氟尿嘧啶诱发人支气管损伤修复过程及支气管干细胞的定位

目的观察离体人支气管损伤修复过程,进行支气管干细胞的定位。方法取肺癌手术切除的人支气管的正常部分进行组织培养,应用氟尿嘧啶(5-FU)诱发支气管上皮损伤,动态观察修复过程,用免疫组化SP法检测PCNA,β1-整合素及CK-19的表达,同时进行Hoechst33342荧光染色。结果1．5-FU作用12h后人支气管上皮细胞绝大部分脱落,可见少量间隔分布的类似裸核的细胞呈钉状位于基底膜上,PCNA染色阴性,证明为G0期细胞。其中部分细胞Hoechst33342染色阴性。2．将5-FU去除3～6h后,细胞形态变为扁平,PCNA染色见核染色阳性的细胞与阴性细胞(Go期细胞)间隔分布;12h后细胞变为立方,细胞数目逐渐增多,到48～72h恢复假复层柱状上皮。3．β1-整合素及CK-19在已分化细胞中呈阳性反应。结论在5-FU的打击下,进入增殖期细胞死亡、脱落,仅余Go期细胞,其中含有支气管干细胞。正是这些干细胞增殖分化使支气管上皮修复。     

5-FU富集人白血病细胞系KG-1a中肿瘤干细胞样亚群细胞

探讨利用细胞周期特异性药物5-氟尿嘧啶(5-fluorouracil,5-FU)从人白血病细胞系KG-1a中富集肿瘤干细胞样亚群细胞.体外药物敏感试验确定5-Fu的最佳作用浓度和作用时间;KG-1a细胞经5-FU药物处理后,流式细胞术检测存活细胞群中CD34+CD38-亚群细胞比例;吖啶橙染色观察细胞内的核酸组成;RT-PCR半定量检测细胞内三磷酸腺苷结合转运蛋白G超家族成员2(ATP-binding cassette superfamily G member 2,ABCG2)mRNA的表达;半固体培养观察细胞的集落形成能力.结果显示,50 μg/ml 5-FU作用KG-1a细胞4天后,CD34+CD38-亚群细胞比例提高10倍以上;吖啶橙染色可见大部分细胞核酸以发出绿色荧光的DNA为主,RNA含量低;此类细胞高表达ABCG2 mRNA水平;而药物处理后细胞集落形成数量较未处理细胞明显减少.研究结果表明,利用5-FU能够杀死增殖期细胞的性质,成功建立体外药物筛选富集人白血病细胞系KG-1a中肿瘤干细胞样亚群细胞的方法.     

利用405nm激光激发Hoechst33342染色细胞DNA的流式细胞技术

目的:探讨流式细胞仪上405 nm激光激发Hoechst33342染色细胞DNA的效果及影响检测结果的因素。方法:SW480和A549两种细胞经Hoechst33342染色后,流式细胞仪405 nm激光激发检测DNA含量,利用软件计算出处于G0/G1期、S期和G2/M期细胞的百分比,以PI染色法结果作为对照。结果:SW480和A549细胞经Hoechst33342染色后各期的细胞百分比与PI染色法基本一致,无明显差异(P0.05)。结论:405 nm激光激发Hoechst33342染色细胞DNA结果可靠,可作为紫外检测的替代方法。     

香烟烟雾提取物对小鼠气管环上皮钙粘附素表达的影响

体外进行器官培养,通过免疫组织化学和原位杂交方法检测香烟烟雾提取物（CSE）作用48、72h,小鼠气管上皮细胞上皮钙粘附素（E－cd）表达的变化。结果表明,正常对照组E－cd分布于气管假复层纤毛柱状上皮细胞连接处的胞膜上,50％CSE作用后,膜上E－cd表达减少,随着作用时间延长,胞浆内E-cd表达明显增加（P＜0.05）,而细胞内E－cd mRNA含量无明显变化。提示,CSE对小鼠气管上皮细胞E-cd表达的调控在翻译后水平,胞膜上E－cd表达的下调与吸烟所致气道上皮细胞的损伤修复过程相关。     

