过表达Bmi-1促进CD133~+CD44~+HCT116结肠癌干细胞发生上皮间质转化

目的探讨Bmi-1促进结肠癌干细胞发生迁移、侵袭的作用及机制。方法常规培养结肠癌HCT116细胞,成球培养基法(SFM)和磁珠分选法(MACS)富集和筛选CD133~+CD44~+HCT116结肠癌干细胞;流式细胞学鉴定结肠癌干细胞CD133和CD44表型,体外平板克隆实验和CCK8法鉴定细胞增殖克隆能力,裸鼠体内成瘤实验鉴定细胞致瘤能力。构建过表达Bmi-1质粒并转染CD133~+CD44~+HCT116结肠癌干细胞,Western blotting验证细胞中Bmi-1、E-cadherin及Vimentin的表达情况。平板划痕实验和Transwell实验评价转染后不同细胞的迁移和侵袭能力。结果 CD133~+CD44~+HCT116结肠癌细胞具有更强的体外克隆增殖能力和体内致瘤能力,可作为结肠癌干细胞研究模型;过表达Bmi-1后促进CD133~+CD44~+HCT116结肠癌干细胞的E-cadherin表达下降,而Vimentin表达上升;过表达Bmi-1后细胞24 h迁移速度为(18.44±0.59)μm/h,明显高于对照组(1.88±0.21)μm/h和普通组(1.92±0.36)μm/h(P0.05),同时其侵袭细胞数(37.67±2.51)也明显高于对照组(9.33±0.58)和普通组(7.67±0.58)(P0.05)。结论 Bmi-1介导CD133~+CD44~+HCT116结肠癌干细胞发生上皮间质转化(EMT)而促进肿瘤侵袭、转移。     

肝癌MHCC97H细胞中CD133~+和CD133~-亚群分选及其生物学特性

目的分选肝癌MHCC97H细胞中的CD133+和CD133-细胞亚群,并初步探讨CD133+细胞亚群的干细胞特性。方法用磁珠分选技术分选MHCC97H细胞株中的CD133+和CD133-细胞亚群,用流式细胞仪检测分选前后CD133的表达,用MTT法检测其增殖能力,比较其体内成瘤能力,用Western印迹法检测其干细胞相关基因蛋白的表达。结果分选前后MHCC97H细胞中CD133表达分别为(1.09±0.43)vs(86.65±6.49)%(P<0.01)。与CD133-亚群相比,CD133+亚群的体外增殖能力较强,成瘤能力高,高表达干细胞相关基因蛋白(P<0.05,P<0.01)。结论肝癌细胞株MHCC97H中的CD133+细胞亚群具有肿瘤干细胞的特性;CD133是人肝癌MHCC97H细胞株的肿瘤干细胞的表型之一。     

卵巢癌CD90^＋肿瘤细胞的分离及其肿瘤干细胞生物学特性观察

目的：分离人卵巢癌细胞系SKOV3和原代卵巢癌细胞中的CD90^＋细胞,并观察其肿瘤干细胞的生物学特性。方法从卵巢癌患者腹水中分离原代卵巢癌细胞,采用流式细胞术检测人卵巢癌细胞系SKOV3和原代卵巢癌细胞的CD133、CD90阳性率。流式分选得到CD90^＋、CD90^-细胞后,采用RT-PCR法检测其干细胞及上皮间质化（EMT）相关基因mRNA相对表达,Transwell小室侵袭试验观察细胞侵袭力,克隆形成试验观察细胞增殖分化能力,悬浮成球试验观察干细胞潜能,免疫缺陷小鼠体内有限稀释成瘤试验观察成瘤时间和成瘤率。结果人卵巢癌细胞系SKOV3的CD133和CD90阳性率均低于原代卵巢癌细胞,P均＜0．05。在人卵巢癌细胞系SKOV3和原代卵巢癌细胞中,CD90^＋细胞干细胞相关基因（CD133、OCT4）和EMT间质标志相关基因（N-cadherin、Vimentine、MMP9）相对表达、穿到膜背面的细胞数、细胞克隆数、悬浮成球数均高于CD90^-细胞,EMT上皮标志相关基因E-cadherin相对表达均低于CD90^-细胞,P均＜0．05。随着接种细胞数目的增加,CD90^＋、CD90^-细胞的成瘤率和成瘤时间均升高,CD90^＋细胞升高更明显。结论卵巢癌细胞中,CD90^＋细胞高表达间质属性基因和干细胞相关基因,具备更高的侵袭力、增殖分化能力、体内成瘤能力和干细胞潜能,CD90^＋细胞分离可能成为卵巢癌干细胞分离的新方法。     

