Location of osteoclast precursors in fetal rat calvaria cultured on collagen gels

Although osteoclasts are derived from hematopoietic cells, the exact identity of their precursors and the mechanism for their recruitment onto bone surfaces remain unclear. We wished to study their differentiation in the fetal rat calvaria and to locate its source of osteoclast precursor cells. Osteoclasts were detected by neutral red staining or cytochemical reaction for acid phosphatase of intact bone (cell number and area measured by computerized image analysis) or in cryostat sections of bone (enzyme activity measured by quantitative cytochemistry). Histology of semithin sections of fixed bones was also examined. The 19 day calvariae contained few mature osteoclasts. After 48 h culture on gels of type 1 collagen (1.5 mg/ml) supplemented with 5 mM calcium beta-glycerophosphate, 10 mM proline, and 2 micrograms/ml ascorbic acid, numerous large osteoclasts were seen on their endocranial surfaces. In contrast, cell morphology and enzyme activity deteriorated in bones cultured in liquid medium. The cells that formed in vitro rapidly responded to calcitonin by contraction. Stripping of endocranial membranes from the calvariae prevented osteoclast formation in culture, but these cells were seen when "stripped" bones had been cocultured with their membranes for 48 h or with intact 16 day calvariae (well before the onset of osteogenesis). Few osteoclasts were found when an 0.22 micron filter was inserted between the stripped calvaria and the endocranial membranes. We conclude that the endocranial membranes, which contain the meningeal blood vessels, are a major source of osteoclast precursors and that these cells are present in calvarial tissue even before the onset of osteogenesis.     

Canine ACL fibroblast integrin expression and cell alignment in response to cyclic tensile strain in three-dimensional collagen gels.

Tissue-engineered ligament substitutes have the potential to become an alternative graft source for ligament reconstruction. If this approach is to become viable, one must first understand and define the mechanisms responsible for creation, maintenance, and remodeling of the native anterior cruciate ligament. It is well accepted that mechanical load alters fibroblast phenotypic expression in a variety of cell sources; however, the mechanosensitive pathways responsible for alteration in matrix production, remodeling, and alignment are unknown. We hypothesize that cell surface integrins play a role in this mechanotransduction process, and as such respond to application of cyclic tensile load. Linear 3D collagen gels containing canine ACL fibroblasts were created in Flexercell Tissue-Train Culture Plates. Gels were untethered (control), tethered without external strain (tethered), or tethered and exposed to 2.5% cyclic strain for 2 h per day for 4 days (strain). Quantitation of alpha1, alpha5, and beta1 integrin subunit was performed using flow cytometry. Cell and matrix alignment was studied using light, polarized light, and fluorescent microscopy. Expression of alpha5 and beta1 integrin subunits was increased significantly in fibroblasts in tethered and strained 3D collagen gels compared with the control, unloaded constructs (p < 0.05). These integrins are known to function as mechanotransducers in other tissues, implicating a similar role in mechanotransduction in ACL fibroblasts. Histologic analysis of the tethered and strained gels demonstrated a linear arrangement of cells and parallel collagen fibril architecture. In contrast, cell distribution and collagen alignment were disorganized in the control, unloaded gels. The alignment of cells and collagen in the 3D gels parallel to applied strain is similar to the in vivo state. These data add to our understanding of the behavior of ACL fibroblasts in vitro. The ability to manipulate signal transduction pathways may enhance our ability to engineer implantable ACL grafts or to modify ACL healing response.     

钙黏蛋白12在结肠癌细胞株与结肠直肠癌组织中的表达及其影响

目的：研究钙黏蛋白12（cadherin-12,CDH-12）在结肠癌细胞株与结肠直肠癌组织中的表达及其对结肠癌细胞株增殖、侵袭、迁移的影响。方法：应用实时PCR和Western印迹法在7株结肠癌细胞株中筛选CDH-12高表达细胞株;shRNA瞬转干扰高表达细胞株SW1116,Western印迹法验证干扰效果;CCK-8检测癌细胞增殖活性变化;transwell实验验证癌细胞迁移、侵袭能力的变化;组织芯片免疫组织化学技术检测CDH-12在结肠直肠癌组织中的表达。结果：实时PCR和Western印迹结果显示CDH-12在细胞株中表达,SW1116和LoVo两个细胞株高表达CDH-12,在成功下调SW1116细胞中CDH-12表达之后,SW1116结肠癌细胞株的增殖、迁移、侵袭能力较对照组明显下降;免疫组化染色结果显示,CDH-12在结肠直肠癌组织中的表达明显高于癌旁正常组织中的表达。结论：CDH-12在结肠直肠癌组织中的表达明显高于癌旁正常组织,证实了CDH-12为癌基因的可能性;下调CDH-12在结肠癌细胞株SW1116的表达可明显抑制细胞的增殖、迁移、侵袭能力,显示其在肿瘤细胞生长和转移中的重要作用。     

Effects of growth factors on cell proliferation and matrix synthesis of low-density, primary bovine chondrocytes cultured in collagen I gels.

