无血清分离人结直肠癌细胞的体外培养形态及标志物表达特征

目的探讨人结直肠肿瘤干细胞在体外分化过程中细胞形态和干细胞相关标志物CD133的表达变化,为进一步研究结直肠肿瘤干细胞分化走向提供实验依据。方法取来源于人结直肠癌的细胞系HCT116,无血清培养分离出CD133+细胞,加血清诱导分化,相差显微镜下观察其形态变化;在未分化状态下无血清培养第7天和14天与血清诱导分化后收集细胞,利用流式细胞仪检测干细胞标志物CD133的表达量,采用激光共聚焦检测CD133表面标记分子的表达。结果 1)细胞形态:无血清培养分离的CD133+细胞,在生长过程中聚集成规则的细胞球,血清诱导后即贴壁生长,贴壁形态与同来源细胞形态一致,且再次无血清悬浮培养后聚集成球稳定生长。2)标志物变化:结直肠肿瘤干细胞未分化时CD133表面标记分子高表达,流式细胞仪检测未分化细胞CD133第7天表达率为(20.4±0.52)%,第14天表达率为(78.5±2.80)%,分化后表达率为(0.50±0.17)%。结论细胞形态和标志物表达改变均表明高表达CD133+的HCT116结直肠癌肿瘤干细胞可定向分化为同源的结直肠癌细胞,CD133+细胞经血清诱导后表达下调而使细胞失去干细胞特性。     

Surface morphology of the subfornical organ: effects of low and high sodium chloride diet

Ependymal cells found in the subfornical organ of the rat were counted. Cells covered by small microvilli, small protrusions and smooth cells were frequently found. Also present were cells with long or short cilia, cels with large protrusions and supraependymal cells. High and low sodium diets reduced the number of cells with large protrusions. Microvilli-covered cells increased after a low sodium diet.     

Age-related differences in the effect of in vivo administration of indomethacin on hemopoietic cell lineages of the spleen and bone marrow of mice

During 21 days of indomethacin treatment, erythroid cells in the spleens of both young adult and older mice, and in the bone marrow of young adult mice, were increased significantly early, in treatment, relative to age-matched control organs, and remained high throughout treatment. During drug exposure, the numbers of myeloid cells in young adult bone marrow, but not spleen, were reduced, but in older mice these cells were elevated in both organs. Lymphoid cells in the young adult and older mouse spleens decreased and increased, respectively, during treatment, but were unchanged and decreased, respectively, in the bone marrow of young adult and older mice. Monocytemacrophage cells in the spleen were elevated but unchanged in the bone marrow of both age groups. During 14 days of indomethacin treatment of houng adult mice, the proportions of precursor cells in DNA synthesis of only the splenic erythroid lineage were increased. Thus, the major hemopoietic lineages in both the bone marrow and spleen are affected by exposure to indomethacin in a time-dependent and age-dependent manner. For all lineages studied, those of the bone marrow were least disturbed, and/or were first to recover, even during continued drug exposure.     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

人结肠癌CW-2干细胞获取方法研究

目的运用3种不同的方法分离纯化人结肠癌CW-2干细胞,并对其分离纯化效率进行比较,探讨获得癌干细胞的有效方法。方法采用单纯无血清悬浮培养、无血清悬浮培养联合化疗药物、流式细胞分选技术分别富集人结肠癌细胞株CW-2干细胞;然后运用流式细胞术、NOD—SCID小鼠致瘤实验和Transwell侵袭实验分析比较3种方法的富集效率。结果无血清悬浮培养细胞．无血清悬浮培养联合化疗药物处理细胞和流式细胞仪分选技术分选后细胞中具有结肠癌干细胞特性的CD44＋EPCAM_细胞分别为（59．39±4．55）％、（74.36±6．78）％、（86．43±8．43）％;3群细胞的成瘤能力和侵袭能力都存在显著统计学差异（P值〈0．05）：流式细胞分选技术分选后细胞〉无血清悬浮培养联合化疗药物处理细胞〉单纯无血清悬浮培养细胞。结论流式细胞分选技术富集癌干细胞的能力强于单纯无血清悬浮培养和无血清悬浮培养联合化疗药物,无血清悬浮培养联合化疗药物又强于单纯无血清悬浮培养。     

Long-term production and culture of clones of human T-lymphocytes

Culture of blood T lymphocytes collected from normal individuals and cancer patients were carried out in presence of T cell growth factor (TCGF); these cultures presented cytotoxic activity directed against different targets (lectin activated cells, autologous cancer cells, antibody coated cells and K 562). In order to study separately the different effector subpopulations, isolation of single cultured cells were performed with the help of a micropipette under microscope and monoclonal cultures were carried out in presence of TCGF. In the preliminary cytotoxic assays performed in the clones: (1) a marked activity directed against lectin targets was observed in many clones and (2) an important N K activity was exhibited by the clone 45 B9 (65% of the tested cells lysed human lymphoma K 562 cells).     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals.