2种气管黏膜上皮细胞体外培养技术的研究

目的为以猪气管黏膜上皮为细胞模型的研究奠定物质基础,进一步探讨猪气管黏膜上皮细胞的体外传统培养和气液界面培养技术,从而使2种培养技术优势互补。方法对猪气管上皮分离、纯化、培养和传代,并探索上皮细胞最佳冻存复苏条件;复苏后的气管上皮细胞进行气液界面培养,绘制细胞生长曲线和观察细胞纤毛生发情况。利用免疫组化法鉴定上皮细胞。结果4步纯化法可以得到高纯度的气管上皮细胞。使用胎牛血清、DMEM/F12培养液和DMSO的体积分数为50%、40%和10%的冻存体系保存的气管上皮,复苏后细胞存活率平均可达89%。优化后的传统方式培养的上皮细胞可连续传代到第8代,但从第2代开始便观察不到纤毛,转换成气液界面连续培养2代后重新生发纤毛,细胞存活期延长。免疫组化结果显示分离培养细胞为上皮细胞。结论成功建立了2种猪气管上皮细胞培养技术,并找到适宜的气管上皮细胞冻存条件,节省了不断原代取材的成本和时间,并成功实现传统培养细胞冻存复苏后很快适应气液界面的培养并恢复细胞的天然结构,为猪气管黏膜上皮相关研究提供丰富的细胞来源。     

紫花牡荆素诱导人肝癌HepG2细胞凋亡的研究

目的：研究紫花牡荆素（Casticin）对肝癌HepG2细胞增殖抑制和凋亡诱导的作用,并探讨其作用机制。方法：用终浓度为0、0.5、1.0、2.0umol/L的Casticin作用于HepG2细胞,于12、24、48h后采用MTT法检测细胞增殖抑制率;Hoechst33342核染色,观察细胞形态学变化;24h后收集各组肝癌HepG2细胞,流式细胞术检测细胞周期及凋亡率;RT-PCR检测survivin mRNA表达。结果：MTT法检测显示,Casticin对肝癌HepG2细胞有增殖抑制作用,并存在浓度和时间依赖关系;Hoechst33342染色后,可见核染色质凝集,凋亡细胞呈致密浓染,与对照组相比,Casticin处理后凋亡细胞比例增加;Casticin作用24h后,细胞被阻滞于G2/M期,随药物质量浓度的增加,细胞凋亡率逐渐增加;RT-PCR结果显示,Casticin下调肝癌HepG2细胞survivin mRNA表达。结论：Casticin在体外对肝癌HepG2细胞有明显的增殖抑制和凋亡诱导作用,初步推断Casticin诱发肝癌细胞凋亡与其对survivin基因表达的抑制有关。     

小鼠受精卵、卵裂球和桑椹胚细胞无SP表型

SP(side population)表型的概念在干细胞研究中用来表示某些细胞具有的将进入细胞的荧光染料如Hoechst 33342排出细胞的特性, 这种表型的形成与ABCG2蛋白有关. 近年研究报告某些干细胞及某些前体细胞具有SP表型, 并提出SP表型可能是干细胞或前体细胞的筛选标志. 在本研究中通过分离了小鼠受精卵、2细胞期及8细胞期卵裂球、桑椹胚和囊胚并直接进行Hoechst 33342染色, 结果发现小鼠受精卵、卵裂球、桑椹胚细胞都不具有SP表型, 但是处于其分化下游的囊胚中的内细胞团细胞却具有明显的SP表型, 而同一囊胚中的滋养层细胞却不具有SP表型. 该结果表明来源于内细胞团的胚胎干细胞具有的SP表型是内在的, 与体外培养条件无关; 另一方面该结果也提示SP表型至少是多能干细胞所具有的独特表型之一, 但并不存在于更早期的全能干细胞. 当在培养液中加入ABCG2蛋白抑制剂后直接导致囊胚中的内细胞团细胞SP表型消失, 提示囊胚内细胞团细胞SP表型的形成与ABCG2蛋白具有密切相关性. 以上结果表明并不是所有的干细胞都具有SP表型, SP表型可能可以作为某些种类的干细胞, 如胚胎干细胞及某些成体组织干细胞的分选标志, 但并不具有普遍性.     