p75NTR阳性人食管鳞癌细胞的干细胞特性研究

目的探讨p75NTR阳性人食管鳞癌细胞的干细胞特性。方法取15例新鲜人食管鳞癌组织制成单细胞悬液,分别获取p75NTR阳性和p75NTR阴性细胞,进行Transwell侵袭试验、耐化疗药物试验及裸鼠成瘤试验。结果15例新鲜人食管鳞癌组织中有7例发现p75NTR表达,其阳性表达率最高为10.37%,p75NTR阳性和p75NTR阴性细胞在侵袭性、对化疗药物的耐受能力及致瘤能力上差异有统计学意义。结论 p75NTR阳性较p75NTR阴性食管鳞癌细胞具有更强的侵袭性、耐化疗能力及致瘤能力,可能富集人食管鳞癌干细胞。     

E1A基因对人肺腺癌细胞增殖和细胞周期的影响

目的 探讨E1A基因对人肺腺癌细胞增殖的抑制作用与细胞周期的关系及作用机制。方法 利用细胞增殖曲线和裸小鼠体内致瘤性实验研究E1A基因对人肺腺癌细胞增殖的影响,采用流式细胞术分析人肺腺癌细胞周期,以蛋白印迹方法分析P16、P21、P53和cyclin B1水平变化。结果 转染E1A基因后,人肺腺癌细胞（Anip973-E1A）生长缓慢,体内致瘤性降低。Anip973-E1A细胞周期出现明显的S期抑制和G2/M阻滞,周期调控蛋白P16、P21、P53水平无明显变化,但cyclin B1表达明显下降。结论 E1A基因能显著抑制人肺腺癌细胞的体内外增殖,降低周期蛋白cyclin B1的表达,使细胞周期阻滞于G2/M,这可能与A1A基因抑制作用有关。     

CD133在人肺腺癌组织中的表达及其临床关系

目的研究CD133在肺腺癌中的表达,探讨CD133表达与患者临床信息的相关性。方法取83例肺腺癌组织石蜡标本,用免疫组化法检测CD133的表达,结合患者年龄、性别、吸烟指数等信息进行统计分析,探讨CD133的表达与患者相关资料的关系。结果 83例中CD133阳性率为81.9%,同时具有表达的不均一性。结论 CD133在肺腺癌中广泛表达,表达强度具有不均一性,其表达与患者淋巴转移及病理分化程度相关,与年龄、性别、吸烟指数未见相关性。     

CD98对肝癌细胞侵袭和转移的影响

目的本研究拟通过RNA干扰技术下调人类肝癌细胞(hepatocellular carcinoma,HCC)CD98基因的表达,探讨CD98对肿瘤侵袭与转移的影响.方法利用CD98 siRNAs瞬时转染人类HCC系(FHCC-98),免疫印迹法证实癌细胞中CD98蛋白表达下调后,通过细胞黏附实验、Transwell侵袭实验及划痕愈合实验,检测CD98对HCC的黏附、侵袭和迁移能力的影响.结果 CD98 siRNAs瞬时转染FHCC-98细胞48 h后,癌细胞CD98的蛋白水平显著下调,干扰CD98蛋白表达水平后,癌细胞黏附能力显著下降,Transwell实验证实与对照组相比,癌细胞的侵袭能力显著下降,划痕实验也证实干涉CD98后癌细胞的迁移能力显著下降.结论 CD98通过增强HCC的黏附及迁徙能力,促进HCC的侵袭和转移,为未来肝细胞癌治疗提供了可能的靶点.     