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Age-related differences in the effect of in vivo administration of indomethacin on hemopoietic cell lineages of the spleen and bone marrow of mice.

During 21 days of indomethacin treatment, erythroid cells in the spleens of both young adult and older mice, and in the bone marrow of young adult mice, were increased significantly early in treatment, relative to age-matched control organs, and remained high throughout treatment. During drug exposure, the numbers of myeloid cells in young adult bone marrow, but not spleen, were reduced, but in older mice these cells were elevated in both organs. Lymphoid cells in the young adult and older mouse spleens decreased and increased, respectively, during treatment, but were unchanged and decreased, respectively, in the bone marrow of young adult and older mice. Monocyte-macrophage cells in the spleen were elevated but unchanged in the bone marrow of both age groups. During 14 days of indomethacin treatment of young adult mice, the proportions of precursor cells in DNA synthesis of only the splenic erythroid lineage were increased. Thus, the major hemopoietic lineages in both the bone marrow and spleen are affected by exposure to indomethacin in a time-dependent and age-dependent manner. For all lineages studied, those of the bone marrow were least disturbed and/or were first to recover, even during continued drug exposure.     

Surface morphology of the subfornical organ: Effects of low and high sodium chloride diet

Summary Ependymal cells found in the subfornical organ of the rat were counted. Cells covered by small microvilli, small protrusions and smooth cells were frequently found. Also present were cells with long or short cilia, cels with large protrusions and supraependymal cells. High and low sodium diets reduced the number of cells with large protrusions. Microvilli-covered cells increased after a low sodium diet.Acknowledgments. We thank Dr A. C. Taquini for his helpful discussions and Servicio de Microscopía Electrónica de Barrido, Conicet, and Facultad de Odontologia for technical assistance.     

Retroviral-mediated gene therapy for the treatment of citrullinemia. Transfer and expression of argininosuccinate synthetase in human hematopoietic cells

Citrullinemia is a recessive genetic disease caused by a deficiency in argininosuccinate synthetase (AS). Retroviruses were used to transduce the human AS gene into cultured human cells. Using amphotropic viruses with high titer (>10⁶ cfu/ml), we were able to correct the defect in cultured fibroblasts from citrullinemic patients. Retroviral transduction of the human AS gene into human bone marrow cells was also studied. Co-cultivation was used to infect the cells and up to 80% of progenitor cells were found to be carrying and expressing the AS retrovirus after infection. When the infected cells were kept in culture, integration and expression of the retrovirus was observed. Retroviral sequences were present and expressed in the cultured bone marrow-derived cells for up to 10 weeks.     

Mitogenicity of autolysates of Trypanosoma congolense

Autolysates of Trypanosoma congolense, in subcytotoxic amounts, were found to be highly mitogenic in vitro for the spleen cells of normal mice. Significant amounts of [3H]-thymidine were also incorporated by the responding spleen cells of nu/nu (athymic) mice. In contrast, the spleen cells of cyclophosphamide-treated mice were unresponsive. The findings suggest that a potent B-cell-mitogen is generated by the autolysing T. congolense organism.     

Effects of mistletoe (Viscum album L.) extracts on cultured tumor cells

Bacterially fermented mistletoe preparations (BFMP) were tested on rat hepatoma tissue culture (HTC) cells and human leukemia Molt 4 cells. A dose-dependent inhibition of the growth rate of the cells was observed. For both cell lines, cytostatic concentrations, expressed in weight of fresh plant, were 0.5 mg/ml culture medium for oak BFMP and 1 mg/ml for apple tree BFMP. However, the action of the two preparations was markedly different on each cell line. Non-viable HTC cells were not stained by trypan blue while non-viable Molt 4 cells were fully colored by this reagent. A lysis of cellular membranes of HTC cells was observed by electron microscopy. Furthermore, oak BFMP inhibited the growth of virus transformed 3T3-SV40 cells more than that of non-transformed 3T3 cells. In contrast to BFMP, non-fermented extracts and a purified mistletoe lectin showed a greater inhibition of the growth of Molt 4 cells than of HTC cells. Samples withdrawn at different times during fermentation gradually lost their inhibitory effect on the growth of Molt 4 cells while their action on HTC cells increased up to the 4th day of fermentation. These results are discussed in relation to the cytotoxic substances of mistletoe already characterized.     