大鼠气管上皮细胞发生过程中Ca~(2+)-ATPase活性变化的形态学观察

本文应用光镜和电镜组化方法,对胚胎晚期至生后早期的 Wistar 大鼠气管上皮细胞Ca~(2+)-ATPase 的动态变化进行了观察。结果证实在胚胎期气管上皮细胞侧面膜和表面膜及纤毛细胞的纤毛干处有 Ca~(2+)-ATPase 活性反应产物,提示胚胎期气管上皮细胞之间可能已存在与气管上皮细胞分化、发育密切相关的钙;在生后早期,纤毛细胞的基粒和纤毛小根处出现活性 Ca~(2+)-ATPase,表明生后早期已出现纤毛运动,其对廓清气道具有重要意义。     

解毒消癞饮逆转氟尿嘧啶诱导的肝癌干细胞耐药的机制研究

目的探讨解毒消瘾饮乙酸乙酯提取物（EE—JXY）能否降低肝癌细胞株Huh7对小剂量氟尿嘧啶（5-FU）化疗耐药的影响,并从肿瘤干细胞的角度探讨可能的机制。方法EE-JXY联合小剂量5-FU体外干预人肝癌细胞株Huh7。MTT法检测细胞存活率;流式细胞仪分析Huh7肝癌细胞株中侧群（sidepopulation,SP）细胞的比例;PCR检测肝癌干细胞相关基因ATP结合盒转运蛋白G2（ABCG2）和八聚体结合转录因子4（Oct4）mRNA的表达。采用单因素方差分析对数据进行统计。结果d,N量5-FU对Huh7细胞的增殖有抑制作用[24h存活率（84．54±2．88）％,96h存活率（58．36±3．52）％],但SP细胞的比例也从24h的4．72％升高至96h的12．07％,同时诱导了ABCG2和Oct4mRNA表达的上调。EE．JXY能够增强小剂量5-FU对Huh7细胞增殖的抑制能力[24h存活率从（84．54±2．88）％下降到（31．23±2．42）％,96h存活率从（58．36±3．52）％下降到（25．37±0．99）％],SP含量从24h的4．72％下降至1．69％,96h的12．07％下降至9．97％,同时也抑制了ABCG2和Oct4 mRNA的表达。结论肝癌Huh7细胞对小剂量5．FU存在化疗抵抗。EE—JXY能通过降低肝癌干细胞相关基因ABCG2 mRNA和Oct4 mRNA水平及SP的比例,增强Huh7细胞对小剂量5-FU的化疗敏感性,这可能是解毒消瘤饮降低肿瘤复发和转移的机制之一。     

当归多糖延缓5-氟尿嘧啶所致人骨髓基质细胞衰老

探讨5-氟尿嘧啶(5-fluorouracil,5-FU)对人骨髓基质细胞(human bone marrow stromal cell,h BMSC)的损伤作用机制;当归多糖(angelica sinensis polysaccharides,ASP)对5-氟尿嘧啶损伤h BMSC的保护作用。采用CCK-8法测定人骨髓基质细胞株HS-5对不同浓度5-FU(0μg/mL,12.5μg/mL,25μg/mL,50μg/mL和100μg/mL)的敏感性。流式细胞术分析细胞周期;β-半乳糖苷酶染色检测衰老细胞;DCFH-DA荧光染色流式检测胞内活性氧(reactive oxygen species,ROS)水平;酶学法检测谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)含量;流式细胞术分析γH2AX表达水平;ELISA检测8-羟基脱氧鸟苷(8-hydroxydeoxyguanosine,8-OHd G)的含量;5-FU 12.5~100μg/mL抑制HS-5增殖,抑制作用具有浓度依赖性和时间依赖性。5-FU组较与对照组相比细胞周期发生G1期阻滞,β-半乳糖苷酶染色阳性率升高;胞内ROS含量显著升高;细胞抗氧化能力降低;γH2AX和8-OHd G表达水平增高。ASP治疗组较5-FU组细胞相比细胞周期阻滞减少;衰老细胞比率降低;胞内ROS含量显著降低;细胞抗氧化能力升高;DNA损伤指标表达水平降低。5-FU可通过增强氧化应激诱发DNA损伤致骨髓基质细胞衰老;ASP对骨髓基质细胞损伤有保护作用,其机制可能与ASP降低损伤后骨髓基质细胞氧化应激减轻DNA损伤从而延缓骨髓基质细胞衰老有关。     

The Recovery Process of Mice Tracheal Epithelium Injured by 5-FU and Its Microarray Analysis