Low cell density cell numbers and dedifferentiation are two major problems of human chondrocyte culture associated with articular cartilage repair. Bovine chondrocytes seeded at low density (3.5 x 10(4) cells/ml of gels) in three-dimensional collagen type I gels do proliferate and maintain their phenotype as shown by cell counts, morphology and matrix synthesis. The combination of three growth factors (3GFs; 10 ng/ml TGF-beta1 + 100 ng/ml IGF-I + 10 ng/ml b-FGF) added to serum-free culture medium in this culture system enhances the mitotic activity of bovine chondrocytes similar to 20% foetal calf serum (FCS). At day 21, cells proliferated by 41 fold in gels-FCS and 37 fold in gels-3GFs. Protein synthesis by gels-3GFs cultures was similar to 20% FCS when cultured for 3 weeks but much less proteoglycan was synthesized. The matrix deposition as observed by light and electron microscopy was quite different. More small diameter branching collagen fibrils and a denser matrix were presented in gels-FCS culture whilst loosely arranged larger diameter collagen fibrils were observed in gels-3GFs.     

Antitumor effects of 5-fluorouracil on human colon cancer cell lines: antagonism by levamisole

BACKGROUND: The addition of levamisole (Lev) to 5-fluorouracil (5-FU) for the adjuvant treatment of stage III colon cancer has been shown to improve 5-year survival in patients. The mechanism of action of Lev remains unknown. Because we showed little in vitro immunological effect of Lev, we asked whether Lev, alone or in combination with 5-FU, had antitumor activity in vitro. METHODS: Proliferation of COLO-205 and HT-29 colon cancer cells incubated for 2 to 3 days in Lev and 5-FU was measured in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium colorimetric assays. Cell cycle analysis was performed by treating tumor cells for 6, 24, and 48 h with Lev and 5-FU, staining cells with propidium iodide, and measuring DNA content by flow cytometry. RESULTS: The addition of Lev to 5-FU did not reduce proliferation below that of 5-FU alone. The inhibitory concentration 50% (IC(50)) for 5-FU was 3.2 x 10(-6) M for COLO-205 and 1.3 x 10(-5) M for HT-29. An IC(50) was not reached for Lev, even at millimolar doses. DNA analysis of cells treated for 48 h revealed significant S-phase accumulation of both HT-29 (from 17% in control cells to 36% in treated cells) and COLO-205 (from 35% in control cells to 59% in treated cells) cell lines at micromolar 5-FU concentrations. In contrast, Lev alone did not affect cell cycle distribution for either cell line. The addition of Lev to 5-FU not only did not augment, but inhibited, the effects of 5-FU. CONCLUSIONS: Levamisole has no direct cytotoxic effect and no additive or synergistic cytotoxic effect when combined with 5-FU on two colon cancer cell lines. Either the observed clinical effects of Lev treatment occur through an as yet unknown mechanism, require longer treatment periods in vitro to become evident, or the results of clinical trials showing its effectiveness should be carefully reexamined.     

大肠癌干细胞研究进展

随着肿瘤下细胞研究的深入,近年来学者提出了大肠癌起源的干细胞学说,并借助干细胞分离技术,将其成功分离.本篇旨在回顾基于肿瘤干细胞概念提出的大肠癌起源假说和大肠癌干细胞在大肠癌进展的作用,大肠癌干细胞分离技术及其在体内和体外的功能特征,与大肠癌干细胞相关的信号通路及大肠癌耐药机制研究.     

人乳头瘤病毒16型E6E7癌基因对结肠癌细胞周期和增殖的影响

目的检测和分析人乳头瘤病毒(HPV)16型E6E7癌基因对结肠癌细胞增殖状况和细胞周期的影响。方法运用含有HPV16 E6E7癌基因的重组腺伴随病毒(rAAV—E6E7)感染HCT116和SW480结肠癌细胞株,应用流式细胞术检测感染效率,Western blot法检测HPV16 E6癌蛋白的表达。MTT法和流式细胞技术分别检测感染前后结肠癌细胞的增殖状况和细胞周期。结果rAAV—E6E7感染结肠癌细胞后24h开始可以检测到HPV16 E6癌蛋白的表达,未感染的细胞不表达HPV16 E6蛋白。rAAV—E6E7感染后,HCT116细胞群体倍增时间缩短为对照组的68．42％,OD540值升高47.48％,G0／G1期细胞比例下降,S期细胞比例增高。SW480细胞群体倍增时间缩短为对照组的77.06％、OD540值升高47.18％。结论高危型HPV的E6E7癌基因能够促进结肠细胞的增殖、干扰其细胞周期,有可能在结直肠癌的发生、发展过程中发挥作用。     