In vitro morphological characterization of the bursal reticuloepithelial (REp) cells of chicken

Summary The REp cells of the bursa follicle medulla of chicken were isolated in vitro. Culture of the REp cells was maintained over a period of 10 days and the cells were observed at 3 and 10 days by means of transmission electron microscopy (TEM) and immunofluorescence. The use of an anticytokeratin monoclonal antibody confirmed their epithelial nature. TEM observations showed the presence of desmonsomes and tonofilaments, which are characteristic of epithelial cells. Furthermore, to some extent the cells regenerated in vitro the network they form in vivo. Though the growth rate becomes slower with time, the features of the REp cells do not significantly change.     

Studies on chemically induced tumors in rats: I. Heterogeneity of tumor cells and establishment of syngeneic, tumor-specific cytotoxic T cell clones

Sarcoma P1 was induced in DA rats by DMBA. Anti-P1 antibodies were produced in DA rats, purified via fixed tumor cells and used to induce anti-idiotypic antibodies in syngeneic rats. The anti-idiotypic antibodies were used to generate cytotoxic, P1 specific DA T cells in vitro. These cytotoxic T cells and P1 tumor cells were cloned by limiting dilution. Using the DA anti-P1 specific cytotoxic T cell clones, we were able to characterize 2 types of P1 tumor cell clones, namely those which were susceptible and those which were resistant to the P1 specific cytotoxic T cells. Cytotoxic T cell injected i.v. into syngeneic DA rats could not prevent the development of lethal P1 tumors.     

The fine structure of walker 256 carcinoma cells

Summary Several ultrastructural features of Walker 256 rat tumor cells which have not been observed before are revealed. Nuclear bodies either singlely or in pairs were found in some cells. Cytofilaments were observed in the cytoplasm and cytoplasmic projections in a few cells. Desmosomes which are usually regarded as a feature of differentiated cells were also observed.     

In vitro morphological characterization of the bursal reticuloepithelial (REp) cells of chicken.

The REp cells of the bursa follicle medulla of chicken were isolated in vitro. Culture of the REp cells was maintained over a period of 10 days and the cells were observed at 3 and 10 days by means of transmission electron microscopy (TEM) and immunofluorescence. The use of an anticytokeratin monoclonal antibody confirmed their epithelial nature. TEM observations showed the presence of desmosomes and tonofilaments, which are characteristic of epithelial cells. Furthermore, to some extent the cells regenerated in vitro the network they form in vivo. Though the growth rate becomes slower with time, the features of the REp cells do not significantly change.     

Immunohistochemical individualization of adenohypophyseal thyrotropic cells in the monkey Macaca irus, using a human anti-beta-TSH antibody

Using the indirect immunofluorescence technique and an antiserum against the specific beta subunit of human TSH, selective immunocytochemical staining was localized in a cell population of the pars distalis of the monkey Macaca irus. These thyrotropic cells, which were positive to aldehyde fuchsin, Alcian blue, and PAS, were localized on the one hand in the ventro-medial zone of the pars distalis, especially alongside the large vessels of this region, and on the other in the zona tuberalis, i. e. at the bottom of the pars tuberalis. Some cells, sparsely distributed, were seen in the posterior part of the lateral lobes of the pars distalis, in the vicinity of the pars intermedia. No thyrotropic cells were encountered in pars tuberalis. Pars intermedia and posterior lobe contained no cells immunoreactive to beta-hTSH. In females, the thyrotropic cells were more numerous and larger than in males. Generally the thyrotropic cells were round or oval in shape, sometimes they were polygonal.     

Primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum

This is the first report on a primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum. These primary cells revealed a low seeding efficiency after 3 days (11.6 ± 4.6%), whereas subcultured cells had a higher seeding efficiency at the same time point (75.5 ± 34.0%) and increased in cell number (150 – 200% of seeded cells after 20 to 30 days). The cells were characterized applying histological, immunocytochemical and ultrastructural methods. The culture consisted of undifferentiated keratinocytes. Mucous cells as well as differentiated epithelial cells were absent. To date the cells were cultured for maximally 9 passages and 402 days and therefore provide the possibility for long-term studies. Received 31 March 1998; received after revision 14 July 1998; accepted 14 July 1998     

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with³H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.     

靶向CD133的RNA干扰对人肝癌SMMC-7721细胞的生长抑制作用

目的利用RNA干扰技术下调SMMC-7721肝癌细胞中CD133的表达,观察其在肝癌细胞增殖和侵袭方面的作用.方法构建CD133特异性的siRNA真核表达载体,转染SMMC-7721肝癌细胞并筛选出稳定表达siRNA的转染克隆;应用RT-PCR和Western blot检测CD133 mRNA和蛋白的表达;流式细胞仪检...