CD133在83例人肺腺癌细胞中的表达研究

目的:研究CD133在人肺腺癌中的表达,观察其表达情况及其临床意义。方法:取83例人肺腺癌石蜡标本,用免疫组化法检测CD133的表达,结合患者性别、淋巴转移等信息进行统计分析,研究CD133的表达与患者相关资料的关系。结果:83例患者中CD133阳性率为81.9%,同时具有表达的不均一性,阳性表达者其表达强度也不同。其表达与患者淋巴转移(χ2=14.01,P<0.01)、病理分化程度(χ2=9.67,P<0.01)及5年存活率(χ2=3.95,P<0.05)相关,与性别(χ2=2.70,P>0.05)、吸烟指数(χ2=0.09,P>0.05)未见相关性。结论:CD133在肺腺癌中的表达具有普遍性和不均一性,高表达患者淋巴转移率高,病理分化差,5年存活率低。CD133的表达对判断肺腺癌临床预后有指导作用。     

血管内皮生长因子C反义脱氧寡核苷酸对肺癌细胞的作用

目的 探讨非小细胞肺癌(NSCC)中血管内皮生长因子C(VEGF-C)信使核糖核酸(mRNA)的表达及其反义脱氧寡核苷酸(antisense oligodeoxyribonucleotide,ASODN)对人肺癌细胞的作用.方法 应用原位杂交技术检测48例新鲜肺癌标本中VEGF-C mRNA的表达,将脂质体介导的VEGF-C ASODN转染人肺癌细胞株A-549,用Western印迹法检测肺癌细胞VEGF-C蛋白的表达.结果 VEGF-C mRNA在肺鳞癌和腺癌细胞中的表达阳性率分别为65.4%(17/26)和59.1%(13/22),经VEGF-C ASODN转染的细胞株A-549中,VEGF-C蛋白表达水平明显下降(P＜0.01).结论 VEGF-C mRNA在NSCC中有一定程度的表达,体外实验VEGF-C ASODN通过下调VEGF-C蛋白的表达抑制肺癌细胞的侵袭作用.     

CD68+巨噬细胞表达与肺腺癌侵袭性及临床病理分期相关性分析

目的通过肺腺癌肿瘤实质中CD68~+巨噬细胞浸润情况的分析,及肿瘤实质中Ki67指数表达情况,探讨肿瘤相关巨噬细胞(TAMs)与肺腺癌临床病理分期及肿瘤侵袭能力相关性。方法选取南京市胸科医院2017年间130例肺腺癌组织切片,应用免疫组织化学技术观察并计数CD68~+巨噬细胞在肺腺癌肿瘤实质及间质浸润情况及Ki67指数表达情况分析其相关性。结果 (1)肺腺癌肿瘤实质内TAMs表达低于肿瘤癌旁间质内TAMs表达(P0.05)。(2)肺腺癌肿瘤实质及间质内TAMs表达与Ki67表达有相关性(P=0.0416)。(3)Ⅲ～Ⅳ期NSCLC肿瘤实质组织中CD68~+阳性TAMs表达显著高于Ⅰ～Ⅱ期,差异有统计学意义(P0.05)。结论结果显示CD68~+巨噬细胞浸润情况与肿瘤TNM分期、及Ki67表达有一定相关性。肿瘤相关巨噬细胞(TAMs)浸润肿瘤实质及间质导致肺内炎症微环境,与肿瘤侵袭能力正相关。     

不同诱导因子下内皮祖细胞具有双向分化的潜能

目的探讨内皮祖细胞(EPCs)在不同诱导因子作用下的体外分化潜能。方法人脐血单个核细胞分别予50ng/ml血管内皮生长因子(VEGF组)或50ng/ml血小板源生长因子(PDGF组)诱导分化。光镜形态观察,免疫荧光鉴定。流式细胞分析CD133+EPCs分化特征。结果新鲜脐血分离单个核细胞培养1周后贴壁细胞的EPCs特异的DiI标记乙酰化低密度脂蛋白鉴定为80%阳性。在VEGF或PDGF诱导下1周,单个核细胞大量贴壁生长多呈圆形,少量梭形生长。2周时,被诱导细胞有近50%贴壁呈梭形生长,VEGF组和PDGF组无明显差异。至4周时两组出现明显的分化差异,其中VEGF组呈"铺路石"样细胞融合,血管性假血友病因子免疫荧光呈阳性;而PDGF组呈梭形或长多角形融合,予α-平滑肌肌动蛋白标记部分呈阳性。单个核细胞磁珠分选后得到CD133+较CD133-更多分化为内皮样细胞(P<0.05);而在PDGF的诱导下CD133-较CD133+更多分化为平滑肌样细胞(P<0.05)。结论单个核细胞在体外不同的诱导因子诱导下可以双向分化,CD133+EPCs具有更强的内皮细胞分化潜能。     

