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1.
We have screened primary cultures of human prostate for the expression of markers reported to be characteristic of specific cell lineages in vivo, in order to ascertain whether human prostate cells in vitro maintain and reflect their in vivo differentiated phenotypes and to evaluate the homogeneity of the populations of cells that can be derived from this tissue. Using single and dual stain immunofluorescent microscopy to analyse very early organoid and subsequently derived monolayer stage cultures, we have observed that expression of markers characteristic of human prostate epithelial cells in vivo is deregulated within 48h, indicating that dissociation of human prostate tissue and cultivation of prostate epithelial cells in culture can result in promiscuous expression of cell type specific markers of prostate epithelial cells. These observations have important implications for studies of cell lineage and differentiation of prostate cells in vitro.  相似文献   

2.
In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.  相似文献   

3.
M M Webber 《In vitro》1979,15(12):967-982
Isolation of normal human glandular epithelia and their growth and maintenance in vitro have been major problems. The primary objective of studies presented here was to isolate postpubertal, normal human, viable prostatic epithelium for in vitro cultivation. The long-term objective of these investigations was to develop an in vitro human cell model system for studies on prostatic carcinogenesis. A method for isolation of viable, normal and benign human prostatic epithelium, using collagenase for tissue dissociation, is described. Intact acini were isolated, which, on plating gave rise to vigorously growing monolayer cultures of epithelial cells. The purity of epithelial cultures partly depended upon the source of tissue. Specimens of normal prostate and those of benign tissue derived from open prostatectomies provided primarily pure epithelial cultures with occasional fibroblast colonies in some cultures, which could be removed. Cultures from some specimens of transurethral resection of the prostate (TURP) contained many fibroblast colonies due to incomplete separation of acini from the stroma. This resulted from incomplete digestion of denatured tissue caused by electrocauterization during surgery. Cultures established in this manner are being used to study the effects of hormones, vitamins and other growth regulators in order to establish growth requirements of these cells in vitro, which would facilitate their long-term maintenance.  相似文献   

4.
In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1–6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiologicalcharacteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferationand invasive tumor growth. J. Cell. Physiol. 171:196–204, 1997. © 1997 Wiley-Liss, Inc.  相似文献   

5.
G E Milo  G A Ackerman  R L Sanders 《In vitro》1984,20(12):899-911
Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro.  相似文献   

6.
The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.  相似文献   

7.
The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.  相似文献   

8.
原代培养大鼠前列腺细胞建立前列腺增生筛药模型   总被引:1,自引:0,他引:1  
目的建立原代培养大鼠前列腺上皮细胞体外筛药模型。方法无菌状态下取雄性SD大鼠腹侧叶前列腺,称量后,剪成1 mm3小块,经Ⅱ型胶原酶消化1 h后,过滤、离心获取前列腺细胞,接种于24孔板培养并对细胞进行形态学和免疫组织化学鉴定。获取的大鼠前列腺上皮细胞分别接种于96孔板和24孔板培养12 d后给予系列剂量(0.1~1000μmol/L)的他莫昔芬和阳性药物爱普列特处理72 h,利用CCK-8法检测前列腺上皮细胞存活率并计算药物IC50值,并进一步通过Giemsa染色后观察细胞生长及形态变化。结果细胞鉴定结果显示,获取的细胞具典型上皮细胞形态特征,细胞角蛋白和前列腺特异性抗原表达阳性,提示所获细胞为前列腺上皮细胞。爱普列特与前列腺上皮细胞孵育72 h后,CCK-8法检测结果显示能够明显抑制前列腺上皮细胞生长,其IC50值为42.7μM,进一步镜下观察结果显示,爱普列特未明显改变大鼠前列腺上皮细胞形态,但能导致存活细胞数减少。他莫昔芬则对大鼠前列腺上皮细胞生长无明显抑制作用,镜下观察前列腺上皮细胞数量和形态未见明显改变。结论利用原代培养SD大鼠前列腺上皮细胞可以成功建立前列腺增生体外筛选模型。  相似文献   

9.
Epithelial cell transformation has been demonstrated in numerous animal models for the study of solid tumor biology. However, little evidence exists for human epithelial cell transformation without previous immortalization via genetic influences such as SV40 T-antigen, thus limiting our knowledge of the events that can transform naive human epithelium. Here we describe a system developed in our laboratory to directly transform freshly isolated primary human prostate epithelial cells without previous culture or immortalization. Prostate tissue is obtained from patients and benign tissue is separated from malignant tissue. Benign and malignant tissues are mechanically and enzymatically dissociated to single cells overnight, and immune cells and epithelial subsets are isolated on the basis of differential expression of surface antigens. Epithelial progenitor cells are transduced with lentiviruses expressing oncogenes and combined with inductive stroma for in vivo studies. At 8-16 weeks after transplantation into immune-deficient mice, the development of lesions, histologically classified as benign prostate, prostatic intraepithelial neoplasia and adenocarcinoma, can be evaluated.  相似文献   