伴侣蛋白CCT6A参与结肠癌细胞迁移及侵袭的作用研究

目的:研究伴侣蛋白CCT6A在结肠癌细胞株迁移和侵袭过程中的作用。方法:培养9株结肠癌细胞,抽取总RNA和蛋白。采用实时定量PCR和Western印迹法筛选CCT6A高表达的细胞株。设计购买3种siRNA片段,转染高表达CCT6A的结肠癌细胞株,利用实时定量PCR和Western印迹法验证干扰效果。选择干扰效果最优的RNA干扰片段,通过Transwell实验来评估转染后结肠癌细胞迁移和侵袭能力的变化。结果:SW1116细胞株中的CCT6A在mRNA及蛋白水平都呈现高表达;3种干扰片段转染SW1116细胞后,CCT6A表达量均有不同程度下降,其中以片段2的干扰效果最优。在迁移实验中,CCT6A干扰组穿膜细胞数为(6±2)个,低于阴性对照组(23±12)个和空白对照组(21±11)个;在侵袭实验中,CCT6A干扰组穿膜细胞数为(9±2)个,低于阴性对照组(40±12)个和空白对照组(43±10)个;上述差异均有统计学意义(P<0.05)。结论:下调CCT6A的表达能显著抑制结肠肿瘤细胞的迁移、侵袭能力,提示CCT6A可能在结肠癌浸润和转移中起到一定作用。     

Human breast cancer cell lines as models of growth regulation and disease progression

The routine isolation and culture of human breast cancer cells from patients samples has been a goal of breast cancer cell biologists for over 30 years. Despite extensive work in this area and the development of many human breast cancer cell lines, the proportion of patient samples that give rise to immortalized breast cancer cell lines is still disappointingly low. The majority of human breast cancer cell lines that have been established were isolated many years ago and have been grown continuously under poorly defined culture conditions. These cell lines have been useful for studies of the estrogen receptor biology in human breast cancer cells, in identifying growth factors synthesized by breast cancer cells, and for the characterization of genetic alterations in oncogenes and tumor suppressor genes present in these cells. More recently, tissue culture methods have improved, resulting in the ability to culture routinely normal human mammary epithelial cells of specific lineages and this has resulted in the development of new human breast cancer cell lines. The ability to isolate and culture normal and neoplastic human mammary epithelial cells under similar culture conditions has improved these models dramatically and has resulted in the identification of altered cellular phenotypes of human breast cancer cells.     

Characterization of in vivo cross-reactive tumor resistance between two cultured murine colon lines

Previous comparative investigations of the in vivo biology of two cultured colon tumors of BALB/c origin, C-C26 and C-C36, demonstrated different biologic activities. To elucidate these differences, the immunogenicity of these lines in providing protection to a subsequent challenge with different doses of tumor cells was investigated. Furthermore, the specificity of this protection was evaluated with cross-protection experiments. The findings demonstrate these lines to be immunogenic and suggest the presence of cross-reactive tumor rejection type antigens on these two cultured colon tumor lines. BALB/c mice immunized with these tumors developed resistance to challenge with the same tumor as well as to challenge with the other colon tumor line. Unexpectedly, BALB/c mice immunized with C-C26 cells and challenged with a syngeneic methylcholanthrene-induced sarcoma, Meth A, exhibited partial resistance to this tumor compared to nonimmunized controls. However, mice immunized with C-C36 cells and challenged with Meth A cells supported the same rate of growth of this tumor as unimmunized controls. These results suggest the presence of at least two different cross-reactive tumor rejection antigens. The first antigen appears to be shared between C-C26 and C-C36 cells while the second antigen may be shared between C-C26 and Meth A. Further definition of these putative antigens could provide us with a better understanding for the diagnosis and treatment of colon tumors.     

CO2对结肠癌细胞表面黏附分子mRNA表达的影响

目的 探讨不同压强持续性CO2压力对结肠癌细胞表面黏附分子mRNA表达的影响。方法 建立体外气腹模型，选用人结肠癌细胞株SW1116，分别在6、9、12和15mmHg压强下，以99％医用CO2气体以及常规培养条件下暴露1h，采用实时荧光定量PCR检测细胞表面黏附分子0，12，24，48和72h的表达变化。结果 SW116细胞株E—cadherin，ICAM-1，CIM4，CIM4v6 mRNA在处理后表达增高，与处理前差异有统计学意义，在处理后的72h之前表达降至或低于处理前水平。E—cadherin、CD44v6和ICAM-1随压强增高其表达量逐渐降低，在处理后0h各压强组差异有统计学意义。结论 持续性CO2压力对结肠癌细胞表面的黏附分子mRNA的表达产生一过性双向影响；随CO2气体压强增高，黏附分子的表达遭到抑制.     