Foxp3调节的CD_4~+CD_(25)~+Treg细胞计数与肺腺癌患者分期之间的关系

目的分析Foxp3调节的CD_C~+CD_(25)~+Treg细胞计数与肺腺癌患者分期之间的关系。方法流式细胞法检测临床各期肺腺癌患者和健康人群血清中表达Foxp3的CD_4~+CD_(25)~+Treg细胞计数,并观察化疗及手术对肺腺癌患者血清中Foxp3水平的影响。结果肺腺癌各期患者血清中CD_4~+CD_(25)~+Treg细胞计数均明显高于健康人群,而且同样与肿瘤分期呈正相关,经手术或化疗干预后患者外周血清中该细胞计数下降明显(P0.05)。结论 Foxp3调控的CD_4~+CD_(25)~+Treg细胞在肺腺癌的发生发展中可能起着重要的作用,对其深入研究将有助于肺腺癌的早期诊断,并为肺腺癌治疗提供新的靶点。     

Generation of effector cells from primary lung cancer patients by stimulation with anti-CD3 monoclonal antibody followed by culture with recombinant interleukin-2]

Peripheral blood mononuclear cells obtained from 24 primary lung cancer patients were stimulated with anti-CD3 monoclonal antibody (alpha CD3MoAb) followed by culture with recombinant interleukin-2. The optimal concentration of alpha CD3MoAb for stimulation was 50 ng/ml in the liquid phase, and the sensitization culture was commenced at a cellular concentration of 1 x 10(6)/ml. Patients entered into this study were 14 cases of adenoca rcinoma, 7 of squamous cell carcinoma, and 3 of small cell carcinoma. After 4-6 days of stimulation with alpha CD3MoAb followed by culture with RIL-2 for 5-7 days, the cellular expansion was 3.7 folds (mean). Surface marker analysis of the cells revealed significant increments of CD3+, CD8+, HLA-DR+, and IL-2R+ cells after sensitization culture. In 2 cases, fresh autologous tumor cells could be obtained from surgical specimens. Effector cells generated in those 2 cases did not show significant cytotoxic activity against autologous tumor cells in 4 hr 51Cr release assay. In 5 cases, cytotoxicity against established lung cancer cell lines, STC-1 and L0301, were analyzed. In all cases, effector cells showed significant cytolytic activity against both targets. The sensitization culture utilizing alpha CD3MoAb was easy to perform and feasible for the majority of patients, and it is considered that utilization of this culture system would be worth while for adoptive immunotherapy in primary lung cancer patients.     

Identification of CD133+ cells in pituitary adenomas

Stem-like cells in tumors are capable of self-renewal and pluri-differentiation; they are thought to play important roles in tumor initiation and maintenance. Stem-like cells in malignant glioma express CD133. We examined samples from human pituitary adenoma, a generally benign neoplasm, for CD133 expression using routine immunohistochemical and biochemical methods. Our study of 70 pituitary adenomas (clinically nonfunctioning adenomas and growth hormone-, prolactin-, adrenocorticotropic hormone-, and thyroid-stimulating hormone-producing adenomas) showed that 18 (25.7%) expressed CD133. This rate was higher in clinically nonfunctioning (33.3%) than functioning adenomas (12.0%) (p = 0.085). Real-time PCR assay revealed the expression of CD133 mRNA in samples immunohistochemically positive for CD133. Neither the patient age and gender, nor the tumor size or postoperative recurrence rate correlated with CD133 positivity. CD133+ cells ubiquitously coexpressed CD34, nestin, and VEGFR2 (KDL1). S-100 and GFAP were not coexpressed with CD133. Chromogranin A, Pit-1, SF-1, and NeuroD1 were immune-negative, indicating that CD133+ cells did not have the potential to differentiate into functional endocrine cells. Our data suggest that the expression of CD133 in pituitary adenomas is related to immature endothelial progenitor cells that may play a role in the neovascularization of pituitary adenomas. Further studies are needed to elucidate the significance of CD133+ cells with respect to neovascularization and their sustainable growth in pituitary adenomas.     