10.
This work reports the isolation and characterization of a line of human, nontransformed and differentiated prostate epithelial cells (EPN) in continuous culture. Primary cultures of epithelial prostate cells were set up using normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer. After 70 passages, EPN cells did not undergo "Hayflike crisis" and were free of fibroblast contamination and were thus subcloned and characterized. EPN cells in culture, as prostate epithelial cells in vivo, express high-molecular weight cytokeratin and Pyk2, whereas they do not express desmin. EPN cells are nontransformed because they do not form colonies in semisolid medium and do not form tumors once injected into nude mice. EPN cells express the functional androgen receptor, which can mediate the mitogenic activity of testosterone. Finally, clonal production of the prostate-specific antigen could be detected in EPN cells. The availability of a line of epithelial nontransformed prostate cell in culture will be useful in investigating the complex process regulating normal prostate physiology as well as the development and progression of prostate tumors.  相似文献   

11.
Recent advances in cell biology and tissue engineering have used various delivery vehicles for transplanting varying cell cultures with limited success. These techniques are frequently complicated by tissue necrosis, infection, and resorption. The purpose of this study was to investigate whether urothelium cells, tracheal epithelial cells, and preadipocytes cultured in vitro could be successfully transplanted onto a prefabricated capsule surface by using fibrin glue as a delivery vehicle, with the ultimate goal for use in reconstruction. In the first step of the animal study, tissue specimens (bladder urothelium, tracheal epithelial cells, epididymal fat pad) were harvested for in vitro cell culturing, and a silicone block was implanted subcutaneously or within the anterior rectus sheath to induce capsule formation. After 6 to 10 days, when primary cultures were confluent, the animals were re-anesthetized, the newly formed capsule pouches were incised, and the suspensions of cultured urothelia cells (n = 40), tracheal epithelial cells (n = 32), and preadipocytes (n = 40) were implanted onto the capsule surface in two groups, one using standard culture medium as a delivery vehicle and the second using fibrin glue. Histologic sections were taken, and different histomorphologic studies were performed according to tissue type. Consistently in all animals, a highly vascularized capsule was induced by the silicon material. In all animals in which the authors used fibrin glue as a delivery vehicle, they could demonstrate a successful reimplantation of cultured urothelium cells, tracheal epithelial cells, or preadipocytes. Their animal studies showed that capsule induction in combination with fibrin glue as a delivery vehicle is a successful model for transplantation of different in vivo cultured tissue types.  相似文献   

12.
Summary Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro. This work was supported in part by grants from EPA, R 806638-01 and 131-640-1599A1  相似文献   

13.
Summary The objective of this work was to establish an in vitro multidimensional culture system for human ovarian surface epithelial (HOSE) cells as a model for ovarian carcinogenesis. The epithelial origin of cell outgrowth from cells obtained from the ovarian surface was confirmed by keratin staining. Two cultures from two different patients were established, HOSE-A and HOSE-B. Cultures were infected with a retrovirus expressing human papillomavirus genes E6 and E7 to extend their life span. HOSE cells were seeded onto collagen gels containing NIH3T3-J2 fibroblasts as feeder cells and grown to confluence submerged in growth medium. The collagen bed was then raised to the air-medium interface for 7 d (organotypic culture). Microscopically, fixed cultures revealed a single layer of flat cells growing on the collagen surface, reminiscent of HOSE cells in vivo. Infected HOSE-A and HOSE-B cells exhibited aberrant growth because they stratified. In addition, established ovarian cancer lines grown in this fashion stratified and showed malignant phenotypes. Thus, cells grown in organotypic culture resemble their in vivo counterparts, providing a basis for establishing a system to study growth, proliferation, differential gene expression, and perhaps malignant transformation of HOSE cells.  相似文献   

14.
Under standard culture conditions, epithelial cells grow with their basal surface attached to the culture dish and their apical surface facing the medium. Morphological and functional markers are located in the appropriate plasma membrane, and transepithelial transport occurs in a variety of cultured epithelia. As a result of the polarity of the cells and the presence of tight junctions between cells, on standard tissue culture dishes there is restricted access of growth medium to the basolateral surface of the epithelium, which is the surface at which nutrient exchange normally occurs. Greater differentiation of epithelial cultures can be achieved by growing primary cultures or continuous cell lines on permeable surfaces such as porous bottom cultures dishes in which the porous bottom is formed by a filter or membrane of collagen, or on floating collagen gels. In many cultures, differentiation varies with the time after the culture was seeded. Certain chemicals that accelerate differentiation in nonepithelial cells also accelerate the differentiation of epithelial cultures. Ultimately, defined media and specific substrates for cell attachment should lead to further differentiation of epithelia in culture.  相似文献   