CO_2对结肠癌细胞表面黏附分子mRNA表达的影响

目的探讨不同压强持续性CO2压力对结肠癌细胞表面黏附分子mRNA表达的影响。方法建立体外气腹模型,选用人结肠癌细胞株SW1116,分别在6、9、12和15mmHg压强下,以99%医用CO2气体以及常规培养条件下暴露1h,采用实时荧光定量PCR检测细胞表面黏附分子0,12,24,48和72h的表达变化。结果SW1116细胞株E-cadherin,ICAM-1,CD44,CD44v6mRNA在处理后表达增高,与处理前差异有统计学意义,在处理后的72h之前表达降至或低于处理前水平。E-cadherin、CD44v6和ICAM-1随压强增高其表达量逐渐降低,在处理后0h各压强组差异有统计学意义。结论持续性CO2压力对结肠癌细胞表面的黏附分子mRNA的表达产生一过性双向影响;随CO气体压强增高,黏附分子的表达遭到抑制。     

The Viennese culture method: cultured human epithelium obtained on a dermal matrix based on fibroblast containing fibrin glue gels

The aim of this study was to develop a new keratinocyte culture system on a dermal equivalent suitable for skin wound closure. Our dermal matrix is based on a fibrin glue gel containing live human fibroblast (from human foreskin). Keratinocytes obtained from primary culture according to the Rheinwald and Green method, were seeded on to the gel. In all cases, the keratinocytes plated on the dermal equivalent grew to confluence and stratified epithelium was obtained. After 10 days an irregular multilayer could be observed. The cells showed active interaction with the fibrin support, presenting as cell formations projecting into the matrix. After 15 days a regular epithelial sheet consisting of three to four layers of cells was formed. A limiting membrane demarcating the keratinocytes from the fibrin matrix was discernible. Squamous differentiation similar to Strata reticulare and corneum found in vivo could be observed. Nuclei of basal cells were regularly spaced from each other and the chromatin was of homogeneous appearance without prominent nucleoli. The last time point (20 days) showed signs of disintegration of the epithelial sheet. A basement membrane-like structure could not be seen any more. Detachment of the basal cells was associated with subepithelial vacuoles. Basal cells contained irregular nuclei. Therefore, we conclude that 15 days of culture were optimal for the generation of a keratinocyte layers with signs of differentiation; this new culture system could be an important step forward in covering severely burned patients due to a number of advantages, as for example a large expansion factor, the shortening of the optimal culture time to 15 days, the usage of commercially available fibrin glue gels and the versatile manipulation of composite cultures.     

非甾体类消炎药抑制结肠癌细胞株细胞生长的实验研究

目的 研究非甾体类消炎药（NASIDs）通过产生一氧化氮的亚硝基谷胱甘肽（GSNO）对结肠癌细胞生长的影响及其机制。方法 采用细胞生长曲线和流式细胞仪方法研究细胞凋亡，竞争酶联免疫分析测定细胞培养上清PGE2的环氧化酶2（PGE2）表达水平，Western Blot法检测COX-1／COX-2蛋白表达。结果 在不同时间和浓度梯度中，GSNO处理可增加PGE2的产量。用500μmol／L处理48h后，COX-1和COX-2的蛋白表达水平增加。NASIDs可以阻断PGE2的产生，但对于GSNO诱导的细胞生长抑制无影响。结论 GSNO可促进细胞产生PGE2增加，并诱导COX-1和COX-2蛋白呈时间依赖和剂量依赖性表达；高浓度的GSNO可抑制细胞生长，并诱导3种细胞株凋亡，而不受COX-2表达及其活性的影响。NADIs能阻断PEG2产生，但对于GSNO诱导的结肠癌细胞株生长抑制并无协同作用。     

Culture of chondrocytes in alginate and collagen carrier gels

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation.

Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation.

The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.     

结肠癌干细胞研究进展

随着肿瘤干细胞学说的提出,人们陆续从白血病、乳腺癌、脑肿瘤、肺癌、前列腺癌等多种恶性肿瘤组织中分离出肿瘤干细胞.有学者研究发现CD133+可能为结肠癌干细胞的表面特异性标志,这一观点的提出引起人们对结肠癌干细胞的研究热潮.本文通过结肠癌干细胞在临床耐药、转移及应用中的作用对近年结肠癌干细胞研究进展进行综述.