%.SP亚群占MKN-28细胞的0.25%,维拉帕米阻断后减少为0.05%.结论:人胃癌细胞株MKN-28中存在SP细胞亚群,提示胃癌干细胞的存在.     

胰腺癌肿瘤干细胞的悬浮培养法

目的 建立无血清悬浮培养分离人胰腺癌细胞系PANC1干细胞球的方法.方法 采用无血清悬浮法培养PANC1细胞,显微镜下观察干细胞球形成率;流式细胞仪检测细胞CD133表达和细胞周期;以含10%FBS培养基诱导干细胞球细胞分化,荧光显微镜下观察细胞形态及CK18的表达;干细胞球细胞接种NOD/SCID小鼠皮下,观察其成瘤能力.结果 在无血清悬浮培养条件下存活的PANC1细胞形成干细胞球,体外连续传代培养20代始终保持4%0～5%0的干细胞球形成率.干细胞球细胞CD133表达率(5.91±0.7)%,G0/G1期细胞占(80.99±2.60)%,与原代PANC1细胞的(1.44±0.52)%和(69.01±5.03)%相差显著(P＜0.05).将于细胞球细胞置含血清培养基中培养.细胞逐渐恢复原代细胞形态,并表达上皮标志蛋白CK18,2×103的干细胞即在NOD/SCID小鼠皮下成瘤.结论 无血清悬浮法培养PANC1细胞可分离出肿瘤干细胞,其具有自我更新、多向分化和成瘤能力.     

侧群细胞与肝癌干细胞

肝癌是严重威胁人类生命和健康的一种疾病.其病因和发病机制尚不完全清楚,治疗缺少有效靶点.对肝癌恶性生长、转移及复发机制的研究正在逐渐深入.近年来的研究认为,肿瘤中存在一小群具有自我更新和分化潜能的细胞,即肿瘤干细胞,可能是肿瘤转移和复发的根源.肝癌中应同样存在这样的一群细胞.侧群(side population,SP)细胞是肿瘤细胞中一小部分,具备干细胞的多种特性且易于分离.肝癌组织中SP细胞的鉴定和分离有可能找到肝癌干细胞,有助于肝癌的转移和复发机制的研究,并为肝癌治疗提供有效治疗靶点.     

原代猪肝细胞无血清培养

目的研究原代猪肝细胞无血清培养及肝细胞的功能。方法采用EGTA和胶原酶P两步法经肝静脉逆行灌注分离乳猪肝细胞,在无血清培养基中培养,并对不同培养时间的肝细胞生化合成及生物转化功能进行检测。结果肝细胞产量为（1．5±0．1）×10^10／肝,活率为（90．3±1．5）％,接种培养后肝细胞增生旺盛,LDH漏出第2天达到高峰,然后逐渐下降并稳定于一定水平。随着时间的延长及肝细胞数量的增加,自蛋白合成及利多卡因转化率逐渐增加,呈时间及肝细胞数量的依赖关系。结论采用本法分离获取的猪肝细胞产率高、活性强、具有良好的生物合成及生物转化功能,可作为生物人工肝较理想的肝细胞来源。     

大肠癌细胞系Lovo中亚群(SP)细胞的分离培养和鉴定

目的:应用无血清培养液(SFM)培养Lovo细胞系,克隆分离具有干细胞样特性SP细胞.方法:应用SFM培养Lovo细胞,分离成球样生长的细胞群作为SP细胞,并通过有限稀释,分化,自我更新,含血清培养基(SSM)和SFM交替培养及Musashi-1化学染色方法来鉴定SP细胞.结果:Lovo细胞中约含有0.54%-0.62%的SP能够在无血清培养基中能够存活、增殖,形成悬浮的肿瘤细胞球;在SFM中加入血清可促使SP细胞分化;SP细胞可以连续传代,并可交替培养于SSM和SFM中,细胞形态无明显变化;SP细胞Musashi-1染色阳性.结论:应用SFM可从Lovo细胞中分离出极少量的具有干细胞特性的SP细胞.     

Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines

BACKGROUNDColon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers. It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-expressing stem cells, these so-called “tumour-initiating” cells, reminiscent in their properties of the normal intestinal stem cells (ISCs), may explain the apparent heterogeneity of colon cancer cell lines. Also, colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5. Furthermore, in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIMTo investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODSA portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines (Caco-2 and LoVo). These cells were then characterized according to their proliferation capacity, gene expression signatures of ISC markers, and their tumorigenic properties in vivo and in vitro.RESULTSThe data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2, but not from the LoVo, cell lines, in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog (+4 crypt) stem cells. This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny. Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein (EYFP) from this cell line exhibit tumorigenic-like properties in vivo and in vitro. In contrast, cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties. Thus, cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells, but not LoVo cells.CONCLUSIONOur findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines. Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data. We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.     

脂联素对结肠癌HT-29细胞的增殖及凋亡研究

目的探讨脂联素对结肠癌HT-29细胞的增殖、凋亡的影响。方法以不同浓度脂联素干预细胞,MTT法检测细胞增殖能力;AnnexinV／PI双染后流式细胞仪检测细胞凋亡。结果随着脂联素浓度的不断升高和培养时间的延长,HT-29细胞的生长逐渐受到抑制,脂联素可以诱导细胞凋亡。结论脂联素可抑制HT-29细胞增殖,诱导细胞凋亡。     

Preliminary study on mechanism of drug resistance in human colon cancer cell line HCTV2000

AIM: To investigate the mechanisms of multidrug resistance in human colon cancer cell line HCT_V2000. METHODS: P-glycoprotein (P-GP) was detected by immunocytochemical staining. Expression and amplification of mdr1 gene were detected by nucleic acid hybridization. Cytotoxicity was measured by MTT method. RESULTS: HCT_V2000 cells showed a positive response to monoclonal antibodies against P-GP encoded by the mdr1 gene. Overexpression of mdr1 mRNA was found, but no evidence of mdr1 gene amplification or rearrangement was suggested. Verapamil increased the accumulation of ³H-VCR inside the cells and partially reversed drug resistance to VCR. CONCLUSION: Overexpression of mdr1 mRNA is one of the important mechanisms of drug resistance in HCT_V2000 cell line; however, there may be some other mechanisms which are also involved in this.     

Liver cancer stem cell markers: Progression and therapeutic implications

Cancer stem cells(CSCs) are a small subpopulation in cancer, have been proposed to be cancer-initiating cells, and have been shown to be responsible for chemotherapy resistance and cancer recurrence. The identification of CSC subpopulations inside a tumor presents a new understanding of cancer development because it implies that tumors can only be eradicated by targeting CSCs. Although advances in liver cancer detection and treatment have increased the possibility of curing the disease at early stages, unfortunately, most patients will relapse and succumb to their disease. Strategies aimed at efficiently targeting liver CSCs are becoming important for monitoring the progress of liver cancer therapy and for evaluating new therapeutic approaches. Herein, we provide a critical discussion of biological markers described in the literature regarding liver cancer stem cells and the potential of these markers to serve as therapeutic targets.