15.
Mini-pig prostate epithelial cells exhibited the unique metabolic characteristics associated with the specialized function of production and secretion of high levels of citric acid. Epithelial cell suspensions from mini-pig prostate were successfully grown in primary and secondary cultures. The cultured epithelial cells exhibited rapid proliferation reaching confluency in approximately 6 days. Growth and proliferation of fibroblasts were markedly restricted by the dominance of epithelial cell growth. Confluent cultures could be maintained for approximately 6 weeks. The epithelial cells retained their polymorphic appearance in primary and secondary cultures and exhibited the characteristic formalin-resistant acid phosphatase reaction. Testosterone stimulated mitochondrial aspartate aminotransferase (mAAT) activity and citrate production by confluent epithelial cell cultures. These initial results indicate that cultured epithelial cells derived from mini-pig prostate might be an excellent model related to human for studies of prostate biology and hormonal regulation.  相似文献   

16.
Summary Reproducible subculture of adult human prostatic epithelial cells from normal, benign hyperplastic and malignant tissue has been achieved. Cholera toxin is the key component in the culture system, but use of an optimal basal medium (PFMR-4) supplemented with a high level of serum in collagen-coated dishes also improves growth and serial propagation. Editor’s statement The critical first step in the development of an optimized culture system for any cell type is to obtain enough growth to perform detailed growth-response studies. Multiplication of human prostatic epithelial cells in primary culture has been possible for several years, but scarcity of tissue specimens and inability to subculture have severely limited the usefulness of such cultures. The procedures described by Peehl and Stamey in this communication make it possible to expand the original inoculum substantially and to use subcultures to perform precisely controlled replicate experiments. This will open the way for rapid progress in the development of practical cell culture model systems for research on normal, hypertrophic, and malignant human prostatic cells. Richard G. Ham  相似文献   

17.
18.
Cells from cranial and spinal arachnoid membranes of humans were grown in culture. Their growth characteristics, morphology and details of their cytoskeletal composition are described. Arachnoid membranes, obtained at autopsy, were finely minced and incubated in tissue culture medium. Monolayers of cells of homogeneous morphology grew from these tissue fragments. The cells were flat and polygonal. They divided slowly to form non-overlapping monolayers of low cell density. Electron microscopic examination of cultured arachnoid cells revealed numerous desmosome-like tight junctions and abundant intermediate filaments (tonofilaments). Both morphological features are characteristic of arachnoid cells in situ, but not of cells in the fibroblast-rich dura mater. Immunofluorescence microscopy with monoclonal antibodies demonstrated cytokeratin in the cytoplasm of primary cultures of arachnoid cells. Thus we demonstrated that these cultured cells retained certain of the specific differentiated properties of arachnoid cells in situ and that they are not fibroblasts (which lack tight junctions and cytokeratins). To our knowledge, there have been no previous reports of in vitro growth of arachnoid cells. This in vitro model should be useful in studying the response of arachnoid cells to a variety of substances thought to be involved in the chronic inflammatory condition of the meninges known as arachnoiditis.  相似文献   

19.
The epididymides of adult male hamsters were surgically ligated at the junction of the distal corpus and proximal cauda regions. After 3 days, spermatozoa recovered from the distal corpus displayed greater progressive motility and head to head agglutination in capacitating medium than did those from intact controls, but had low fertilizing ability (3% fertilization rate) in vitro or in vivo. When these spermatozoa were incubated for 6 h with epithelial cells from the proximal cauda epididymidis, previously cultured for 3 days, they maintained motility and exhibited a significant increase in fertilizing ability (30% and 29% in vitro and in vivo respectively). The fertilizing ability of distal corpus spermatozoa incubated with 3-day-old cultures without androgens, or 8-12-day-old epithelial cells with fibroblast overgrowth, or without epithelial cells, remained low (5%). Increase in sperm fertilizing ability was associated with increased sperm binding to the zona pellucida in vitro. These results demonstrate that, under suitable culture conditions, the final stages in the development of hamster sperm fertilizing ability can be achieved in vitro. Factors secreted by cultured epithelium from the proximal cauda epididymidis are implicated in this maturation process.  相似文献   

20.
Summary— A mini organ culture of mouse gallbladder was developed as an alternative to primary cultures of epithelial cells of this organ. Small pieces of tissue were prepared and maintained in minimum essential Eagle medium with 10% foetal calf serum, for as long as 7 days. Qualitative and quantitative ultrastructural studies have been performed using electron microscopy. The viability of cells was evaluated by stereological quantification of endocytotic vesicles containing horseradish peroxidase and labelling of exocytotic glycoproteins with tannic acid. The morphology of tissue pieces during the 1st h of culturing and tissue isolated directly from animals exhibited no significant differences. However, after 4 h in culture degradative changes became evident in many cells. At that time, endo- and exocytosis were both dramatically reduced. After 24 h, the morphology, as well as endo- and exocytosis recovered and were comparable to the parameters of the tissue in vivo or after 1 h in culture. The endocytotic activity remained unchanged from day 1 to 7 of culturing, while the number of exocytotic vesicles gradually decreased after 2 days in culture. Our results prove that mini organ culture of gallbladder is morphologically and functionally comparable with the tissue in vivo and for studies of epithelium in culture it is more convenient than primary cultures.  相似文献   

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