目的利用无血清条件悬浮培养人大细胞肺癌H460系,从中分离到肺癌干细胞并进行其生物学特性研究。方法无血清条件培养法悬浮培养H460细胞形成悬浮细胞球,通过qPCR、Western blot、流式细胞术、平皿克隆形成等实验,比较H460细胞和H460细胞球中干性相关分子表达水平及细胞增殖能力强弱,并利用裸鼠移植瘤形成实验研究肺癌细胞干细胞球的体内生物学特性。结果利用无血清条件培养法成功从H460细胞中分离出悬浮生长的肿瘤干细胞样细胞,其增殖能力强于H460贴壁细胞(P0.05),干细胞相关转录因子Sox2、Oct4和Nanog表达在mRNA及蛋白水平均有增加(尤以Nanog提升水平明显(P0.01)),细胞球在裸鼠体内具有较强成瘤能力。结论无血清条件培养法可以有效富集到H460细胞系中的肺癌干细胞,Nanog可能与H460细胞系中CSLCs的干性特征维持相关。

Objective To isolate lung cancer stem-like cells (LCSCs) from human large-cell lung cancer cell line NCI-H460 (H460) and explore their biological characteristics. Methods H460 cells were cultured in serum-free medium in the presence of specific growth factors. Quantitative PCR (qPCR), flow cytometry and colony formation assay were performed to characterize the stemness of H460 spheres. Adherent H460 cells and H460 cell spheres were inoculated subcutaneously in nude mice and the tumor growth was assessed. Results The isolated LCSCs from H460 cells in serum-free medium grew as floating cell spheres and exhibited stronger proliferative activity than H460 cells. Compared with H460 cells, H460 cells spheres showed higher expressions of stem cell markers Sox2, Oct4, and especially Nanog, and possessed a stronger tumorgenicity in nude mice. Conclusion The serum-free culture system can effectively enrich lung cancer stem cells from human lung cancer stem cell line H460, and the high expression

目的采用血清饥饿法诱导K562细胞凋亡,比较K562细胞与人白血病骨髓间充质干细胞(LMSC)共培养前后,K562细胞凋亡的变化,并比较在常氧和低氧条件下,K562细胞凋亡情况的变化。方法通过在细胞培养体系中加入150μmol/L氯化钴(CoCl2)模拟低氧环境。采用Annexin V/碘化丙啶(PI)荧光标记法检测不同氧条件下,血清饥饿后及与LMSC共培养后K562细胞的凋亡情况。结果在单独悬浮培养组,10%胎牛血清(FBS)培养条件时,K562细胞凋亡率为(5.00±0.04)%,FBS饥饿培养(无FBS)24h后,K562细胞凋亡率为(11.40±0.63)%,较10%FBS培养明显升高(P0.05)。常氧条件下与LMSC共培养后,K562细胞凋亡率为(7.43±0.86)%,细胞凋亡率较血清饥饿培养下降(P0.05),在150μmol/L CoCl2存在时,细胞凋亡率下降更明显(5.91±0.35)%(P0.05)。结论①LMSC能够抑制血清饥饿诱导的K562细胞凋亡。②在CoCl2模拟的低氧条件下,LMSC抵抗血清饥饿诱导的K562细胞凋亡的能力增强。

Objective To shed light on the effects of human leukemic bone marrow mesenchymal stem cells (LMSC) on the pathogenesis and prognosis of leukemia ,we compare the apoptosis of K562 cells that cultivated with or without LMSC under normoxia or hypoxia .Methods The hypoxia environment was achieved by treating cells with 150 μmol/L cobalt chloride(CoCl2 ) .Annexin V/PI binding assay was applied to investigate apoptosis of K562 cells cultured in FBS-starvation and with LMSC .Results Apoptosis of K562 cells was significantly in-creased while treated with 0% FBS[(11.40 ± 0.63)% ] than that treated with 10% FBS[(5.00 ± 0.04)% ] ,but it was redecreased when they were exposed to LMSC either in normoxia [(7.43 ± 0.86 )% ] or hypoxia [(5.91 ± 0.35)% ] condition .Conclusion ① LMSC can resist FBS-starvation-induced apoptosis of K562 cells . ② The effects of LMSC on K562 cells is enhanced under hypoxia condition .

目的 建立牛血清中牛病毒直接免疫荧光检测方法,并进行验证.方法 建立直接免疫荧光法检测牛血清中牛病毒,对细胞接种浓度和血清浓度、病毒接种量、荧光抗体浓度、染色温度及时间进行优化;对优化的方法进行重复性、特异性、灵敏度验证,并与细胞培养法的检测结果进行比较.结果 优化的试验条件为:Vero和BT细胞的接种浓度分别为0.5×105和1.0× 105个/ml,血清浓度为5％,病毒接种量为100～300 CCID50,荧光抗体1∶10稀释,4℃染色12h以上,但不超过24 h.2名实验人员按建立的方对3批牛血清分别进行3次检测,均未检出6种牛病毒;每种病毒仅在相应抗体进行染色时出现荧光;该方法检测6种牛病毒的灵敏度较高.分别采用细胞培养法和直接免疫荧光法检测15批新生牛血清和5批胎牛血清,结果均未检出牛病毒.结论 建立的牛血清中牛病毒直接免疫荧光检测方法重复性好,特异性强,灵敏度高,操作简便,与细胞培养法的检测结果无差异,可应用于牛血清中牛病毒的检测.