Although the research on the localization of trachea stem cells has made a rapid progress, the mechanism of proliferation and differentiation of trachea stem cells remains unclear. The objective of this study is to observe and analyze the recovery process of mice tracheal epithelium injured by 5-FU, and to investigate the mechanism involved in the regulation of tracheal stem cells proliferation and differentiation through morphological, immunofluorescence, and microarray analysis. After treatment with 5-FU, the mature cells were dead and desquamated. Only a few G₀ phase cells remained on the basement membrane. When supplied with normal culture media, the cells eventually became flat, cubic, and restored as pseudostratified epithelium. These G₀ phase cells were ABCG2 positive. It suggested that these cells could differentiate into cilia cells or Clara cells, and had the multi-differentiation ability of stem cells. We examinated the expression profile of genes involved in the stem cell differentiation in normal tracheal epithelial cells and the regenerated epithelial cells at 24 and 48 h after injured by 5-FU using gene microarray. After 24 h treatment, 8 genes were up-regulated and 31 genes were down-regulated. After 48 h treatment, 5 genes were up-regulated and 42 genes were down-regulated. The differential gene expressions in gene microarray analysis focused on cell cycle regulation, intercellular junction, fibroblast growth factors, bone morphogenetic protein, Notch and Wnt-signaling pathways, which suggested that the differential gene expressions might be closely associated with the proliferation and differentiation of tracheal stem cells.     

Molecular and functional properties of lung SP cells

Previous analysis of lung injury and repair has provided evidence for region-specific stem cells that maintain proximal and distal epithelial compartments. However, redundant expression of lineage markers by cells at several levels of the stem cell hierarchy has complicated phenotypic and functional characterization of clonogenic airway cells. Based on the demonstration that rapid efflux of the DNA dye Hoechst 33342 can be used to prospectively purify long-term repopulating hematopoietic stem cells, we hypothesized that lung cells with similar biochemical properties would be enriched for clonogenic progenitors. We demonstrate that Hoechst-dim side population (SP) cells isolated from proximal and distal compartments of the mouse lung were relatively small and agranular, exhibited low red and green autofluorescence, and that the SP fraction was highly enriched in clonogenic cells. Quantitative RT-PCR indicated that vimentin mRNA was enriched and that epithelial markers were depleted in these preparations of SP cells. Bleomycin exposure was associated with decreased clonogenicity among alveolar SP and suggested that SP cell function was compromised under profibrotic conditions. We conclude that the SP phenotype is common to clonogenic cells at multiple airway locations and suggest that Hoechst efflux is a property of cells expressing a wound-repair phenotype.     

Presence of side-population cells in an immortalized nontumorigenic human liver epithelial cell line

Side-population (SP) cells have been shown to be highly enriched stem cells. We investigated whether an immortalized, nontumorigenic human liver cell line, THLE-5b, contains SP cells. Flow cytometry analysis after Hoechst 33342 staining demonstrated that the THLE-5b line contained a small component of SP cells. These SP cells were essentially eliminated by treatment with verapamil and expressed higher levels of ABCG2 mRNA than non-SP cells. In addition, the level of these SP cells detected by Hoechst 33342 staining was affected by the experimental conditions including the incubation medium. This is the first report of the presence of SP cells in the immortalized, nontumorigenic human liver cell line.     

Akt signaling regulates side population cell phenotype via Bcrp1 translocation

Akt is an important regulator of cell survival, growth, and glucose metabolism in many cell types, but the role of this signaling molecule in hematopoietic stem cells is poorly defined. Side population (SP) cells are enriched for hematopoietic stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342. Bone marrow from Akt1-null mice exhibited a reduced SP fraction. However, bone marrow cellularity, growth factor-responsive progenitor cultures, and engraftable stem cells were normal in these mice. Treatment of bone marrow with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol 3-kinase, led to a reversible loss of the SP fraction. Bcrp1, which encodes the Hoechst dye transporter, was translocated from the membrane to the intracellular compartment under conditions that promote the SP-depleted state. Lentivirus-mediated overexpression of Akt1 in bone marrow markedly increased the SP fraction, whereas there was no effect on bone marrow from Bcrp(-/-) mice. These data suggest that Akt signaling modulates the SP cell phenotype by regulating the expression of Bcrp1.     