Strong expression of CD133 is associated with increased cholangiocarcinoma progression

AIM:To determine the role of CD133 in cholangiocarcinoma progression. METHODS:CD133 protein expression was evaluated by immunohistochemistry in 34 cholangiocarcinoma specimens.In addition,proliferation,chemoresistance and invasive properties of CD133-enriched(CD133 + ) and CD133-depleted(CD133 )RMCCA1 cholangiocarcinoma cells were studied and compared. RESULTS:Strong CD133 expression was observed in 67.6%(23/34)of the cholangiocarcinoma specimens. Strong expression of CD133 was significantly associated with...     

Interleukin-13 and -4 expression in the central airways of smokers with chronic bronchitis.

The aim of this study was to determine whether the T-helper 2-type cytokines interleukin (IL)-13 and -4 are involved in mucus hypersecretion, the hallmark of chronic bronchitis (CB). Surgical specimens were examined from 33 subjects undergoing lung resection for localised peripheral malignant pulmonary lesions: 21 smokers with symptoms of CB, 10 asymptomatic smokers (AS) and two nonsmokers with normal lung function. The number of IL-4 and -13 positive (+) cells in the central airways was quantified. To better assess the cytokine profile, a count was also made of IL-5+ and interferon (IFN)-gamma+ cells. Compared to AS, the CB group had an increased number of IL-13+ and -4+ cells in the bronchial submucosa, while the number of IL-5+ and IFN-gamma+ cells were similar in all the groups. No significant associations were found between the number of cells expressing IL-13 or -4 and the number of inflammatory cells. Double labelling showed that 13.2 and 12.9% of IL-13+ cells were also CD8+ and CD4+, whereas 7.5 and 5% of IL-4+ cells were CD8+ and CD4+, respectively. In conclusion, T-helper-2 and -1 protein expression is present in the central airways of smokers and interleukin-4 and -13 could contribute to mucus hypersecretion in chronic bronchitis.     

Viable CD34+/CD133+ blood progenitor cell dose as a predictor of haematopoietic engraftment in multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation

Both CD34 (cluster of differentiation 34) and the more recently described CD133 are markers of primitive stem cells with haematopoietic repopulating ability. Most transplanting centres use a minimum number of CD34+ cells as the requirement for a transplant and consider this a predictor of haematopoietic engraftment. However, transplanted CD34+ cell dose does not always give a close correlation with time to engraftment nor explain delayed engraftment in some patients. We have retrospectively evaluated the potential of measuring viable CD133+ cell numbers in the autograft as an alternative predictor of haematological engraftment after autologous stem-cell transplantation in a cohort of patients with multiple myeloma (MM). We found an average 32% loss of viability of CD34+ cells in the post-thaw sample compared with the fresh sample. Of the original estimated CD34+ cell numbers transplanted per kg, 43% of the thawed samples were double positive for CD34+/CD133+. In this patient group, the CD34+/CD133+ subset gave the closest statistical correlation with time to neutrophil engraftment (p < 0.05), particularly for patients given above median (1.8 × 10⁶/kg) dose of the double-positive cells. The CD34+/CD133+ population was the only parameter to give a significant correlation with white cell engraftment in this patient cohort (p < 0.05). There was no significant correlation between CD34+, viable CD34+ or viable CD34+/CD133+ cells/kilogram with platelet engraftment. Determination of viable CD34+/CD133+ progenitor cell dose in the autograft may be a useful tool to predict neutrophil recovery after autologous transplantation than conventional assessment of CD34+ numbers. These results warrant further investigation of the role of CD133 in haematopoietic engraftment.     

Upregulated CD133 expression in tumorigenesis of colon cancer cells

AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging dro...