Objective To develop and verify a direct immunofluorescence assay for bovine virus in bovine serum.Methods A direct immunofluorescence assay was developed for determination of bovine virus in bovine serum,of which the cell concentration for inoculation,serum concentration virus inoculation quantity,fluorescent antibody concentration as well as time duration and temperature for staining were optimized.The optimized method was verified for reproducibility,specificity and sensitivity,by which the determination result was compared with that by cell culture method.Results The test condition was optimized as follows:the concentrations of Vero and BT cells for inoculation were 0.5 × 105 and 1.0 ×105 cells / ml,the serum concentration was 5％,the virus inoculation quantity was 100 ～ 300 CCID50,the dilution of fluorescent antibody was 1 ∶ 10,while the temperature for staining was 4 ℃,and the time for staining was more than 12 h but not more than 24 h.Three batches of bovine sera

采取无血清培养法培养出SW620细胞球,并对细胞球细胞进行干细胞鉴定;在细胞水平研究n-3多不饱和脂肪酸(n-3 PUFAs)对结肠癌干细胞样细胞的作用。方法:正常培养人结肠癌细胞株SW620并使其逐步适应无血清培养条件,经无血清培养1周后收集SW620细胞球。用免疫荧光法检测胚胎干细胞标志物SSEA-1和TRA-1-81;采用real-time PCR的方法检测干细胞相关基因Sox-2和Oct-4的表达情况;对比SW620贴壁细胞和干细胞样细胞(CSCLC)在软琼脂上的克隆形成能力;采用裸鼠移植瘤模型比较2种细胞的成瘤能力;用MTS法对比2种细胞在递增浓度的5-氟尿嘧啶(5-FU)或mitomycin C处理下的生长抑制情况;用MTS法、Annexin V/PI染色和台盼蓝染色分别观察递增浓度二十二碳六烯酸(DHA)和二十碳五烯酸(EPA)作用于SW620 CSCLC后细胞生长抑制情况、凋亡情况和死亡情况;MTS法检测5-FU或mitomycin C联合n-3 PUFAs对结肠癌CSCLC增殖的影响。结果:无血清培养法成功从SW620中培养出细胞球。细胞球细胞高表达SSEA-1和TRA-1-81并一过性表达Sox-2和Oct-4基因;对5-FU及mitomycin C相对抵抗;在软琼脂上克隆形成率及在裸鼠皮下的成瘤率均显著高于贴壁细胞,表明这些细胞具有干细胞特性,即为来自于SW620的CSCLC。DHA和(

[ ABSTRACT] AIM:To cultivate stem-like spheres from SW620 cell line in the specific serum-free medium and evaluate the features of the cancer stem cells, and to investigate the effects of docosahexaenoic acid ( DHA) and eicosapen-taenoic acid ( EPA) on the growth of SW620 stem cell-like cells.METHODS: Human colon cancer stem cell-like cells ( CSCLC) were obtained from SW620 spheres cultured in serum-free medium.These cells were tested for the expression of SSEA-1 and TRA-1-81 by immunofluorescence staining.The mRNA expression of Sox-2 and Oct-4 was detected by real-time PCR.The efficiency of colony formation on a soft agar gel and tumor formation in the nude mice was compared between SW620 adherent cells and CSCLC.The inhibitory effects of 5-fluorouracil (5-FU) and mitomycin C on both types of cells were measured by MTS assay.MTS assay, Annexin V/PI staining and trypan blue staining were used to determine the effects of DHA and EPA on both types of cells.MTS assay was also us

背景：以往研究是关于含磷复合物对动物源性或人源性其他干细胞的影响，三维情况下磷离子对人骨髓间充质干细胞作用研究尚未见。目的：探讨磷离子对三维培养条件下人骨髓间充质干细胞的影响。方法：实验分为6组，将人骨髓间充质干细胞接种在三维聚苯乙烯培养支架上，细胞分别在无血清生长培养基和添加4，8 mmol/L磷离子的无血清生长培养基中培养21 d；在二维聚苯乙烯培养板上培养的细胞作为相应的对照组。CCK8法检测细胞增殖情况，RT-PCR 检测成骨分化标记性基因的表达，茜素红染色检测人骨髓间充质干细胞成骨分化生成的矿化结节。结果与结论：培养4，7，14，21 d，相对于二维的细胞培养，磷离子的促细胞生长作用在三维聚苯乙烯培养支架上表现更明显(P <0.05)。相对与三维培养对照组，培养7，14 d时，胶原蛋白Ⅰ基因的表达在4，8 mmol/L磷离子三维培养组显著增加(P <0.05)；培养14 d时，碱性磷酸酶基因的表达在4，8 mmol/L磷离子三维培养组显著增加(P <0.05)；培养14，21 d时，骨钙素基因的表达在4，8 mmol/L磷离子三维培养组显著增加(P <0.05)。培养21 d时，在4，8 mmol/L磷离子三维培养组可生成矿化结节，在三维培养对照组无矿化结节生成。结果说明，磷离子可以促进

BACKGROUND:Previous researches have focused on the effect of phosphorus compounds on stem cels from animals or from human. But there is no study on the effect of phosphorus ions on human bone marrow mesenchymal stem cels under three-dimensional culture. OBJECTIVE:To explore the effect of phosphorus ions on human bone marrow mesenchymal stem cels under three-dimensional culture. METHODS:There were six groups in the experiment. Human bone marrow mesenchymal stem cels were inoculated in three-dimensional polystyrene scaffolds and then subjected to serum-free growth medium (group 3-GM) or serum-free growth medium containing 4 mmol/L (group 3-4P), 8 mmol/L (group 3-8P) phosphorus ions for 21 days, respectively. Cels cultured on the two-dimensional polystyrene scaffolds were used as control groups (groups 2-GM, 2-4P, 2-8P). Celular proliferation was examined by cel counting kit-8; the mRNAexpressions of osteogenic marker genes were assessed by RT-PCR; the formation of mineralized n