Expression and role of Oct3/4, Nanog and Sox2 in regeneration of rat tracheal epithelium

Objectives:  To explore the role of Oct3/4, Nanog and Sox2 in regeneration of rat tracheal epithelium.
Materials and methods:  An ex vivo model of rat tracheal epithelial regeneration using 5-fluorouracil (5-FU) was developed, to induce injury. Expression levels of Oct3/4, Nanog and Sox2 were examined using Western blot analysis, RT-PCR or microscopically observed immunofluorescence, and cell morphological changes were observed using HE staining, during the recovery process.
Results:  Oct3/4, Nanog and Sox2 were not detectable in normal tracheal epithelium. After treatment with 5-FU, the normally proliferating tracheal epithelium desquamated and only a few cells in G0 phase of the cell cycle were left on the basement membrane and Oct3/4, Nanog and Sox2 could be observed at this time. Thereafter, the number of Oct3/4-, Nanog- and Sox2-positive cells increased gradually. When the cells differentiated into ciliate cells, mucous cells or basal cells, and restored pseudostratified mucociliary epithelium, the number of Oct3/4-, Nanog- and Sox2-positive cells decreased and gradually disappeared.
Conclusions:  G0 phase cells with resistance to 5-FU damage expressed Oct3/4, Nanog and Sox2. This indicated that these cells were undifferentiated, but had the ability to terminally differentiate into downstream-type cells. They possessed stem cell properties. The results are consistent with Oct3/4, Nanog and Sox2-expressing cells being considered as tracheal stem cells.     

Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma cell line

“Side population” (SP) cells, which pump out the fluorescent dye H33342 via the ABCG2 transporter, define a putative stem/progenitor cell population in the mammary gland. Breast cancer SP cells recently isolated from the MCF-7 cell line possess similar properties and may represent stem cell-like cancer cells. This study extends SP cell analysis to a broad panel of human breast cancer cell lines and investigates the expression of differentiation-associated markers in isolated cancer SP cells. Expression of ABCG2 was determined in 16 breast cancer cell lines by quantitative RT-PCR, Western blotting and immunohistochemistry. Subsequently, all cell lines were screened for the presence of SP cells. Human breast cancer cell lines commonly express ABCG2. ABCG2-immunoreactivity was clearly restricted to rare cancer cells in several cell lines including Cal-51. Analysis of H33342-labeled Cal-51 cells revealed a small fraction of putative SP cells accounting for one percent of all cells. The genuine nature of Cal-51 SP cells was unambiguously verified by demonstrating a 30-fold increased ABCG2-expression in isolated Cal-51 SP cells. During in vitro expansion, Cal-51 SP cells generated heterologous non-SP (NSP) cells and ABCG2-expression declined dramatically. In contrast, NSP cells failed to sustain proliferation. Freshly isolated Cal-51 SP cells also exhibited increased expression of Muc1 and CALLA. Noteworthy, non-malignant mammary epithelial SP cells lack these differentiation markers, highlighting fundamental differences between non-malignant and breast cancer-derived SP cells. In summary, we established Cal-51 SP cells as a novel in vitro model to study differential gene expression in breast cancer-derived SP and NSP cells.     

ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate

The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the “side population” (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145, and RWPE-1, but not in the BPH-1, LAPC-4, or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) human benign prostate tissue, and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population.     

The presence of a side population and its marker ABCG2 in human deciduous dental pulp cells

Stem cells have been identified using the DNA-binding dye Hoechst 33342 and flow cytometry (FCM) in various tissues known as the side population (SP). The present study shows, for the first time, the presence of side population cells in human deciduous dental pulp cells (DPCs). Flow cytometric identification revealed that 2% of human deciduous DPCs were SP cells and that this SP profile disappeared in the presence of verapamil. The SP marker ABCG2 protein was localized to DPCs in the cell membrane by immunofluorescence staining, and flow cytometric analysis demonstrated that 3.6% of DPCs were ABCG2-positive. Furthermore, quantitative real-time PCR proved that ABCG2 mRNA expression in DPCs isolated from human exfoliated deciduous teeth was higher than in DPCs from permanent teeth. Our findings demonstrate that DPCs from human exfoliated deciduous teeth contain a higher proportion of the SP phenotype than permanent teeth and that they may constitute a stem cell population.